Invited Oral Abstract
Quantifying glycogen in waste activated sludge from municipal wastewater treatment facilities
Raymond Red Corn and Abigail Engelberth, Purdue University, West Lafayette, IN, USA
39th Symposium on Biotechnology for Fuels and Chemicals
Waste Activated Sludge (WAS), produced from Municipal Wastewater Treatment, contains glycogen, which if separated, could be a viable source of glucose for the production of various biobased chemicals. Prior work, utilizing varied methods applied to lab scale sludge reactors, indicates glycogen comprises greater than 10% of WAS solids. However, full scale WAS systems have yet to be evaluated, and methodologies have yet to be compared or refined. In the present work we compare previously defined glycogen assays then apply a refined method to multiple full scale wastewater treatment operations that utilize either a traditional Activated Sludge system or an Enhanced Biological Phosphorus Removal system. Results indicate that Pfluger’s method (5M KOH at 100°C) releases greater glycogen from sludge than sonication up to 7 minutes, when both were followed by enzymatic hydrolysis for final measurement of monomeric glucose. Treatment using 6M HCl effectively released and hydrolyzed glycogen in a single step, but also hydrolyzed cellulosic pools of glucose resulting in an overestimate of glycogen. A refined version of Pfluger’s method was applied to multiple treatment facilities to provide a macro view of the resource potential of glycogen in WAS.
Invited Oral Abstract
Green synthesized copper nanoparticles into granular activated carbon of babassu coconut by Hibiscus Sabdariffa flowers
Rebecca Paixo1, Isabela Reck1, Prof. Marcelo Vieira2, Prof. Rosngela Bergamasco3 and Anglica Marquetotti Salcedo Vieira1, (1)State University of Maringa, Maringa, Brazil, (2)State University of Maring, Maring, Brazil, (3)State University of Maring, Maring, PR, Brazil
39th Symposium on Biotechnology for Fuels and Chemicals
Adsorption processes are widely used for the purification and separation of products and contaminants. The inclusion of nanoparticles on the surface of the adsorbents changes as their physicochemical properties. In this study, granular activated carbon (GAC) of babassu coconut was used as adsorbent and it was developed a green synthesized method for the impregnation of copper nanoparticles into its surface using Hibiscus Sabdariffa flower extract. The impregnated carbon was characterized using scanning electron microscopy (SEM), X-ray diffraction (XRD) and Total Reflection X-ray fluorescence (TRXF). After the characterization, the nitrate was used as the standard contaminant for the evaluation of the new adsorbent. For the batch adsorption studies, solutions containing 100 mg / L of nitrate were used. The characterization confirmed the successful synthesis of copper nanoparticles since peaks related to the copper oxides were detected by XRD and copper quantity was determined by TRXF. Also, SEM confirmed the difference between the surface of both pure and impregnated GAC, suggesting the presence of some nanoparticles. The results of NO3- adsorption on impregnated GAC demonstrated that is exothermic process and favorable at lower temperature. The present work indicated that the impregnated GAC is a promising material for removal of nitrate, where the impregnation of copper oxide increased the efficiency of nitrate removal by more than 240%, compared with the pure GAC, at same experimental conditions.
Invited Oral Abstract
The effects of coumaryl alcohol on lignin dehydrogenation polymer synthesis, composition and structure
Dr. Anne Harman-Ware1, Renee Happs1, Prof. Brian H. Davison2 and Mark Davis2, (1)National Renewable Energy Laboratory, Golden, CO, USA, (2)Oak Ridge National Laboratory, Oak Ridge, TN, USA
39th Symposium on Biotechnology for Fuels and Chemicals
Lignin dehydrogenation polymers (DHPs) were synthesized using a Zutropfverfahren method with horseradish peroxidase and three lignin monomers, sinapyl (S), coumaryl (H) and coniferyl (G) alcohol, in the presence of hydrogen peroxide. The H monomer was incorporated in to S, G and S/G =1 polymers at molar compositions of 0, 10 and 20 % to study how the presence of the H monomer affected the structure and composition of the polymers. The DHPs were analyzed using GPC, GC/MS, thioacidolysis, pyrolysis-Molecular Beam Mass Spectrometry and NMR. GPC analysis suggests that the H monomer has an effect on the molecular weight of the polymers. Thioacidolysis showed higher recoveries of thioethylated products from polymers with higher H content, indicating an increase in the abundance of reactive ether bonds with an increase in H content. NMR spectra of solvent-soluble oligomers showed that the H monomer caused minor changes in the bond distributions of the polymers. Overall, the experimental results generally agree with theoretical predictions for the reactivity and structural influences of the H monomer on lignin-like polymers. The results suggest that the incorporation of the H monomer in the lignin structure may influence the recalcitrant properties of biomass and may help guide genetic modifications of biomass to improve conversion technologies.
Invited Oral Abstract
Simultaneous saccharification and fermentation (SSF) of draught resistant California agave spp. to fuel ethanol
May-Ling Lu, Bourns College of Engineering, University of California Riverside, Riverside, CA, USA and Dr. Charles E. Wyman, Center for Environmental Research and Technology, Bourns College of Engineering,University of California Riverside, Riverside, CA, USA
39th Symposium on Biotechnology for Fuels and Chemicals
With the signing of Assembly Bill (AB) 32 in 2006 to cut greenhouse gas emissions 80% below the 1990 level by the year 2050, California thrust itself into the forefront of reducing greenhouse gas (GHG) emissions. This aggressive reduction in GHG emissions requires blending of cellulosic ethanol into the gasoline pool as cellulosic ethanol can have a much greater impact on carbon footprint reduction than first generation starch-based ethanol. In light of the importance of meeting this goal, energy crops must be identified that are suitable for cultivation in the State without affecting the State’s status as a major breadbasket for the Nation. Drought-resistant agave, with its low lignin content and high water use efficiency, along with productivity comparable to woody and herbaceous energy crops, appears ideal for the State. This presentation presents results for optimization of enzyme loading in simultaneous saccharification and fermentation (SSF) of the unpretreated California agave natives A. Americana and A. deserti into fuel ethanol using Saccharomyces cerevisiae D5A at 1% solid loading.
Invited Oral Abstract
Storage moisture content impacts on the rates and extents of aerobic degradation of high-biomass sorghum
William A. Smith, Lynn M. Wendt and J. Austin Murphy, Idaho National Laboratory, Idaho Falls, ID, USA
39th Symposium on Biotechnology for Fuels and Chemicals
Utilization of high-biomass sorghum for conversion to fuels and chemicals requires stable storage to enable year-round use of this seasonally-harvested feedstock. As with other herbaceous biomass feedstocks such as corn stover and switchgrass, outdoor storage of baled sorghum represents a cost-effective starting point for this emerging industry. However, sorghum harvest moisture often exceeds 50%; a challenge to long-term aerobic bale storage. Field drying of sorghum to an industrially-accepted stable moisture content of <20% with its thick stems and high soluble sugar content is difficult but may not be necessary. Previous results from our work using corn stover show that dry matter loss rates and extents decrease non-linearly as moisture content drops from 40% to 30%. This study shows the rates and extents of dry matter loss (DML) in sorghum stored aerobically at a range of moisture contents from 80% to 30% using 100-L reactors to control moisture, oxygen flow, and heat loss and compares DML and composition to anaerobically-stored sorghum as a baseline. At the extremes, DML under fully anaerobic and aerobic conditions at 80% moisture result in losses of 5% and >60%, respectively. This work will report the results from fixed intermediate moisture conditions (30%, 40%, 50%, and 60%) to show the impact of partial drying on the stability and practical shelf-life of aerobically stored high-biomass sorghum and the effect on its composition after storage as it relates to soluble and structural sugar contents.
Invited Oral Abstract
Alternative feedstock supplementation can add value for lignocellulosic bioethanol production from wheat straw
Sune Tjalfe Thomsen1, Dr. Heng Zhang1 and Prof. Claus Felby2, (1)University of Copenhagen, Frederiksberg C, Denmark, (2)University of Copenhagen, Frederiksberg, Denmark
39th Symposium on Biotechnology for Fuels and Chemicals
All cheap local biomasses that contain unexploited fermentable sugars should be included in biorefineries. Yet, many current and projected biorefineries have relatively narrow feedstock portfolios. Increasing the feedstock diversity will create value to several stakeholders in the supply chain, by expanding the feedstock market to enter the large-scale 2G bioethanol plants. In turn, this will increase feedstock resource security, creating more competition at the supplier side, and consequently lower overall prices for 2G bioethanol.
In the current study whey, whey permeate, beer production mash, deep litter, potato pulp, rape seed press cake, and saw dust are all investigated as feedstock supplements on 2G bioethanol production of wheat straw. These biomasses can all be found in close proximity to a projected full-scale 2G ethanol plant in Denmark.
The impact of including these alternative biomasses is assessed on the basis of composition, high dry matter enzymatic hydrolysis yields, fermentations yields, and assessment of selected inhibitors and potential fermentation nutrients. The impact of different blend-in ratios for blending in the alternative biomasses in a wheat straw based ethanol production will also be presented, while the optimal point of including the different biomasses in the production will be discussed.
Invited Oral Abstract
Engineering lignocellulose to deliver an improved hexose:pentose ratio
Amir Mahboubi, Thea Pick, Jeemeng Lao and Jenny Mortimer, Lawrence Berkeley National Laboratory, Berkeley, CA, USA
39th Symposium on Biotechnology for Fuels and Chemicals
A successful lignocellulosic biofuel industry will require specialized crops (feedstocks) that are optimized for deconstruction into sugars and bioconversion into fuels and other co-products. The cell walls of many crop species have a large proportion of pentose sugars, mostly in the form of xylose. Most microbes preferentially metabolize hexoses over hexoses, leading to an inefficient utilization of the biomass. Our goal is to engineer plant cell walls with an increased hexose:pentose ratio by increasing glucomannan biosynthesis. Glucomannan is a soluble polysaccharide composed of glucose and mannose. Here we will describe our approach to achieving an increase in total cell wall mannan content in the model plant Arabidopsis thaliana, and present some preliminary data.
Invited Oral Abstract
Production of acetate from different metabolic node in cyanobacterium Synechococcus elongatus PCC7942
Mr. Ken Lu, National Tsing Hua University, Hsinchu, Taiwan, 30013, Taiwan and Prof. Claire Shen, National Tsing Hua University, Hsinchu, Taiwan
39th Symposium on Biotechnology for Fuels and Chemicals
As a photosynthetic autotroph, cyanobacterium has been engineered to synthesize a wide variety of chemicals directly from CO2 such as bioplastic precursors, biofuel and other bio-chemicals. To increase target productivity, many efforts have been invested to re-distribute metabolic partitioning in the photosynthetic system. It has been consistently observed that production efficiency of pyruvate-derived metabolites is generally higher than that of acetyl-CoA derived compounds in cyanobacteria, suggesting greater availability of pyruvate intracellularly. In this work, acetate production is used to exemplify the effect of carbon hijacking from different metabolic node in cyanobacteria. Several natural and synthetic pathways were overexpressed heterologously in Synechococcus elongatus PCC 7942 to synthesize acetate from pyruvate, acetyl-CoA, or fructose-6-phosphate. Based on the preliminary production results, biosynthesis of acetate from pyruvate reached the highest titer of 0.8 g/L in 10 days, followed by 0.5 g/L of acetate from acetyl-CoA. Whereas cell growth remained normal during acetate production from pyruvate or acetyl-CoA, introduction of the non-oxidative glycolysis (NOG) by phosphoketolase overexpression led to severe growth retardation and leakage of acetate throughout lag phase prior to induction, accumulating about 0.1 g/L of acetate at the end. These observations strengthen the conclusions made previously: 1) higher production efficiency is achieved using pyruvate as a metabolic precursor and 2) phosphoketolase plays a significant role in the central metabolism of cyanobacteria.
Invited Oral Abstract
Evaluating the impact of biomass pelletization for cellulosic ethanol and animal feed from sugarcane harvest residues
Thapelo Mokomele1, Johann Gorgens1, Leonardo da Costa Sousa2, Venkatesh Balan2 and Prof. Bruce E. Dale2, (1)University of Stellenbosch, Stellenbosch, South Africa, (2)DOE Great Lakes Bioenergy Research Center, Lansing, MI, USA
39th Symposium on Biotechnology for Fuels and Chemicals
Sugarcane bagasse and cane leaf matter (CLM) are major agricultural residues from the sugarcane industry, postulated to be available for cellulosic biofuels and/or animal feed in many tropical regions. However, unfavorable physical properties of these feedstocks, such as low bulk density and poor flowability, pose a challenge to biomass supply chain logistics and downstream processing. A promising solution to this challenge is regional pre-processing depots, which collect and densify biomass locally before transportation to biorefineries for upgrading to biofuels or valuable commodities such as animal feed. This concept can be easily adapted to sugar mills, where sugarcane bagasse is produced and potentially pre-processed before being shipped to large-scale biorefineries.
In this study, we compared the properties of AFEX and steam-exploded (StEx) pre-treated sugarcane bagasse and CLM pellets, as well as their subsequent feasibility to being used as input commodities to cellulosic ethanol refineries and the animal feed industry. The pellet properties of these feedstocks were studied to provide an understanding of their potential logistical benefits. Furthermore, the impact of pelletization on enzymatic hydrolysis and fermentation for AFEX and StEx pretreated sugarcane harvest residues was evaluated. The results provide greater insight into the impact of biomass densification on ethanol yields that could be obtained in the centralized biorefinery. Lastly, we compared the in-vitro rumen digestibility of AFEX and StEx-treated sugarcane bagasse and CLM relative to untreated controls, underlining the potential of these depots to co-produce feedstocks that would supply the animal feed and/or cellulosic ethanol industries.
Invited Oral Abstract
A thioacidolysis method tailored for higher‐throughput quantitative analysis of lignin monomers
Cliff Foster, Michigan State University, Lansing, MI, USA and Dr. Anne Harman-Ware, National Renewable Energy Laboratory, Golden, CO, USA
39th Symposium on Biotechnology for Fuels and Chemicals
Thioacidolysis is a method used to measure the relative content of lignin monomers bound by β-O-4 linkages. Current thioacidolysis methods are low-throughput as they require tedious steps for reaction product concentration prior to analysis using standard GC methods. A quantitative thioacidolysis method that is accessible with general laboratory equipment and uses a non-chlorinated organic solvent and is tailored for higher-throughput analysis is reported. The method utilizes lignin arylglycerol monomer standards for calibration, requires 1–2 mg of biomass per assay and has been quantified using fast-GC techniques including a Low Thermal Mass Modular Accelerated Column Heater (LTM MACH). Cumbersome steps, including standard purification, sample concentrating and drying have been eliminated to help aid in consecutive day-to-day analyses needed to sustain a high sample throughput for large screening experiments without the loss of quantitation accuracy. The method reported in this manuscript has been quantitatively validated against a commonly used thioacidolysis method and across two different research sites with three common biomass varieties to represent hardwoods, softwoods, and grasses.
Invited Oral Abstract
The multinuclear strains composed of autopolyploid nuclei in a cellulolytic fungus, Trichoderma reesei and an edible fungus, Lentinula edodes
Dr. Hideo Toyama, Minamikyushu University, Miyazaki, Japan
39th Symposium on Biotechnology for Fuels and Chemicals
When the mycelial mat of
T. reesei was treated with mitotic arrester under some conditons, the multinuclear strain composed of autopolyploid nuclei was obtained. This multinuclear strain could make the agar plate containing microcrystalline cellulose more transparent comparing with the original strain.
Moreover, such multinuclear strain could be produced also in the Basidiomycete, Lentinula edodes. This multinuclear strain enhanced laccase production in additon to the microcrystalline cellulose degrading ability. From the above results, it was concluded that the multinuclear strain composed of autopolyploid nuclei is useful for the application of fungi.
Invited Oral Abstract
Engineering of industrial yeast cell factories for production of dicarboxylic acids
Vratislav Stovicek, Ksenia Chekina, David Caldern Franco and Irina Borodina, Technical University of Denmark, Kongens Lyngby, Denmark
39th Symposium on Biotechnology for Fuels and Chemicals
Yeast Saccharomyces cerevisiae is one of the most promising hosts for production of bio-based chemicals from biomass. To achieve an economically viable industrial process, industrial strains tolerant to harmful large-scale environment and capable of efficient conversion of biomass sugar content to valuable chemicals must be generated. Lignocellulose forestry residues, rich in C5 sugars that are not naturally fermented by yeast are among the cheapest and abundant biomass materials. In second-generation biorefineries, such non-food biomass feedstocks are supposed to be efficiently utilized for generation of multiple products of societal interest using a suitable cell factory. Here, we demonstrate development of cell factories based on a diploid industrial S. cerevisiae strain with a biorefinery application. Using an advanced molecular toolbox employing the CRISPR-Ca9 system, we engineered the strains for consumption of xylose. Various genome-wide evolutionary engineering approaches were applied to obtain a set of strains capable of efficient xylose fermentation and/or tolerance to a hardwood lignocellulose hydrolysate. Lastly, such strains were engineered for production of selected dicarboxylic acids that can find application as precursors for production of bio-based plastics. The applied metabolic engineering strategies will be shown in more detail. This project is part of BioREFINE-2G (www.biorefine2g.eu), which is co-funded by the European Commission in the 7th Framework Programme (Project No. FP7-613771).
Invited Oral Abstract
Crude glycerol as feedstock for microbial biodiesel production
Mr. Mikolaj Chmielarz, Dr. Johanna Blomqvist, Mats Sandgren and Dr. Volkmar Passoth, Swedish University of Agricultural Sciences, Uppsala, Sweden
39th Symposium on Biotechnology for Fuels and Chemicals
Crude glycerol (CG) is a major by-product of biodiesel transesterification and due to constantly increasing global biodiesel production, also a constantly increasing surplus of CG is being generated. Therefore, conversion of CG to any commercially interesting product would greatly improve the economic and environmental sustainability of biodiesel production. One obstacle of CG is that it contains several toxic or inhibitory substances, making it problematic to convert CG to higher value products. One potential use of CG is its use as feedstock for microbial biodiesel production. In this study, CG from a Swedish biodiesel production plant was tested as a potential substrate for oleaginous yeasts’ cultivation. Twenty-seven oleaginous yeast strains were screened for growth using CG as carbon source on cultivation plates, and subsequently in shake flask cultivations. After the screening tests, the yeast strain with highest growth rate and glycerol consumption was selected - Rhodosporidium toruloides. After growth in single batch setups the lipid content in lower concentration of CG was 4.1 g/L with a yield of 0.16 g.g-1, in higher concentration of CG lipid content was 5.7 g/L with a yield of 0.171 g.g-1. Most dominant fatty acids were oleic acid, followed by palmitic acid, linoleic acid, stearic acid and α-linolenic acid. All these fatty acids are commonly found in vegetable oils used for biodiesel production leading to conclusion that crude glycerol has a very good potential as carbon source to produce biodiesel by oleaginous red yeast.
Invited Oral Abstract
Performance of Zymomonas mobilis C5/C6-sugar assimilating strains in ethanol production from Greek lignocellulosic biomass
Giannis Savvakis1, Konstantina Moysi1, Matina Roussou1, Min Zhang2, Milton Typas1, Dimitris Hatzinikolaou1 and Katherine M. Pappas1, (1)National and Kapodistrian University of Athens, Athens, Greece, (2)National Renewable Energy Laboratory, Golden, CO, USA
39th Symposium on Biotechnology for Fuels and Chemicals
Τhe bacterium Zymomonas mobilis is regarded as a platform organism for large scale bioethanol production due to its ability to ferment glucose into ethanol, at rates and yields reaching the theoretical maximum. Genetically engineered strains of Z. mobilis, able to co-ferment pentoses and hexoses, were among the first microbial strains to be produced for high-yield lignocellulosic fermentations. In this work, two C5/C6-sugar fermenting Z. mobilis strains constructed at NREL, 8b and AX101, were employed in ethanol fermentations of biomass streams derived from crop farms in central Greece. Corn, barley and wheat straw hydrolysates served as source for Z. mobilis growth. Biomass was initially subjected to weak acid or base pretreatment, followed by enzymatic hydrolysis by commercial (hemi)cellulases cocktails. The effect of separate hydrolysis and fermentation (SHF) or simultaneous saccharification and fermentation (SSF) regimes was assessed at various operating conditions. Strain 8b proved to be superior in all conditions, with ethanol yields ranging from 14 g/L in wheat straw SSF, to 23 g/L in wheat straw SHF. Hydrothermally pretreated biomass, depleted for most of its cellulosic content and with or without delignification, was especially inhibiting for Z. mobilis. MNNG-mutagenesis and evolutionary adaptation on such inhibiting biomass hydrolysates led to Z. mobilis 8b and AX101 derivatives with enhanced growth capabilities. The latter are promising for superior fermentations in sugar-rich same-source substrates. Lastly, plasmid pZB301, designed in NREL to enable C5-sugar catabolism in Z. mobilis, was evaluated for use in Z. mobilis wild-type strains ZM4 and CP4.
Invited Oral Abstract
Neurospora intermedia pellets for enhanced ethanol and fungal biomass production from wheat straw
Ramkumar B. Nair, Vamsi K. Ravula, Patrik R. Lennartsson and Mohammad J. Taherzadeh, University of Borås, Boras, Sweden
39th Symposium on Biotechnology for Fuels and Chemicals
Recent studies at our research group have described an ‘integrated-biorefinery’ model for the existing 1st generation wheat-based ethanol facilities, by using edible filamentous fungus, Neurospora intermedia. The process focuses on the production of 2nd generation ethanol together with fungal biomass (for animal or aquaculture feed applications) from wheat straw. A final ethanol yield of 94% (theoretical maximum based on substrate glucan content) was obtained with N. intermedia fermentation in dilute phosphoric acid pretreated (0.7%w/v acid, 7min at 201±4°C) and enzymatically hydrolyzed (10FPU cellulase/g substrate) straw. Fungal cultivation in liquid straw hydrolysate resulted in a maximum of 3.71±0.11g/L dry fungal biomass. Considering the industrial significance of the fungal process, attempts were made to manipulate N. intermedia to grow as pellet forms in the straw hydrolysate, for the first time. Of the various culture conditions screened, stable pellet morphology was obtained at pH 3.0 to 5.5, resulting in uniform pellets with size ranging from 2.5 to 4.25mm. Fermentation using N. intermedia pellets in liquid straw hydrolysate, resulted in about 31% increase in the ethanol yield, with an improved glucose assimilation by the pellets (82% reduction) as opposed to filamentous forms (51% reduction), at similar culture conditions. The growth of fungal pellets in presence of inhibitors (at different concentrations of acetic acid and furfural) resulted in about 11% to 45% increase in ethanol production as compared to filamentous forms, at similar growth conditions in liquid straw hydrolysate. Detailed results on N. intermedia pelletization in liquid straw hydrolysate will be discussed in this presentation.
Invited Oral Abstract
Identification of a novel Issatchenkia orientalis GPI-anchored protein involved in tolerance to acid and salt stress
Dr. Akinori Matsushika1, Dr. Toshihiro Suzuki1, Dr. Tetsuya Goshima2 and Dr. Tamotsu Hoshino1, (1)National Institute of Advanced Industrial Science and Technology (AIST), Hiroshima, Japan, (2)National Research Institute of Brewing (NRIB), Hiroshima, Japan
39th Symposium on Biotechnology for Fuels and Chemicals
Environmental stress, especially acid and salt stress, is the most serious problem in industrial bioproduction of ethanol and of lactic acid. To develop robust yeast strains with high stress tolerance, we isolated and identified a novel gene by screening a genomic library of a multiple-stress-tolerant yeast, Issatchenkia orientalis. This gene, named IoGAS1, allowed Saccharomyces cerevisiae cells to grow under acid stress (pH 2.0) and under combined acid and salt stress (pH 2.5, 7.5% Na2SO4) conditions. The predicted amino acid sequence of IoGAS1 was 60% identical to the S. cerevisiae Gas1 protein, a glycosylphosphatidylinositol (GPI) -anchored protein essential for maintaining cell wall integrity, and 58-59% identical to Candida albicans Phr1 and Phr2, pH-responsive proteins implicated in cell wall assembly and virulence. Transcriptional analysis indicated that the endogenous expression of IoGAS1 was pH dependent, with maximum expression at pH 2.0, suggesting that IoGAS1 represents a novel pH-regulated system required for the adaptation of I. orientalis to environments of diverse pH. Heterologous expression of IoGAS1 complemented the growth and morphological defects of an S. cerevisiae gas1Δ mutant, showing that IoGAS1 is both structurally and functionally similar to the S. cerevisiae gene. Furthermore, overexpression of IoGAS1 in S. cerevisiae remarkably improved the ethanol fermentation ability under acid stress (pH 2.0−2.5) and under combined acid and salt stress (pH 2.0−2.5, 5.0% Na2SO4) conditions, compared to that of a reference strain. Our results strongly suggest that constitutive expression of the IoGAS1 gene in S. cerevisiae could be advantageous for several fermentation processes under these stress conditions.
Invited Oral Abstract
Heterogenous expression of chaperonin in Clostridium acetobutylicum strain to enhance butnol production
Prof. Chia-Wen Hsieh, National Chiayi University, Chiayi City, Taiwan
39th Symposium on Biotechnology for Fuels and Chemicals
Solvents toxicity is a major limiting factor hampering the production of chemicals by fermentation. A butanol-tolerant Clostridium acetobutylicum MFY105 mutant was screened throughout continuous adaption culture, which can grow well in 10 g/L butanol contained medium and can produce about 18.4 g ABE/L during 28hrs. The C. acetobutylicum MFY105 strain was able to produce 192 mM butanol with 52 mM acetone at pH 4.8, resulting in a butanol selectivity (a molar ratio of butanol to total solvents) of 0.71, which is much higher than that (0.6) of the wild-type strain Clostridium acetobutylicum MFY. The acetate accumulation was not observed during fermentation of the MFY105 strain. A hyper-butanol producing C. acetobutylicum MFY105 (pSOSCPN), which was created to overexpress the Pyrococcuss horikoshii OT3 originated specific group II chaperonin gene, from a clostridial phosphotransbutyrylase promoter, was able to produce final butanol titers 45% and 137% higher than those of the wild-type and plasmid control strains, respectively. The remarkable butanol-tolerance of strain MFY105 (pSOSCPN) demonstrates that overexpression of heterogenous chaperonin, could help C. acetobutylicum to effectively produce high concentration of butanol.
Invited Oral Abstract
Enhanced production of cis-cis muconic acid through phosphate-limited fed-batch and continous fermentations of recombinant Corynebacterium glutamicum cells
Gie-Taek Chun and Sungju Byun, College of Biomedical Science, Kangwon National University, Chuncheon, Korea, Republic of (South)
39th Symposium on Biotechnology for Fuels and Chemicals
Cis-cis muconic acid (MA), an unsaturated dicarboxylic acid with six carbon atoms was reported to be easily converted to adipic acid, a valuable commodity chemical for bio-plastics including nylon 6-6 and polyurethane and polyethylene terephthalate (PET). As a first step to mass production of MA, a recombinant C. glutamicum cells (a strain transformed with clustered BYD genes from Klebsiella pneumoniae) were developed through metabolic engineering approaches. During the statistical medium optimization process in shake-flask fermentations with the MA high-yielding recombinant strains, it was found that sufficient supply of dissolved oxygen(DO) during the whole fermentation period is also very important for enhanced production of MA. Therefore, intensive studies were carried out in order to develop fed-batch fermentation processes that could efficiently overcome DO-limited conditions, and also the catabolite repression phenomena caused by high levels of residual nutrients in the fermentation broth. Notably, under the phosphate-limited fed-batch operating conditions, MA was observed to be biosynthesized almost in a growth-associated mode, thus resulting in remarkable enhancement in MA productivity (i.e., approximately 4 fold increase as compared to the parallel batch bioreactor fermentation performed under the identical environments). In this paper, the experimental results from MA bioreactor scale-up studies performed with the recombinant mutant cells of C. glutamicum will be presented as well.
Invited Oral Abstract
Cellobiose fermenting yeast produces varied forms of native β-glucosidase
Xu Wang1, Z. Lewis Liu2 and Scott A. Weber2, (1)Henan Agricultural University, Zhengzhou, China, (2)USDA-ARS, Peoria, IL, USA
39th Symposium on Biotechnology for Fuels and Chemicals
The rapid growing yeast strain NRRL Y-50464 is robust to environmental stress and resistant to 2-furaldehyde (furfural) and 5-[hydroxymethyl]-2-furaldehyde (HMF). It is able to utilize cellobiose as its sole source of carbon and produces ethanol from lignocellulosic biomass by simultaneous saccharification and fermentation (SSF) without supplemental β-glucosidase. Beta-glucosidase is a key member of the cellulase enzyme complex that hydrolyzes glycosidic bonds from glycosides and oligosaccharides. Eliminating the need for external β-glucosidase should directly reduce enzyme costs. Here we characterize three native β-glucosidases (BGL1, BGL2 and BGL3) of NRRL Y-50464. Specific activities of all three β-glucosidases were intact in the presence of furfural and HMF. With a molecular weight ranging from 93 to 104 KD, these BGLs showed an optimum temperature from 45 to 55°C and optimum pH from 5 to 6. All forms of β-glucosidase displayed a relatively stronger affinity toward cellobiose and higher levels of product inhibition against glucose with the highest Ki of 61.97 mM for BGL2. BGL2 was also tolerant to 16% ethanol, which was the highest for the three enzymes. Amino acid sequence analysis indicated a closer relatedness between BGL2 and BGL3 than for either with BGL1. These enzymes were active against at least 14 oligosaccharides associated with lignocellulosic hydrolysates used for ethanol production. Results of this study confirmed the cellobiose hydrolysis function of strain NRRL Y-50464 with a family of at least three β-glucosidases.
Invited Oral Abstract
Genetically-modified microbes for polyhydroxyalkanoate production
Andrew Cal, Dirk Sikkema, Maria Ponce, William Orts and Charles Lee, USDA-ARS, Albany, CA, USA
39th Symposium on Biotechnology for Fuels and Chemicals
Polyhydroxyalkanoates (PHA) are biologically-produced polyesters first discovered in the 1920s in the gram positive bacterium Bacillus megaterium. These polymers are of great interest due to both their potential production from renewable substrates and their inherent biodegradability, but industrial efforts have focused on gram negative bacteria for their production. In gram positive bacteria, the PHA polymer is degraded during sporulation. Additionally, B. megaterium only produces poly-3-hydroxybutyrate (P3HB), a polymer with a limited range of applications. We have focused on genetically modifying B. megaterium to produce increased amounts of polymer while producing PHA copolymers with monomer units other than 3-hydroxybutyrate. The physical properties of these polymers were tested and will be described.
Invited Oral Abstract
Effect of the physical complexity of lignin on its recalcitrance to biological depolymerization
Chijioke J. Joshua, Blake A. Simmons and Steven W. Singer, Joint BioEnergy Institute, Biological Systems and Engineering, Lawrence Berkeley National Laboratory, Emeryville, CA, USA
39th Symposium on Biotechnology for Fuels and Chemicals
Lignin recalcitrance to biological depolymerization is generally attributed to the complex chemical structure of the polyphenol component of plant cell wall, but the complex physical structure resulting from self-assembly of its amphiphilic molecules also plays a role. To elucidate the mechanism of microbial consumption of lignin, we compared the growth of Streptomyces viridosporus and two isolates, Bacillus circulans and Penicillium sp. on insoluble lignin from ionic pretreated switchgrass. We observed that S. viridosporus grew better on the lignin than the isolates, but there was no observable differences in residual lignin each culture relative to control (uninoculated). The results also indicated that the organisms primarily consumed soluble lignin-derived monomers and small molecules, rather than lignin polymer. This observation was further supported by the very low level of enzyme secretion during growth of S. viridosporus on insoluble lignin compared to pretreated switchgrass, which contained both lignin and polysaccharides. We also determined that lignin aggregates sequester large amount of water-soluble polydispersed lignin moieties that may be released abiotically during microbial growth. To further understand microbial catabolism of polymeric lignin, we extracted water-soluble lignin polymers from the insoluble aggregates using a novel strategy and evaluated utilization of these soluble molecules by a new Penicillium isolate. The Penicillium species grew on these soluble lignin polymers (3-10 kDa) as the sole carbon source, while Phaenerochaete chrysosporium (white-rot fungus) was unable to grow on this polymeric fraction. Our results indicate that using soluble polymeric lignin moieties of defined will enhance our understanding biological lignin depolymerization and metabolism.
Invited Oral Abstract
Fatty alcohol production from oleaginous yeast Lipomyces starkeyi
Wei Wang1, Eric Knoshaug1, Hui Wei1, Stefanie Van Wychen1, Todd Vander Wall1, Min Zhang1 and Michael E. Himmel2, (1)National Renewable Energy Laboratory, Golden, CO, USA, (2)National Renewable Energy Laboratory, Biosciences Center, Golden, CO, USA
39th Symposium on Biotechnology for Fuels and Chemicals
Current research on oleaginous yeasts for biofuels applications has mainly focused on lipid production, which is considered as alternatives to plant oils for biodiesel production. However, yeasts store neutral lipids intracellularly making recovery difficult and expensive. We have, therefore, begun to investigate secreted fatty acid-derived products which can be easily recovered and upgraded to fuels.
Lipomyces starkeyi, considering its strong
de novo synthesis of fatty acids compared to
Yarrowia lipolytica, might be a better source for fatty alcohol production despite of the challenge in genetic engineering. In the present study, we investigated the fatty alcohol production capacity of
L. starkeyi, following transformation with a heterologous
far gene from
M. aquaeolei VT8. Our results show that
L. starkeyi far gene transformant was able to produce fatty alcohols from both glucose and xylose, providing a basis for further engineering aimed at increasing fatty alcohol titer on cellulosic biomass feedstocks. 4.2 g/L of fatty alcohols were produced on glucose in 3-L bioreactor via fed-batch fermentation. In addition to high fatty alcohols production capacity,
L. starkeyi also showed good secretion for fatty alcohols, i.e. half of the total fatty alcohols were secreted into the fermentation broth. Undoubtedly, secreting out fatty alcohols could be beneficial for downstream product processing. Different strategies towards further enhancing fatty alcohols production were also discussed. Taken together, our work demonstrates that in addition to
Y. lipolytica,
L. starkeyi can also serve as a platform organism for production of fatty acid-derived biofuels and bioproducts via metabolic engineering.
Invited Oral Abstract
Regulation of lipid metabolism in oleaginous industrial yeast
Kyle Pomraning1, Erin Bredeweg1, Rosalie K. Chu1, James R Collett1, David Culley1, Ziyu Dai1, Beth Hofstad1, Joonhoon Kim2, Young-Mo Kim1, Carrie Nicora1, Samuel Purvine1, Jon K. Magnuson2, Tom Metz1 and Scott E. Baker1, (1)Pacific Northwest National Laboratory, Richland, WA, USA, (2)Pacific Northwest National Laboratory, Joint BioEnergy Institute, Richland, WA, USA
39th Symposium on Biotechnology for Fuels and Chemicals
Oleaginous yeast accumulate a high quantity of storage lipids in a manner dependent on nutrient availability. While the enzymatic pathways responsible for neutral lipid accumulation are well characterized, regulation of these pathways has received little attention in industrial yeasts. We have examined lipid accumulation using transcriptomics, proteomics, and phospho-proteomics to better understand multi-tiered regulation of lipid accumulation in response to nitrogen quantity and quality in Yarrowia lipolytica and during growth on biomass feedstocks in Lipomyces starkeyi. Our results indicate carbon and nitrogen catabolite repression as central regulators of metabolism during lipid accumulation. We identified and characterized GATA transcription factor genes with nitrogen source specific growth defects and regulators of central carbon metabolism by gene deletion and RNA-sequencing. Both GATA transcription factor mutants exhibit decreased expression of genes predicted to be controlled by carbon catabolite repression, including genes for beta-oxidation, highlighting the complex interplay between regulation of carbon, nitrogen, and lipid metabolism.
Invited Oral Abstract
Current updates of Toyota yeasts for bio-ethanol production, an enhancement of arabinose conversion
Hirokazu Kikuta1, Nobuki Tada1, Junji Ito1, Rie Hirao1, Noriko Yasutani1, Masao Hayashida1, Satoshi Katahira2, Akinori Ikeuchi2, Nobuhiko Muramoto2, Kenro Tokuhiro2 and Toru Onishi1, (1)Toyota Motor Corporation, Aichi, Japan, (2)Toyota Central R&D Labs Inc., Nagakute, Aichi, Japan
39th Symposium on Biotechnology for Fuels and Chemicals
For cost-efficient lignocellulosic ethanol production, enhancing the xylose and arabinose uptake ability and the tolerance to the hydrolysate inhibitors (e.g. acetic acid, furfural and 5-hydroxymethyl furfural) of yeast strains are the important property. We have been developing the yeast strains by introducing and tuning of each gene expression level encoding the xylose and arabinose metabolic pathway enzymes, pentose phosphate pathway enzymes, and the acetate metabolic pathway enzymes. Evolutionary engineering have been adapted to such genetically modified yeast strains to improve the consumption rate of xylose, arabinose and acetic acid and to strengthen the inhibitor tolerance.
We evaluated for inserted genes stability and various property of the developing yeast strains. All inserted genes were stably integrated in the genome of the yeast strains. Developed yeast strains showed the ability of tolerance to inhibitors and higher consumption rate of xylose, arabinose and acetic acid, the ethanol yield was significantly improved consequently.
Moreover we had confirmed the robustness of fermentation ability of developed yeast strain at 46kL tank scale. The latest scientific results will be presented as well as updates on the strain’s development towards commercial production.
Invited Oral Abstract
Pre-culture conditions for ethanol production with Xylose-fermenting yeastCandida intermedia 4-6-4T2 part 1
Hiroshi Nagasaki, COSMO OIL Research & Development Center, Satte, Japan
39th Symposium on Biotechnology for Fuels and Chemicals
We have cloned a new xylose-fermenting yeast Candida intermedia 4-6-4T2 by adaptive mutation. C. intermedia 4-6-4T2 could ferment both of C5 (xylose) and C6 (glucose) simultaneously and produce ethanol. For non-GMO xylose-fermenting yeasts, fermentation of cellulosic and hemicellulosic sugars from biomass is problematic because of glucose or catabolite repression. In order to reduce these phenomena, we found the efficient pre-culture conditions for five strains that can convert xylose to ethanol, including C. intermedia 4-6-4T2. A tentative solution for fermentation containing high concentrations of glucose plus xylose and acetic acid as inhibitor was used for the evaluation of ethanol production with either of five strains. Among them, C. intermedia 4-6-4T2 produced the highest concentration of ethanol when fermented in the tested sugar solution. We determined the material balance between the consumed sugars and ethanol with the other by-products in detail.
Invited Oral Abstract
Pre-culture conditions for ethanol production with Xylose-fermenting yeastCandida intermedia 4-6-4T2 Part 2
Masaru Saito, COSMO OIL Research & Development Center, Satte, Japan
39th Symposium on Biotechnology for Fuels and Chemicals
We have cloned a new xylose-fermenting yeast Candida intermedia 4-6-4T2 by adaptive mutation. C. intermedia 4-6-4T2 could ferment both of C5 (xylose) and C6 (glucose) simultaneously and produce ethanol. We have set the conditions for pre-culture and fermentation with C. intermedia 4-6-4T2. C. intermedia 4-6-4T2 needs microaerobic conditions in the pre-culture phase for consuming xylose sufficiently. After pre-culture C. intermedia 4-6-4T2 could convert both of C5 (xylose) and C6 (glucose) efficiently to ethanol in the fermentation phase. Controlling agitation speed and aeration rate in the pre-culture phase, we found the effect of kLa for ethanol production from sugars (xylose and glucose) in the hydrolysate of lignocellulosic biomass. The xylose consuming ability of C. intermedia 4-6-4T2 was strongly dependent on kLa values. The optimum kLa in the pre-culture phase led to the maximal ethanol production from the hydrolysate containing xylose and glucose in lignocellulosic biomass.
Invited Oral Abstract
Synergistic effect of engineering lipid biosynthesis pathway and supplementing fermentation medium for constitutive production of organic acid and lipid in Yarrowia lipolytica
Dr. Ali Abghari, Washington State University, Pullman, WA, USA and Prof. Shulin Chen, Professor/Washington State University, Pullman, WA, USA
39th Symposium on Biotechnology for Fuels and Chemicals
Development of a safe oleaginous yeast platform is required to meet increasing demand for plant and fuels. The large scale production of single cell oil is challenging because it has high trading volumes but low profit margins. Therefore, it is required to make cellular level improvements for higher production level. We have engineered Yarrowia lipolytica as an oleaginous cell factory platform whose potential for production of lipid and oleochemicals has been the focus of many recent studies. Identification of key genes in the lipid pathway along with understanding of regulatory mechanisms and their interaction with fermentation medium is necessary for overproduction. Thus we have taken the advantage of synthetic biology tools for simultaneous knock out of target gene and knock in of multiple genes. We engineered strain at various metabolic levels of lipid biosynthesis, degradation, and regulation. This was coupled to supplementation of fermentation medium with a signaling molecule under nitrogen limiting conditions. The strains obtained have enhanced capability for constitutive lipid accumulation and citric acid secretion into the production medium. This platform can play dual role as lipid producer in the upstream of biodiesel industry and as glycerol based producer of citric aicd and lipid in the downstream. Our genetic engineering strategies and their synergistic effect with medium modification for enhancing the production level are the subjects of our presentation.
Invited Oral Abstract
L-Arabinose transport and fermentation by Candida succiphila
Boris U. Stambuk1, Mary Ann Franden2, Min Zhang2 and Arjun Singh2, (1)Centro de Ciências Biológicas, Universidade Federal de Santa Catarina, Florianópolis, Brazil, (2)National Renewable Energy Laboratory, Golden, CO, USA
39th Symposium on Biotechnology for Fuels and Chemicals
For cost-effective and efficient ethanol production from lignocellulosic biomass the conversion of not only major constituents, such as D-glucose and D-xylose, but also of less predominant sugars, such as L-arabinose, is required. Candida succiphila is one of the few yeast species known to produce ethanol from this pentose. Our results show that C. succiphila produces ethanol (plus L-arabitol and acetate) from L-arabinose only during growth under microaerobic conditions, while under fully aerobic conditions sugar consumption and cell growth is faster, but no ethanol (or L-arabitol and acetate) are produced, and all carbon is directed into biomass production. Cells grown under microaerobic conditions have a L-arabinose transport system with low affinity (Km ~60 mM) and high capacity (Vmax ~15 nmol mg-1 min‑1), probably mediated by a facilitated diffusion mechanism, while during growth under aerobic conditions C. succiphila cells express a very high-affinity transporter (Km ~75 µM) with low capacity (Vmax ~2.5 nmol mg-1 min‑1) for L‑arabinose, in addition to the low-affinity transport system. Since both protonophores and H+-ATPase inhibitors significantly reduced the rates of transport by this high-affinity transporter, the electrochemical H+ gradient across the plasma membrane is required for the active uptake of L‑arabinose by this transport system. Incubation of C. succiphila cells with 14C-L-arabinose and further extraction of the intracellular metabolites revealed all intermediates of L-arabinose catabolism, but the accumulation of L-arabitol and intracellular xylitol indicates that the two NAD+-dependent dehydrogenases (L-arabitol 4-dehydrogenase and xylitol dehydrogenase) are the limiting steps in L-arabinose catabolism by C. succiphila.
Invited Oral Abstract
Superior yeasts for biotechnical production
Marja Ilmn, Outi Koivistoinen, Maija-Leena Vehkomki, Dominik Mojzita, Anssi Rantasalo, Joosu Kuivanen, Jussi Jntti, Kari Koivuranta, Dr. Mervi Toivari, Laura Ruohonen and Prof. Merja Penttil, VTT Technical Research Centre of Finland Ltd, Espoo, Finland
39th Symposium on Biotechnology for Fuels and Chemicals
Currently, only a limited number of yeast species are used in industrial processes to produce biofuels and chemicals. One reason for this are the demanding process conditions (e.g. low pH and high temperature), which set tough requirements for the production host.
Also, genetic engineering of novel species for production of desired products may be challenging.
To discover yeast species/strains which can tolerate different process conditions and which can be easily genetically modified, we have screened several VTT proprietary yeast species in different conditions: low pH, organic acids, different temperatures, and lignocellulosic hydrolysates. We also tested genetic engineering tools (promoters, markers) and transformations methods for these strains. With the most promising strains we studied the possibility to use CrispR technology and the synthetic promoter tools recently developed at VTT.
As an example of possibilities to carry out genetic modifications we established lactic acid production and the ability to utilize xylose as a carbon source into selected strains.
Invited Oral Abstract
Metabolic flexibility of Yarrowia lipolytica strains cultivated on glycerol
Michael Egermeier1, Hans Marx1, Hannes Russmayer1 and Michael Sauer2, (1)CD Laboratory for Biotechnology of Glycerol, Vienna, Austria, (2)Austrian Center of Industrial Biotechnology, Vienna, Austria
39th Symposium on Biotechnology for Fuels and Chemicals
Yarrowia lipolyticais becoming one of the best studied yeast species and used as model organism in various fields, like the degradation of n-alkanes, protein secretion, dimorphism and many more. It is generally regarded as safe (GRAS) and therefore an interesting host for various biotechnological applications. The type-strain NRRL YB-423 (CBS 6124) was isolated in 1945 from a maize-processing plant. Two other well-characterized strains are the sewage-derived W29 (CBS 7504) used for lipid production and H222 isolated from soil and investigated for the production of organic acids. The sources of isolation clearly has an influence on the metabolic capability of a microorganism, but in literature these primary lab-strains are, however, rarely used in the same experiments. Our goal was to create a comprehensive dataset based on strain screenings through bioreactor cultivations, which would represents the first step in the identification of novel hosts for industrial applications and the basis for strain engineering and evolution.
Therefore we investigated the ability of 20 strains of Y. lipolytica, including the above mentioned primary lab strains as well as 12 completely new isolates from dairy products, to convert glycerol into citric acid and polyols in a bioreactor under different pH conditions. We could show, that strains from a common origin behave comparably, even though completely unlike their relatives from diverging habitats. While some strains convert the excess glycerol to polyols, other strains of the same species shift their metabolism from polyol production towards the secretion of citric acid.
Invited Oral Abstract
Pdu microcompartments: Important intracellular structures for conversion of glycerol to 1,3-propanediol in Lactobacillus diolivorans
Hannes Russmayer, Hans Marx and Michael Sauer, CD Laboratory for Biotechnology of Glycerol, Vienna, Austria
39th Symposium on Biotechnology for Fuels and Chemicals
Lactobacillus diolivorans was already identified as an efficient producer of 1,3 propanediol from crude glycerol under industrial relevant conditions. In a Fed Batch cultivation with a constant glycerol feed in combination with glucose pulsing 1,3 propanediol titers of 90 g/L were reached.
To allow such high conversion rates the flux through the respective pathway has to be at an optimum. L. diolivorans ensures this optimized pathway flux via encapsulation of the needed enzymes into intracellular macromolecular structures, also referred as pdu (propanediol utilization) microcompartments (pdu MC).
Pdu MCs are composed of a semi porous protein shell that encases the enzymes for 1,3-propanediol production. The targeting of the enzymes is ensured via leader sequences found on the N-terminus of the respective enzyme. All necessary genes for expression of pdu MCs are clustered within an operon like structure. A further characterization of this microcompartment is very important in helping to understand more about the organism and how microcompartments can be employed as a tool for metabolic engineering. However, tools for genetic manipulation are rare and characterization of microcompartments in LAB is difficult.
Therefore, Escherichia coli was chosen as expression host due to the availability of a broad genetic toolbox and established protocols for characterization of microcompartments. The expressed pdu MCs were visualized via transmission electron microscopy. Furthermore, via fluorescence microscopy a targeting of GFP into the microcompartment was shown. The gained knowledge will be transferred back to L. diolivorans to further increase the efficiency of 1,3-propanediol formation from glycerol.
Invited Oral Abstract
Characterizing a promising methanotroph strain through a complete carbon and growth analysis
Kyle Stone, Q. Peter He and Jin Wang, Auburn University, Auburn, AL, USA
39th Symposium on Biotechnology for Fuels and Chemicals
Over the past 30 years, methanotrophs have moved from a “black box” organism to being on the cusp of becoming the next biocatalyst in a promising biotechnical world. Of the many different methanotrophs,
Methylomicrobium buryatense 5GB1 has been shown as a promising industrial strain because of its fast growth rate in medium that is resistant to contaminants and due to its potential to generate organic acids, etcoine, and desirable lipids for biodiesel (1-3). One important, controllable factor for methane fermentation to valued products with 5GB1 is the transfer and subsequent uptake of methane and oxygen.
In this work, we utilize a safe gas mixing system to create custom mixtures of oxygen, methane and nitrogen that were then introduced to batch cultures of 5GB1. Analysis of growth and a complete carbon balance (excreted products, biomass, and CO2) was conducted. The approach used to characterize 5GB1 were designed to take into account under pressurized batch vials, gas-liquid equilibrium, effective Henry’s constant for CO2 with various pH conditions, and the accurate quantification of inorganic/organic liquid products. Through this analysis, insights on uptake rates, specific growth rates, and on conversion yields allow for a complete phenotype characterization of 5GB1under different oxygen/methane ratios. The study contributes to the much needed knowledge base to design processes for improved conversions with these promising biocatalysts.
1. Gilman A, et. al.. 2015. Microb. cell factories 14.
2. Kalyuzhnaya MG, et. al. 2015. Metab. Eng. 29:142–152.
3. Strong PJ, et. al. 2016. Bioresour. Technol. 215, 314-323.
Invited Oral Abstract
Limiting steps for xylose metabolism in yeast:comparative fermentation performance and metabolomics
Mr. Henrique Veras1, Ms. Christiane G. Campos1, Jos Antonio Ribeiro2, Dr. Patrcia Verardi Abdelnur1, Prof. Ndia S. Parachin3 and Prof. Joo R. M. Almeida1, (1)Embrapa Agroenergy, Braslia, Brazil, (2)Embrapa Agroenergy, Brasilia, Brazil, (3)Universidade de Braslia, Braslia, Brazil
39th Symposium on Biotechnology for Fuels and Chemicals
Yeasts species naturally capable to ferment xylose, the second most abundant sugar in nature, have being identified. Ethanol production by these yeasts is greatly influenced by oxygen availability. In this work, the capacity to ferment xylose of Scheffersomyces stipitis, Spathaspora passalidarum, S. arborariae and Candida tenuis was compared under different aeration conditions. Furthermore, the effects of oxygen on yeast intra- and extracellular metabolites concentration were investigated through metabolomics. S. stipitis and S. passalidarum were the best ethanol producing yeasts, with yields of 0.45 g.g-1 and 0.42 g.g-1, respectively, under oxygen-limited condition. However, S. passalidarum showed better performance than S. stipitis under anaerobiosis. S. arborariae and C. tenuis showed the lower xylose consumption rates among the four yeasts evaluated. In addition, C. tenuis produced xylitol as main conversion product instead of ethanol. Measurements of enzymatic activity in crude-cell extracts of S. stipitis, S. passalidarum and S. arborariae showed that xylose reductase (XR) of these yeasts are able to use both NADH and NADPH as cofactors, however at different rates. C. tenuis XR appeared as strictly NADPH-dependent. Xylitol dehydrogenase activity was NAD+- dependent for all yeasts. Measurements of metabolites involved in central carbon metabolism, such as glycerate-3-P, phosphoenolpyruvate, acetyl-CoA and malate, confirmed significant differences of yeast metabolism under aerobic and oxygen-limited conditions. In addition, our results indicate accumulation of some metabolites at pentose phosphate pathway under oxygen-limited fermentation. Our data will lead to a better understanding of xylose metabolism limiting steps.
Invited Oral Abstract
A new lipase for biodiesel synthesis
Pedro Alves Martins, Dbora Lo Sciuto, Lia Ceclia de Lima Favaro, Thlyta Fraga Pacheco and Thas Fabiana Chan Salum, Embrapa Agroenergy, Braslia, Brazil
39th Symposium on Biotechnology for Fuels and Chemicals
Biodiesel is a fuel produced industrially through alkaline chemical transesterification. Lipases can be used to produce biodiesel with the advantage of being more environmentally friendly. However, the enzymatic process is still expensive compared to the chemical process. In this context, new lipases discovery and the utilization of low-cost cultivation media are necessary to reach the economic viability of enzymatic biodiesel production. Hence, this study aimed to prospect new microorganism strains and to produce lipases by solid-state fermentation in wheat bran. Then, 298 microorganisms (bacteria, yeasts and filamentous fungi) were isolated from oil palm fruits. They were evaluated by qualitative tests in solid media containing triolein and olive oil as a carbon source and quantitative tests in solid-state fermentation using wheat bran. We selected the strain BDA-FI 7, an Aspergillus sp., to optimize lipase production. A Central Composite Rotatable Design (CCRD) was performed. The variables evaluated were: temperature (20-35 °C), moisture (45-65 %) and inoculum concentration (106-108 spores/g). Flasks were incubated, and after 168 h, lipase was extracted from the solid substrate and its pNPP-hydrolyzing activity was determined. The best lipase activity obtained was 55.9 U/g dry substrate. The conditions that maximize the lipase activity are 35 °C, 65 % moisture and an inoculum of 5.05 x 107 spores/g. At this combination, the maximum predicted yield was 58.4 U/g. Preliminary studies showed that this lipase can catalyze biodiesel synthesis (ethyl esters) in n-heptane as a solvent.
Invited Oral Abstract
Xylitol production: physiological and genetic characterization of new yeast strains
Dr. Debora Trichez, Dr. Andrei Stecca, Mr. Carlos E. Soares, Dr. Eduardo F. Formighieri and Prof. Joo R. M. Almeida, Embrapa Agroenergy, Braslia, Brazil
39th Symposium on Biotechnology for Fuels and Chemicals
Growing demand for chemical products and environmental friendly processes has motivated the search for alternative solutions and renewable raw materials to replace some conventional processes. In this way, the lignocellulosic biomass has a great potential to be used for production of biofuels and value added chemicals. Xylitol is a five-carbon polyol with a wide-range of applications in odontologic pharmaceutical and food industries. At industrial scale, it is produced chemically by an expensive process of xylose hydrogenation at high temperatures and pressures, using nickel metal as catalyst. Since some microorganisms are able to convert xylose to xylitol, microbiological fermentation processes can be an interesting alternative to produce xylitol with lower cost. Considering this context, we herein described the physiology of two yeast strains isolated from Brazilian Cerrado ecosystem. Sequencing of 26S r-DNA demonstrated that these strains are close related to the clade Spathaspora sp. and Meyerozyma sp. Xylose consumption profile and xylitol production by these cells were determined during cultivation in bioreactors using mineral medium and sugarcane bagasse hydrolysate under different aeration conditions. The new strains did not produce significant quantities of ethanol from xylose, but the xylitol production reached yields up to 0,70 g.g-1, showing similar performance with strains commonly used for xylitol production. To better understand the genetic background of these yeasts, whole genome sequencing was performed and a comparative analysis was carried out. Data on genomes size, annotation and gene synteny will be presented and discussed.
Invited Oral Abstract
Transcriptome analysis of Candida intermedia sugar metabolism
Fbio Faria-Oliveira, Cecilia Geijer and Lisbeth Olsson, Chalmers University of Technology, Gothenburg, Sweden
39th Symposium on Biotechnology for Fuels and Chemicals
The dependence on fossil fuels has severe consequences to the worldwide environment and economy. In the transition to a fossil fuel-free society the cellulosic bioethanol plays an important role. Currently, in the bioethanol industry, the yeast Saccharomyces cerevisiae is the main fermentative microorganism mostly due to its superior fermentation capacity of hexose sugars, particularly glucose. However, S. cerevisiae utilization for lignocellulosic bioethanol production is hindered by its inability to ferment most of the sugars present in the biomass. Using metabolic and evolutionary engineering, several industrial strains are now able to utilize xylose. Nevertheless, these strains still present an inadequate utilization of xylose, mostly due to enzyme co-factor unbalance and glucose repression over xylose genes. Hence, there is still a need for new strains capable of efficiently using all the available sugars in order to increase the ethanol yield and productivity. In this study, the yeast Candida intermedia CBS141442, which grows in a wide range of sugars, including cellobiose, lactose, and especially xylose, was submitted to a transcriptome analysis. Using RNA-Seq, we studied the gene expression during growth in several carbon sources, including glucose, xylose and lactose. Several genes with significantly increased expression were identified, contributing to elucidate the metabolic and physiological mechanisms underlying the efficient utilization of these carbon sources. The heterologous expression of such genes could help improve the S. cerevisiae xylose fermentation capacity, as well as increase its substrate range, and contribute to a more cost efficient production of bioethanol.
Invited Oral Abstract
The lower limits of pH for Clostridium thermocellum
Jason M. Whitham, Ji-Won Moon, Miguel Rodriguez Jr., Nancy L. Engle, Zamin Koo Yang, Thomas Rydzak, Dr. Timothy J. Tschaplinski and Steven D. Brown, Oak Ridge National Laboratory, Oak Ridge, TN, USA
39th Symposium on Biotechnology for Fuels and Chemicals
Clostridium thermocellum strain LL1210 contains gene deletions limiting acetate, lactate, and formate production to < 0.03 g/g cellobiose, which facilitated investigation into the effects of lower pH decoupled from organic acid accumulation. The lower limit of pH for strain LL1210 in a carbon limited chemostat (3g/L) at a dilution rate of 0.1 –h was approximately 6.24. At pH 6.24, strain LL1210 secreted significantly higher concentrations of overflow metabolites valine, alanine, and threonine (α = 0.05) compared to pH 6.98 and culture optical density decreased by more than 10%. While these amino acids also accumulated intracellularly, there was more than a 266-fold higher concentration of glutamic acid at pH 6.24. Purines and pyrimidines, including adenine, thymine, and guanine; fatty acids, palmitic acid, stearic acids, and heptadecanoic acid and their iso- counterparts and their glycerol conjugates; phosphorylated metabolites, including 3-phosphoglyceric acid and glucose 6-phosphate were also higher at pH 6.24. At pH values lower than 6.24, spores greatly outnumbered vegetative cells. Transcriptomics analysis of chemostat cultures at pH 6.24 compared to pH 6.98 revealed upregulation of 154 genes at lower pH with annotated protein functions including amino acid transport, tRNA synthesis and modification, sporulation, spermidine/putrescine biosynthesis and transport, ATP synthesis, cobalt transport, and cobalamin biosynthesis. Of the 297 down-regulated genes, functions included motility, chemotaxis, cell envelope biosynthesis, cell division, and the citric acid cycle. This study provides a global analysis of the effects of sub-optimal pH on C. thermocellum metabolism.
Invited Oral Abstract
A factorial design analysis of growth kinetics of Pichia stipitis
Renan Stefanini1, Thlyta Fraga Pacheco2, Dr. Silvia B. Gonalves2 and Fabricio Machado3, (1)Universidade de Braslia, Brasilia, Brazil, (2)Embrapa, Braslia, Brazil, (3)University of Brasilia, Brasilia, Brazil
39th Symposium on Biotechnology for Fuels and Chemicals
Pichia stipitis is one of the most important host organisms used for high-level heterologous protein expression due to its capacity to promote proper protein folding and post-translational modification, especially for proteins from eukaryotic organisms. For this reason, as advancement of research in lignocellulosic ethanol occurs, Pichia stipitis has increasingly being used for expression of enzymes used in cellulose hydrolysis. In order to understand some of the factors that may affect the growth kinetics of Pichia, a 23 factorial design experiment, with three replicates in the central point, was performed to study the effects of inoculum concentration, agitation and pH of medium on the growth kinetics of Pichia stipitis. The effect of those three factors was studied on the yields of biomass and ethanol productions, as well as glucose consumption. Results showed that after 24 h of fermentation, the initial concentration of inoculum was significant in determining the biomass and ethanol yield. According to results, an increase in the inoculum meant an increase in biomass yield, and a decrease in ethanol yield. Concerning the glucose consumption rate, all of the factors, along with its binary and tertiary interactions, showed to be significant at some point during fermentation, with inoculum and agitation being the most significant. Inoculum concentration was the only factor that affected all of the kinetics parameters studied, and it was the most significant of all the factors.
Invited Oral Abstract
Unraveling cellulase and xylanase induction in Thermoascus aurantiacus for improved enzyme production
Raphael Gabriel1, Timo Schuerg2, Jan-Philip Prahl2, Simon Harth2, Lauren M. Tom2 and Steven W. Singer2, (1)Lawrence Berkeley National Laboratory, Emeryville, CA, USA, (2)Joint BioEnergy Institute, Biological Systems and Engineering, Lawrence Berkeley National Laboratory, Emeryville, CA, USA
39th Symposium on Biotechnology for Fuels and Chemicals
The thermophilic fungus Thermoascus aurantiacus is an intriguing host for cellulase production; the wild type strain ATCC 26904 produces considerable quantities of cellulases and xylanases which retain high activity above 65°C. Heat-stable enzymes confer numerous advantages for cost efficient lignofuel production. Understanding the regulation of CAZyme gene activity is crucial for the design of a genetic engineering and cultivation strategy, which aims to achieve highest possible CAZyme yields. Here we report on initial RNASeq experiments, which revealed a strong induction of cellulase and xylanase genes upon cultivation in medium containing beechwood xylan. In contrast to the complex carbon source xylan, simple C5 and C6 sugars, such as D-xylose, L-arabinose, and cellobiose failed to significantly induce CAZyme expression in batch cultures, presumably due to carbon catabolite repression (CCR). The development of a novel continuous feed setup allowed us to circumvent CCR while feeding low amounts of potential inducers into 50 mL shake flask cultures. With this system, we were able to show that D-xylose is indeed a strong inducer of the entire repertoire of plant biomass degrading enzymes resulting in up to 5x higher protein titers when fed at low levels compared to the same amount of inducer added at once. We are currently investigating the transcriptional response to the continuous feed of simple sugars, such as D-xylose, L-arabinose, and cellobiose. Our data analysis is focusing on revealing insights into differently regulated genes, predominantly in sugar utilization and plant biomass deconstruction.
Invited Oral Abstract
Metabolic engineering of the ß-ketoadipate pathway towards the bioproduction of high-value compounds
Sandra Notonier, Christopher W. Johnson, Davinia Salvachúa, Nicholas Rorrer, Brenna A. Black and Gregg Beckham, National Renewable Energy Laboratory, Golden, CO, USA
39th Symposium on Biotechnology for Fuels and Chemicals
The ß-ketoadipate pathway (β-KAP) widely found in bacteria and fungi consists in sequential enzymatic reactions for the catabolism of aromatic compounds. Protocatechuate and catechol are central intermediates in the β-KAP, leading to the production of ß-ketoadipic acid, which is of interest as a novel polymer precursor. Additionally ß-ketoadipate has the potential to be biologically converted into adipic acid, a commodity chemical used in the production of coatings, detergents, and for the synthesis of nylon.
Metabolic engineering in the Gram-negative bacterium Pseudomonas putida KT2440 recently enabled the production of another intermediate from the β-KAP, namely cis,cis-muconic acid, using sugar as starting feedstock.[1] Based on such achievements, P. putida KT2440 was engineered to accumulate ß-ketoadipate from D-glucose. The genetically engineered strain performance was evaluated in 2.5 L bioreactors under continuous feeding. Successive optimizations of the fermentation process led to increase in the ß-ketoadipic acid titer as well as enhanced metabolic yields.
[1] C. W. Johnson, D. Salvachúa, P. Khanna, H. Smith, D. J. Peterson, G. T. Beckham, Metab. Eng. Commun. 2016, 3, 111–119.
Invited Oral Abstract
Aromatic metabolism by oleaginous yeast
Allison Yaguchi, Alana Robinson and Mark Blenner, Clemson University, Clemson, SC, USA
39th Symposium on Biotechnology for Fuels and Chemicals
Oleaginous yeasts have long been a target for developing industrial-scale biochemical processes due to their ability to accumulate high amounts of lipids on a per cell basis, synthesize complex chemicals and proteins, and robustly metabolize diverse feedstocks. In parallel, interest in lignocellulosic biomass as a feedstock has gained traction. Unfortunately, metabolism of lignin and lignin-derived aromatics is slow, and phenolic compounds are often toxic. Trichosporon oleaginosus, previously known as Cryptococcus curvatus, is a non-model, non-conventional, oleaginous yeast that we have discovered both tolerates and metabolizes phenolic compounds derived from lignin. BLAST analysis suggests a potential putative pathway for metabolism of aromatic compounds, but qPCR results show the mechanism could involve a cryptic pathway or an alternative mechanism of aromatic metabolism. We have characterized biomass accumulation, lipid profiles, and gene expression patterns for T. oleaginosus on both model monoaromatics and sugars under different nitrogen conditions. Together our data suggest that T. oleaginosus may be a good model oleaginous system for aromatic metabolism.
Invited Oral Abstract
Enabling bioconversion of biorefinery wastes to lipids via Rhocococcus jostii RHA1
Xiaolu Li1, Zhangyang Xu1, Matthew Gaffrey2, Shihui Yang3, Libing Zhang1, Lindsay D. Eltis4, Dr. Weijun Qian5, Dr. Joshua S. Yuan6 and Bin Yang1, (1)Washington State University, Richland, WA, USA, (2)Pacific Northwest National Lab, Richland, WA, USA, (3)National Renewable Energy Laboratory, Golden, CO, USA, (4)University of British Columbia, Vancouver, BC, Canada, (5)Pacific Northwest National Laboratory, Richland, WA, USA, (6)Texas A&M University, College Station, TX, USA
39th Symposium on Biotechnology for Fuels and Chemicals
R. jostii RHA1 has advantage of tractable genetics for metabolic engineering and robust growth on biorefinery wastes, including lignin and other biomass degraded products, to accumulate triacylglycerols (TAGs) for biofuel production. In this study, genomics, transcriptomics, and proteomics analysis were carried out to help establish how the catabolic pathways are arranged in R. jostii RHA1 for funneling biosynthesis of TAGs from a range of biorefinery waste compounds. Several catabolic pathways in R. jostii RHA1 and its mutants, which are responsible for growth and TAGs accumulation on different biomass derived compounds such as lignin-derived aromatics (benzoate, vanillin), furans (furfural, 5-HMF), weak acids (acetic acid) as well as ethanol refinery residues from dilute-acid pretreated corn stover, were then characterized in details. In addition, several putative redox-regulated proteins in R. jostii RHA1, which affected the cellular catabolism network, were identified.
Invited Oral Abstract
Upscaling production of thermostable cellulases using the thermophilic filamentous fungus Thermoascus aurantiacus
Timo Schuerg1, Raphael Gabriel2, Jan-Philip Prahl1, Simon Harth1, Lauren M. Tom1 and Steven W. Singer1, (1)Joint BioEnergy Institute, Biological Systems and Engineering, Lawrence Berkeley National Laboratory, Emeryville, CA, USA, (2)Lawrence Berkeley National Laboratory, Emeryville, CA, USA
39th Symposium on Biotechnology for Fuels and Chemicals
Thermophilic filamentous fungi are a valuable source of thermostable cellulases. Here we report successful scale-up of enzyme production by T. aurantiacus to 20 L scale. We demonstrated that MXP (Methyl beta-D-xylopyranoside), a non-metabolizable xylose analog, strongly induces cellulases in batch cultivations, whereas metabolizable xylose is much less potent. Based on our observations we hypothesized that carbon catabolite repression (CCR) might be responsible for the inhibited induction of xylose. To test our hypothesis, we established an easily deployable fed-batch system for the 50 mL shake flask scale, which allowed us to circumvent CCR by feeding only small amounts of xylose over time. When continuously feeding only 78 mg xylose h-1 L-1, a 5x increase in cellulase induction was achieved as compared to feeding the same amount of xylose in one pulse. The system proved to be successful in optimizing fed-batch cultivation conditions at 50 mL scale and was successfully transferred to 2 L and 20 L pilot scales with crude enzyme titers ranging from 1.1 - 3.2 g/L. Feeding a xylose-rich fraction solubilized from dilute acid pretreated corn-stover resulted in cellulase production of comparable magnitude to the pure xylose feeds. The use of this xylose-rich soluble fraction to produce cellulases, which will then be used to hydrolyze the remaining glucose-rich solid from dilute acid pretreatment, has great potential to link pretreatment and on-site enzyme production at a biorefinery.
Invited Oral Abstract
The secretome analysis and characterization of thermophilic lignocellulolytic fungus Malbranchea pulchella
Carlaile Nogueira1, Juliana Velasco C. Oliveira2, Andr Ferraz1 and Fernando Segato1, (1)Escola de Engenharia de Lorena, Universidade de So Paulo, Lorena, Brazil, (2)Laboratrio Nacional de Cincia e Tecnologia do Bioetanol, Campinas, Brazil
39th Symposium on Biotechnology for Fuels and Chemicals
Enzymatic depolymerization of biomass components is key process for bioenergy industries. The search for new enzymes for this task has increased secretome studies in biomass-degrading microorganisms. Filamentous fungi are important players in carbon recycling in nature, presenting potential enzymes for biomass conversion. Malbranchea pulchella is a thermophilic ascomycete bioprospected in Brazilian Rainforest whose biodegradation ability and enzymatic profile were focus of this study. Semi-solid cultivation of this fungus on lignified (eucalyptus wood) and non-lignified (bleached eucalyptus pulp) substrates for 60 days resulted in 5% and 52% weight losses, respectively. To understand the diversity of the enzymes produced by M. pulchella, the secretome was evaluated in 3-day submerse cultures using the previously mentioned carbon sources. LC-MS/MS results showed that the fungus was able to secrete an array of lignocellulolytic enzymes. The prominent enzymes were glycosyl hydrolases with a total of 29 and 37 CAZymes in lignified and non-lignified substrates, respectively, with GH3 and GH18 families found in greatest number. Considering the oxidative enzymes, a total of 12 and 13 CAZymes were found in lignified and non-lignified substrates, respectively, being AA7 and AA9 the most expressive families. Non-CAZymes proteins showing allergen domains, which are related with expansin-like proteins from plants, were also detected. The secretome produced with bleached eucalyptus pulp substrate was assayed for hydrolysis of Kraft pulp and also in contribution with Celluclast for dissolving pulp and Avicel hydrolysis. Results indicated that M. pulchella produce an interesting machinery that can disrupt both amorphous and crystalline cellulose. Acknowledgements: CNPq, CAPES, FAPESP 14/18714-2.
Invited Oral Abstract
Characterization and development of Cryptococcus curvatus ATCC 20509 as a platform host for bio-hydrocarbon chemicals
John Ojumu1, Richard J. Giannone2, Nancy L. Engle1, Dr. Timothy J. Tschaplinski1, Daniel Jacobson1 and Dan Close1, (1)Oak Ridge National Laboratory, Oak Ridge, TN, USA, (2)BioEnergy Science Center, Oak Ridge National Laboratory, Oak Ridge, TN, USA
39th Symposium on Biotechnology for Fuels and Chemicals
Cryptococcus curvatus ATCC 20509 is an oleaginous yeast strain capable of assimilating xylose, lactose, glucose, and sucrose, as well as a variety of agricultural and food processing wastes, as carbon sources, which has highlighted its potential as a candidate host for the production of hydrocarbon chemicals. For this reason, C. curvatus ATCC 20509 has a long history of investigation. However, it has failed to achieve traction as an oleaginous model because of a lack of modern omics-based assessments and reliable transformation systems for genetic modification. Therefore, to investigate the potential for overcoming these hurdles, our group is characterizing the genomic, lipidoimc, transcriptomic, and proteomic profiles of this organism throughout the growth to lipid synthesis transition to develop metabolic models describing how it regulates its onset of lipid synthesis. Lipidomic analysis shows that C. curvatus ATCC 20509 favors the production of 18 carbon fatty acids throughout its lifecycle, and nitrogen limitation has been shown to trigger the onset of lipid accumulation metabolism as is common in other oleaginous strains. A full transcriptomic analysis of gene expression throughout this metabolic transition is currently ongoing to identify the regulatory controls governing flux switching. To prepare for genetic modification, the genome has been sequenced and various techniques have been evaluated for introducing exogenous DNA. A rapid, bead beating-based protocol has been established for plasmid introduction and work is currently ongoing to support improved expression of introduced gene sequences through the competitive evaluation of exogenous and native promoter sequences.
Invited Oral Abstract
Ionic liquid tolerance of 39 oleaginous yeast species
Kyria L. Boundy-Mills1, Irnayuli Sitepu1, Lauren Enriquez2, Luis Garay2, John Butler2, Russell Fry2, Atit Kanti3, Agus Joko Nugroho3, Sarah Faulina4, Blake A. Simmons5, Steven W. Singer5 and Christopher W. Simmons2, (1)University of California, Davis, Davis, CA, USA, (2)University of California Davis, Davis, CA, USA, (3)Indonesian Institute of Sciences, Cibinong, Indonesia, (4)Agency for Research, Development and Innovation, Bogor, Indonesia, (5)Joint BioEnergy Institute, Emeryville, CA, USA
39th Symposium on Biotechnology for Fuels and Chemicals
Pretreatment of lignocellulosic biomass with ionic liquids (IL) such as 1-ethyl-3-methylimidazolium ([C2C1Im]+) chloride or acetate is an effective method for deconstruction. However, residual IL can be inhibitory to microbial growth and enzyme activity. Although very few of the 90+ oleaginous yeast species have been characterized for IL tolerance to date, studies indicate that oleaginous yeasts are sensitive to IL. The aim of this study was to identify oleaginous yeasts that are tolerant of ionic liquids. Forty-six strains of oleaginous yeasts belonging to 39 taxonomically diverse species were cultivated in laboratory medium supplemented with 0, 1, 2 or 4% (w/v) [C2C1Im][OAc] or [C2C1Im]Cl. While growth of most yeasts was inhibited, growth of some species was detected in IL concentrations up to 4% IL. The six most tolerant yeasts strains, belonging to five different species, were then cultivated in laboratory medium containing no IL, 242mM [C2C1Im][OAc], or 242mM [C2C1Im]Cl. The effects of IL exposure on cell mass production and oil accumulation varied by species and by strain. The acetate IL generally decreased cell biomass and lipid production quite severely, with the effect depending on the yeast species, ranging from a small decrease in growth to undetectable growth. The chloride salt resulted in much milder effects, from 22% decrease to 22% increase in lipid output. The highest lipid outputs in the presence of 242mM [C2C1Im]Cl were 8.3 g/L and 7.9 g/L for Vanrija humicola UCDFST10-1004 and V. humicola UCDFST 12-717, respectively. These two strains are appealing candidates for strain and process development.
Invited Oral Abstract
Deletion of Lipomyces starkeyi Ku70 homologue augments platform stain generation for gene expression and functional analysis
Ziyu Dai, Shuang Deng, Kyle Pomraning, David Culley and Jon K. Magnuson, Pacific Northwest National Laboratory, Richland, WA, USA
39th Symposium on Biotechnology for Fuels and Chemicals
Due to interest in productions of renewable fuels and chemicals, various microorganisms are being explored for their potential use in those applications. Numerous lipid-producing yeasts, e.g. Lipomyces starkeyi, are being actively investigated for their potentials in chemical and biofuel production utilizing various carbon and nutrient sources. Recently, several transformation methods have been established for L. starkeyi, however, the efficiency of targeted gene replacement to generate knock-outs and knock-ins, which is a commonly used method to study the function of genes of interest, has been impeded by inefficient mechanisms for accurate integration. To improve gene targeting efficiency in L. starkeyi, the L. starkeyi Ku70 homologue was identified and deleted. We observed a substantial increase in the targeting efficiency in selected genes. The minimal length of flanking homologous DNA for high efficiency of gene targeting, the Ku70 deletion strain sensitive to UV rays, and targeting deletion or over-expression of several selected genes related to lipid production will be also demonstrated in this study.
Invited Oral Abstract
Real-time H2 and CO2 off-gas analysis of complex carbon sources by Caldicellulosiruptor bescii
Todd Vander Wall, Lauren Magnusson, Yannick Bomble, Stephen R. Decker and Michael E. Himmel, National Renewable Energy Laboratory, Golden, CO, USA
39th Symposium on Biotechnology for Fuels and Chemicals
Caldicellulosiruptor bescii is a an extremely thermophilic, cellulolytic anaerobe with a growth temperature range of 42-90°C with a Topt=78° It can utilize unpretreated cellulose of high lignin and both low and high lignin grasses as well as nearly any cellulosic constitutive sugars. Combine these two attributes with it’s secretion of the enzyme CelA; a cellulase of the highest order of activity, and C. bescii begs to be an organism of substantial additional investigation. However, C. bescii has been known to be a sensitive and difficult organism to grow well, and sometimes to grow at all. Serum bottles allow for easy propagation of the organism, as well as an ease of producing replicates, but growth performance is poor, and the work load to generate enough bottles is high. Fermentation in bioreactors produces substantially better results of cell density and presumably; protein production and biomass degradation, but are extremely time and energy consuming for set-up, operation, and cleanup. Running multiple bioreactors at the same time further complicates issues by a proportional magnitude. However, bioreactors allow for off-gas collection and analysis of H2 and CO2 which yield a direct analog to traditional growth curves. Furthermore, as complex carbon sources and/or different biomass are not only of interest, but may also be the only way to truly induce C. bescii’s full proteome; off-gas analysis may be the only reasonable method available to track growth on these insoluble substrates.
Invited Oral Abstract
Evaluation of metabolic engineering strategies for Clostridium thermocellum
Evert Holwerda1, Daniel G. Olson2, Shuen Hon2, Marybeth Maloney2, Anthony Lanahan2 and Lee R. Lynd2, (1)Dartmouth College;Center for Bioenergy Innovation, Hanover, NH, USA, (2)Dartmouth College, Hanover, NH, USA
39th Symposium on Biotechnology for Fuels and Chemicals
The anaerobic thermophile
Clostridium thermocellum is considered to be one of the best model organisms for studying cellulose utilization as applied in consolidated bioprocessing (CBP) because of its innate cellulolytic and ethanologenic capabilities. It has one of the highest rates of cellulose utilization, and it employs a traditional fermentative pathway resulting in mixed acids and ethanol as products.
One of the first attempts to engineer C. thermocellum into an ethanologen involved knocking out branches of its native metabolism in order to reduce acid production. Here we closely follow what happened when two similar but different sets of gene-knockouts generated two potential ethanologen lineages resulting in four end-strains. Each end-strain was subsequently subjected to a different evolutionary approach (long residence chemostat, serial transfers or long residence pH-auxostat).
Not all the four end-strains displayed an increase in ethanol production. To gain more insight into reasons why, every step of the two engineering strategies was evaluated by resequencing the individual strains (15 strains), and growing it under strictly controlled conditions in a chemostat. Results include fermentation profile; steady state biomass concentration, fermentation products and gas evolution, as well as transciptome analysis (RNAseq).
We also show what happens when the end-strains were grown under high-cellulose loadings and are compared to the wild-type organism. Three end-strains utilized increased amounts of cellulose and had higher rates of cellulose solubilization. Surprisingly valine was the second largest fermentation product after ethanol and further studies into nitrogen metabolism resulted in interesting physiological effects under nitrogen limitation conditions.
Invited Oral Abstract
Characterizing transport of lignin breakdown products into P chrysosporium S. cerevisiae, and E. lignolyticusSCF1
Michael Kent, Meghan C. Barnhart-Dailey, Dongmei Ye, Troy Simoes, Bryan Carson, Leah Appelhans, Dulce Hayes, Kenneth L Sale and Jerilyn A. Timlin, Sandia National Laboratories, Albuquerque, NM, USA
39th Symposium on Biotechnology for Fuels and Chemicals
Valorization of lignin has the potential to significantly improve the economics of lignocellulosic biorefineries. However, efficient conversion of lignin to useful molecular building blocks has been elusive. Microbial conversion of lignin in nature is efficient, but occurs very slowly. Engineering microbes to produce more efficient and more directed lignin conversion is a promising strategy. One aspect of that engineering effort is to optimize transport. However, very little is currently known about lignin transport into microbes. Indirect evidence such as growth and toxicity studies suggests lignolytic organisms may transport a wide range of mono-, di-, and possibly even higher molecular weight lignin breakdown products across the cellular membrane, but direct measurements of the substrate range and specificity are lacking except for a few select studies. To that end we are characterizing the uptake and consumption of lignin-like substrates by P. chrysosporium, S. cerevisiae, and E. lignolyticus SCF1. Internalization is measured directly using imaging and mass spectrometry, and uptake and consumption is followed through depletion of the substrates from the media using HPLC. We report initial results for a range of mono- and di-aryl compounds.
Invited Oral Abstract
Development of a high efficiency integration system and promoter library for rapid modification of Pseudomonas putida KT2440
Joshua Elmore, Anna Furches, Gara Wolff, Kent Gorday and Adam Guss, Oak Ridge National Laboratory, Oak Ridge, TN, USA
39th Symposium on Biotechnology for Fuels and Chemicals
Pseudomonas putida are highly robust bacteria known for their ability to efficiently utilize a variety of carbon sources, including aliphatic and aromatic hydrocarbons. Recently, P. putida has been engineered to valorize the lignin stream of a lignocellulosic biomass pretreatment process. Nonetheless, when compared to platform organisms such as Escherichia coli, the toolkit for engineering P. putida is underdeveloped. Heterologous gene expression in particular is problematic. Plasmid instability and copy number variance provide challenges for replicative plasmids, while using homologous recombination for insertion of DNA into the chromosome is slow and laborious. Further, most heterologous expression efforts to date typically rely on overexpression of exogenous pathways using a handful of poorly characterized promoters. To improve the P. putida toolkit, we developed a rapid genome integration system using the site-specific recombinase from bacteriophage Bxb1 to enable rapid, high efficiency integration of DNA into the P. putida chromosome. We also developed a library of synthetic promoters with various UP elements, -35 sequences, and -10 sequences, as well as different ribosomal binding sites. We tested these promoters using a fluorescent reporter, mNeonGreen, to characterize the strength of each promoter, and identified UP-element-promoter-ribosomal binding sites combinations capable of driving a ~150-fold range of protein expression levels. An additional integrating vector was developed that confers more robust kanamycin resistance when integrated at single copy into the chromosome. This genome integration and reporter systems are extensible for testing other genetic parts, such as examining terminator strength, and will allow rapid integration of heterologous pathways for metabolic engineering.
Invited Oral Abstract
Production of fragrant α-santalene from metabolically engineered E. coli
Hyo-Jung Han, Chonglong Wang, Sin-Young Kim and Prof. Seon-Won Kim, Gyeongsang National University, Jinju, Korea, Republic of (South)
39th Symposium on Biotechnology for Fuels and Chemicals
Sandalwood oil possesses a very pleasant and long lasting scent and is widely used in food and cosmetic industries. It is traditionally obtained from the heartwood and roots of mature sandalwood trees (>25 years) via steam distillation. This traditional method of extraction suffers from low yields and over-harvesting the sandalwood trees. α-Santalene is the precursor of α-santalol, which represents up to 50% of natural sandalwood oil. It is a sesquiterpenoid derived from the universal C5 precursors, isopentenyl diphosphate (IPP) and dimethylallyldiphosphate (DMAPP), which are generated via either the 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway or the mevalonic acid (MVA) pathway. Farnesyl pyrophosphate (FPP) synthase then catalyzes the assembly of IPP and DMAPP to the linear FPP, which undergoes rearrangement and cyclization by santalene synthase to form santalene. Here, we constructed a heterologous biosynthesis pathway for α-santalene production. Manipulation of ribosome binding sites (RBS) or other RNA regulators offer a very effective alternative to tune the expression of multiple genes. A set of synthetic RBSs were utilized to modulate diverse expression levels of FPP synthase and α-santalene synthase for the pathway optimization. By this approach, we have successfully produced α-santalene of > 400 mg/L, several folds higher than the previously reported production. Indole synthesis pathway was also removed because indole has been known to inhibit isoprenoids production and have unpleasant odor. Thus, we could produce pure santalene with an enhanced yield. This work was supported by a grant (NRF-2016R1A2B2010678) from the National Research Foundation, MSIP, Korea.
Invited Oral Abstract
Physiological and systems level characterization of Caldicellulosiruptor bescii acid stress responses
Punita Manga1, Kyle B. Sander2, Miguel Rodriguez Jr.3, Dawn M. Klingeman4, Nancy L. Engle4, Suresh Poudel1, Dr. Timothy J. Tschaplinski5, Robert L. Hettich4 and Steven D. Brown3, (1)University of Tennessee/Oak Ridge National Laboratory, Knoxville, TN, USA, (2)Oak Ridge National Laboratory/University of Tennessee, Oak Ridge, TN, USA, (3)Oak Ridge National Laboratory, Oak Ridge, TN, USA, (4)Biosciences Division and BioEnergy Science Center, Oak Ridge National Laboratory, Oak Ridge, TN, USA, (5)BioEnergy Science Center, Oak Ridge National Laboratory, Oak Ridge, TN, USA
39th Symposium on Biotechnology for Fuels and Chemicals
Caldicellulosiruptor bescii is an anaerobic hyper thermophile (70-80oC) that can utilize wide range of substrates. However, inhibitors released from biomass can result in unfavorable growth conditions and limit bioconversion to products. Medium as well as intracellular pH is a condition critical for growth and prone to change in effect of such compounds. Growth pH for C. bescii is currently reported as a narrow range between 6.8-7.3. In this study, we examine the physiological and systems level acid stress responses in C. bescii. Samples were collected from bottles, controlled batch and chemostat systems to be subjected to growth, product and integrated omics profiling. Cultures grown in unbuffered medium bottles showed growth at pH values as low as 5.5 with a lag of ~50 hours. C. bescii also displayed the ability to maintain growth at this low pH at 0.1 hr-1 dilution rate in chemostat on avicel. In batch reactors, with controls maintained at pH 7.2, a significant increase in growth and product yield was observed in treated reactors where pH was lowered from 7.2 to 6.0 at mid-log growth. Time course transcriptomics data from these reactors revealed a set of sugar ABC transporter genes to be consistently over-expressed through time in the treated cells. Amino acid transport and metabolism; carbohydrate transport and metabolism; energy production and conversion; cell envelop biogenesis; inorganic ion transport and metabolism were the major differentially expressed COG categories in this data set. Proteomics and metabolomics data for these batch fermentations is underway and will also be discussed.
Invited Oral Abstract
Sequencing the largest existing collection of historic commercial solventogenic clostridia strains to dissect industrial acetone-butanol-ethanol (ABE) fermentations
Steven D. Brown1, Dr. Rasmus Jensen2, Vinicio Reynoso2, Dawn M. Klingeman3, Sashini DeTissera2, Wayne Mitchell2, Marcel Huntemann4, Alicia Clum4, Manoj Pillay4, Krishnaveni Palaniappan4, Neha Varghese4, Natalia Mikhailova4, Dimitrios Stamatis4, T.B.K. Reddy4, Chew Yee Ngan4, Chris Daum4, Nicole Shapiro4, Victor Markowitz4, Natalia Ivanova4, Nikos Kyrpides4, Tanja Woyke4, David Jones5, Sean Simpson2 and Dr. Michael Kopke6, (1)Oak Ridge National Laboratory, Oak Ridge, TN, USA, (2)LanzaTech, Skokie, IL, USA, (3)Biosciences Division and BioEnergy Science Center, Oak Ridge National Laboratory, Oak Ridge, TN, USA, (4)US Department of Energy Joint Genome Institute, Walnut Creek, CA, USA, (5)University of Otago, Dunedin, New Zealand, (6)LanzaTech, Inc., Skokie, IL, USA
39th Symposium on Biotechnology for Fuels and Chemicals
The David T. Jones collection of clostridia dates back to 1944 and contains over 300 unique strains used in various commercial operations, including the South African ABE fermentation process at National Chemical Products (NCP). The collection includes some of the earliest spore stocks sent from both Commercial Solvents Corporation (CSC, USA) and Commercial Solvents Great Britain (CS-GB, UK) and comprises of mainly Clostridium acetobutylicum, C. beijerinckii, C. saccherobutylicum and C. saccheroperbutylacetonicum strains. The collection also includes strains from Japanese and Taiwanese commercial operations as well as strains for various research labs. The collection also includes a number of strains resulting from research into solving specific problems such as phage infections or lower solvent yield when the characteristics of the molasses changed on an annual basis. In collaboration with the US Department of Energy Joint Genome Institute (JGI), this historic collection of commercial solventogenic clostridia are having their genome sequences determined and analyzed. To date, the genomes for one Clostridium acetobutylicum, 18 Clostridium beijerinckii, two Clostridium butyricum, four Clostridium saccharoperbutylacetonicum and one Clostridium tetanomorphum have been sequenced using PacBio single molecule sequencing resulting in between 1-7 contigs per genome. Among the Clostridium beijerinckii strains, genome sizes range between 5.6 and 6.5 Mb, the number of predicted gene models range from 5,058 to 5,966 and 16S rDNA copy numbers are between 14 and 21 per genome. An overview of the project will be presented and initial sequence data will be discussed.
Invited Oral Abstract
Efficient estimation of maximum theoretical productivity from batch cultures via dynamic optimization of flux balance models
Peter St John, Michael F. Crowley and Yannick Bomble, National Renewable Energy Laboratory, Golden, CO, USA
39th Symposium on Biotechnology for Fuels and Chemicals
Production of chemicals from engineered organisms in a batch culture typically involves a trade-off between productivity, yield, and titer. However, strategies for strain design typically involve designing mutations to achieve the highest yield possible while maintaining growth viability. One can use multi-stage fermentations to increase either productivity or yield. Such strategies would range from simple manipulations (aerobic growth phase, anaerobic production phase), to more complex genetic toggle switches. One can assume an initial control strategy (i.e., a single reaction target) in maximizing productivity - but it is unclear how close this productivity would come to a global optimum. The calculation of maximum theoretical yield in metabolic engineering can help guide strain and pathway selection for static strain design efforts. Here, we present a method for the calculation of a maximum theoretical productivity of a batch culture system. This method follows the traditional assumptions of dynamic flux balance analysis: that internal metabolite fluxes are governed by a pseudo-steady state and external metabolite fluxes are represented by dynamic system including Michealis-Menten or hill-type regulation. The productivity optimization is achieved via dynamic programming, and accounts explicitly for an arbitrary number of fermentation stages and flux variable changes. We have applied our method to succinate production in two common microbial hosts: E. coli and A. succinogenes. The method can be further extended to calculate the complete productivity versus yield Pareto surface. Our results demonstrate that nearly optimal yields and productivities can indeed be achieved with only two discrete flux stages.
Invited Oral Abstract
High-throughput screening platform to identify strategies for improved anaerobic digestion
Lyndsey Marsh, University of California, Davis, Davis, CA, USA and Dr. Matthias Hess, University of California, Davis, CA, USA
39th Symposium on Biotechnology for Fuels and Chemicals
Anaerobic digestion (AD) of plant material and organic waste and their subsequent conversion into energy or value-added chemicals has significant ecological and socio-economical implications. The efficiency of the fermentation process during AD and the resulting product profile depend on various parameters, ranging from feedstock composition to process conditions. Although these parameters can vary significantly in nature, they have in common that they affect the composition of the microbial community (commonly referred to as the microbiome) and the complex interactions between the individual microorganisms that thrive in the fermentation vessels. The cow’s rumen, the compartment of the cow’s digestive system where plant material is converted into methane, carbon dioxide, volatile fatty acids and other intermediates, is one of the most efficient anaerobic digestions systems known. We designed and established an artificial (in-vitro) laboratory-scale rumen system that takes advantages of the native rumen microbiome and its superior fermentation efficiency. Since our in-vitro system also allows monitoring biological, physical and chemical parameters during the anaerobic fermentation process, it represents an economical platform to quickly screen and evaluate feedstock composition, biological and chemical compounds, as well as process conditions on the fermentation efficiency. Results obtained using our in-vitro screening system will be presented. We anticipate that these results will provide the framework to develop improved strategies for reducing the environmental impact of AD processes and for increasing the yield of desired fermentation end products.
Invited Oral Abstract
RetroSynth: A tool for identifying best metabolic routes for production of a target compound
Dr. Leanne Whitmore, Dr. Anthe George and Dr. Corey Hudson, Sandia National Laboratories, Livermore, CA, USA
39th Symposium on Biotechnology for Fuels and Chemicals
Cost-effective production of chemical compounds, i.e. chemicals found in fuels or plastics, is essential for increasing independence from foreign oil. Biologically synthesizing compounds within microbial organisms is an attractive solution as this method can be cost and time efficient for certain classes of molecules. However, determining the necessary and most effective reactions/enzymes to add to an organism to achieve maximum production of a target chemical is a lengthy and expensive process. Herein, we present an open-source software, which – given a target compound and bacterial host organism – will calculate the optimal number of reactions/enzymes that need to be inserted into the host organism to produce the target chemical. We developed an integer program to succinctly identify the minimal number of reactions, given a set of internal (native to the host organism) compounds and reactions and external (from other organisms) compounds and reactions. This program also finds multiple pathways consisting of the minimal number of reactions if they exist. Output from the program includes not only the external reaction steps, but also all external reactants and products for each identified reaction. Additionally, using flux balance analysis, our software simulates the metabolism of the host organism with the added external reactions, reactants and products which provides insight into validity and success of target compound production using the shortest paths identified by our integer program. Our software streamlines an arduous and complex method, and may replace these costly techniques enabling scientists to inexpensively expedite the scientific process and production of target compounds.
Invited Oral Abstract
Metabolic engineering the thermophilic fungus Myceliophthora thermophila for cellulases and biobased chemicals production through genome editing tools of CRISPR/Cas9 system
Jingen Li, Qian Liu, Xuerong Xing, Liangcai Lin and Chaoguang Tian, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, China
39th Symposium on Biotechnology for Fuels and Chemicals
The thermophilic fungi such as Myceliophthora species are excellent organisms for ligocellulose utilization, this great characteristics make this genus fungi can be used for cellulases and other enzymes production through metabolic engineering. However, the genetics tools for metabolic engineering of these thermophlic species are very limit. Recently, we have developed a high efficiency Agrobacterium tumefaciens mediated transformation (ATMT) system for Myceliophthora thermophila, using green fluorescence protein (GFP) as auxiliary marker. Based on this tool, the CRISPR/Cas9 system for efficient multiplexed genome engineering was also successfully developed in this thermophilic fungus. As a proof of principle, the genes of the cellulase production pathway were chosen as editing targets. Simultaneous disruptions of up to four different loci were accomplished via a single transformation using the CRISPR/Cas9 system. Multiple strains exhibiting pronounced hyper-cellulase production were generated, in which the extracellular secreted protein and lignocellulase activities were significantly increased (up to 5- and 13-fold, respectively) compared with the parental strain. In addition, the engineering of the strain for organic acids production from lignocellulose were also tried, the results was pretty encouraging. Successful expansion of this system without modification to M. heterothallica indicates it has wide adaptability and flexibility for use in other thermophilic fungal species. This system could greatly accelerate strain engineering of thermophilic fungi for production of industrial enzymes, such as cellulases and bio-based fuels and chemicals in the future.
Invited Oral Abstract
Exploring gut microbiome of herbivores animals for biofuels
Dr. Naseem Gaur, Mr. Jakeer Shaik and Mahendra Varma, DBT-ICGEB Centre for advanced bioenergy research, International Centre for Genetic Engineering and Biotechnology, New Delhi, India
39th Symposium on Biotechnology for Fuels and Chemicals
Herbivores animals consume bulk amount of lignocellulosic material, which is subsequently converted into simple sugars by the gut microbes. The genome of such microbes is rich in carbohydrate active enzymes (CAZymes) and enzymes involved in C5/C6 assimilation. We explored elephant (Elephas maximus), rhinoceros (Rhinoceros unicorni), and nilgai (Boselaphus tragocamelus) dung microbiome to evaluate its potential in lignocellulosic biomass degradation and assimilation. Metagenomic analysis showed 93.7% bacterial and 1.08% fungal populations in elephant dung microbiome. Proteobacteria (73.68%) and Ascomycota (75%) were predominantly present among bacterial and fungal species, respectively. Unlike bacterial sp. fungal sp. were equally distributed in elephant dung microbiome. CAZymes were identified by dbCAN analysis and each member of glycoside hydrolase (GH) family was looked for its abundance with in the family. Comparison among GH families of elephant dung with the GH families in the gut microbiome’s of other herbivorous animals revealed that GH 24, 102 and 108 GH families are elephant gut specific, while GH3 family is common among all herbivores. Additionally, three aerobic (Aspergillus flavus, Aspergillus niger, Aspergillus fumigatus) and two anaerobic (Piromyces sp. and Anaeromyces sp.) fungal isolates were isolated from elephant and rhinoceros dung. Aspergillus sp. and Piromyces sp. of fungi are known to produce efficient biomass degrading and assimilating enzymes. Aspergillus sp. isolates secreted maximum amount of proteins with xylan and wheat straw as a carbon source and in-vitro cellulases and xylanases activities were detected in these secretome.
Invited Oral Abstract
Engineering methylotrophic bacteria for the production of itaconic acid from renewable simple carbon sources
Chee Kent Lim, Juan Villada and Patrick K.H. Lee, City University of Hong Kong, Kowloon Tong, Hong Kong
39th Symposium on Biotechnology for Fuels and Chemicals
Itaconic acid is a high-value chemical building block with broad industrial applications. Currently, the production of this compound is by fungal fermentation via
Aspergillus species using carbohydrates as the input carbon sources.
In order to develop a sustainable and cost-effective method to produce itaconic acid, we explored a bacterial system utilizing renewable simple carbon sources. In this study, we employed Methylobacterium extorquens AM1, which is a facultative methylotrophic bacterial species capable of using multiple types of carbon compounds including various C-1 carbon compounds such as methanol and formate but not methane, as the host for producing itaconic acid. A codon optimized cis-aconitate decarboxylase-encoding gene derived from Aspergillus terreus, which encodes an enzyme to convert cis-aconitic acid to itaconic acid, was chemically synthesized and cloned into an expression plasmid. The introduction of this recombinant plasmid into M. extorquens AM1 successfully enabled the production of itaconic acid from C-1 carbon sources.
To further increase the versatility of this production platform, we also applied a modular co-culture of the engineered M. extorquens AM1 with another methylotroph, Methylomicrobium album BG8, which is able to utilize methane as a carbon source. M. album BG8 is engineered to be dependent on M. extorquens AM1 for a key co-factor, while M. album BG8 provides carbon sources to M. extorquens AM1. This scheme permits the mutualistic cooperation of the two species to convert methane to itaconic acid.
Invited Oral Abstract
Reinvigorating Yarrowia lipolytica as synthetic biology chassis for biofuel and bioproducts production
Xiaochao Xiong and Prof. Shulin Chen, Washington State University, Pullman, WA, USA
39th Symposium on Biotechnology for Fuels and Chemicals
The oleaginous yeast Yarrowia lipolytica with long history for industrial application has attracted growing attention due to its capability of accumulating microbial lipid. In our previous studies, we developed of a set of toolbox for genetic manipulation of this important strain, and successfully used these developed molecular tools to metabolically engineer Y. lipolytica for overproducing lipid-derived products such as free fatty acid, wax esters and fatty alcohol. However, construction of a more complex biosynthesis pathway consisting of a number of genes with the large size remains challenging in Y. lipolytica. Additionally, one of the major obstacles for synthetic biology of Y. lipolytica is integration of the large biochemical pathways into yeast genome with high copy numbers for stable industrial production. To address these challenges, we introduced a novel methodology that could generate the individual active enzymes from the polycistronic mRNA of a gene cluster under a single promoter in Y. lipolytica by using viral 2A peptide mediated cleavage. We further developed a versatile genome editing technology for markerless and multiple-copy chromosomal integration of the full multiple-gene pathways with high efficiently in Y. lipolytica. As proof of the concept, the entire carotenoid biosynthesis pathway was assembled and engineered into the heterologous host, Y. lipolytica. By using the developed synthetic biology approaches, we also engineered the pathways for biosynthesis of other valuable products such as sphingolipids in Y. lipolytica. These combined efforts result in a transformative platform technology for producing biofuels and renewable chemicals by synthetic biology of Y. lipolytica.
Invited Oral Abstract
Wild yeast isolates growth in different media
Dr. Sandra H Cruz, Ms. Nadia Viana and Dr. Caur Portugal, University of Sao Paulo, Piracicaba, Brazil
39th Symposium on Biotechnology for Fuels and Chemicals
Wild yeasts can be introduced into the fermentation industrial process through substrates or water due the difficulties to sterilize large volumes of juice. Identifying and exploring the potential of those yeast isolates for ethanol production is a valuable tool for the ethanol producing industry, since the most successful commercial strains available are product of selection processes. Fourteen yeast isolates provenient from a bioethanol plant in the State of Sao Paulo, Brazil, and two reference Saccharomyces cerevisiae strains (CAT-1 and PE-2) were grown in a microplate reader (Tecan Infinite M200) using 96 wells microplate. The cultivation was carried at 30ºC for 24 hours, in four different growth media: YPD, YPSac2, MCC5 (sterilized sugar-cane juice) e MMel5 (clarified molasses). The UV absorbance (600 nm) readings were obtained every two hours, and the cells were grown in triplicates. The results allowed comparison between the wild isolates and the reference strains, showing different responses to the growth media utilized. Some isolates performed better than the reference yeast strains when grown in YPSac, MCC and MMel. Only on YPD medium the reference strains showed an overall better performance. The growth rates and speed showed changeability among the isolates, a signal of the variety of the microbiota found in the Brazilian fermentation process.
Invited Oral Abstract
Evaluation of alcoholic fermentation VHG in fed-batch system with recycle of yeast cells
Dr. Sandra H Cruz and Mrs. Graziela Francischetti, University of Sao Paulo, Piracicaba, Brazil
39th Symposium on Biotechnology for Fuels and Chemicals
The bioethanol production potential can be maximized by using high alcohol fermentation known as Very High Gravity (VHG). The main focus of VHG fermentation is the reduction of production costs and reduction of waste generated. However, the wort containing higher concentration of total sugars causes an increase in osmotic pressure, affecting cell growth and viability, as well as impacting the fermentative yield. In order to evaluate a VHG fermentation, yeast Saccharomyces cerevisiae strain CAT-1 was subjected to fermentation of must (350 g.L-1 total sugars) in Bioflo® / CelliGen® 115 bioreactor with consecutive cell recycling and fed batch system, at 30°C and no aeration. The time for sugar consumption was 16 hours. Total reducing sugars were depleted in all cycles, with the exception of the first fermentative cycle. The ethanol concentration varied from 14.2 to 16.6° GL, with a maximum of 16.6° GL (131.1 g.L-1), in the eighth fermentative cycle. Cell viability remained above 90%. The results obtained in these conditions demonstrate that the increase of scale and the process conditions are determining factors for an industrial process.
Invited Oral Abstract
Rapid measurement of residual ethanol in corn stillage beer during distillation
Amanda Huber, Front Range Energy, LLC, Windsor, CO, USA and William Miller, YSI Life Sciences, Yellow Springs, OH, USA
39th Symposium on Biotechnology for Fuels and Chemicals
The loss of ethanol to waste (by-product) from distillation bottoms in bioethanol production plants can significantly affect the efficiency of fuel-grade ethanol production. Monitoring residual ethanol levels during distillation allows operators to adjust control parameters to minimize ethanol to waste and improve energy efficiency in plant operation. In this study ethanol was measured in both early and late stage distillation beer bottoms and side stripper bottoms, respectively, of a corn-based ethanol fermentation production process at a 40 MMGY bioethanol plant. The target operational value in both stages was 0.05% w/v ethanol. Samples of beer bottoms and side stripper bottoms were collected on thirty different days over a six week period. Each sample was measured with the YSI biochemistry analyzer and with an HPLC system. Comparability of the two analytical methods was evaluated with regards to precision and analysis time. A strong, positive correlation of the two methods was demonstrated. YSI analyzer results were obtained within 2 minutes after sample filtration, while HPLC results were available 35-40 minutes post-filtration. The YSI biochemistry analyzer provided rapid ethanol analysis allowing timely in-process data for improving plant efficiency and reducing operational cost.
Invited Oral Abstract
Facile production of water soluble flavonoids by using steviol glucosides prepared with thermostable lactase and characterization thereof
Dr. Thi Thanh Hanh Nguyen1, Ms. Nahyun Kim2, Ms. So-Hyung Kwak1, Mr. Jin-Beom Si1 and Prof. Doman Kim1, (1)Seoul National University, Pyeongchang, Korea, Republic of (South), (2)University of Southern California, Los Angeles, CA, USA
39th Symposium on Biotechnology for Fuels and Chemicals
Curcuminoids from rhizomes of Curcuma longa possess various biological activities. However, low aqueous solubility and consequent poor bioavailability of curcuminoids are major limitations to their use. In this study, curcuminoids extracted from turmeric powder using stevioside (Ste), rebaudioside A (RebA), or steviol glucosides (SG) were solubilized in water. The optimum extraction condition by Ste, RebA, or SG resulted in 11.3, 9.7, or 6.7 mg/mL water soluble curcuminoids. Curcuminoids solubilized in water showed 80% stability at pH from 6.0 to 10.0 after 1 week of storage at 25°C. The particle sizes of curcuminoids prepared with Ste, RebA, and SG were 110.8, 95.7, and 32.7 nm, respectively. The 2,2-diphenyl-1-picrylhydrazylradical scavenging (SC50) activities of curcuminoids prepared with Ste, RebA, and SG were 33.3, 47.5, and 64.7 µg/mL, respectively. Inhibition activities (IC50) of curcuminoids prepared with Ste, RebA, and SG against NS2B-NS3pro of dengue virus type IV were 3.7, 4.9, and 4.7 µg/mL, respectively. Steviol is a diterpene isolated from the plant Stevia rebaudiana that has a potential role as an antihyperglycemic agent by stimulating insulin secretion from pancreatic beta cells and also has significant potential to diminish the renal clearance of anionic drugs and their metabolites. In this study, the lacS gene, which encodes a thermostable β-glycosidase (SSbgly) enzyme from the extremely thermoacidophillic archaeon Sulfolobus solfataricus, hydrolyzed steviol glycosides to produce steviol with a yield of 99.2%. The optimum conditions for steviol production were 50 U/ml SSbgly and 90 mg/ml Ste at 75°C as determined by the response surface method.
Invited Oral Abstract
Integrated biogas and yeast single-cell protein production
Mr. Jonas A. Ohlsson, Dr. Matilda Olstorpe, Dr. Volkmar Passoth, Mats Sandgren and Dr. Su-lin L. Leong, Swedish University of Agricultural Sciences, Uppsala, Sweden
39th Symposium on Biotechnology for Fuels and Chemicals
Biogas production by anaerobic digestion (AD) is widely deployed in Europe, used both for renewable energy production and as a waste management strategy. Substrate utilization in the AD process is not complete, however, as demonstrated by the contents of C and N in the digestate. Furthermore, AD digestate is typically sold as fertilizer, a low-value commodity. Due to dwindling wild fish stocks and increasing aquaculture production, the demand for sustainable fish meal replacements is growing, and their value is expected to increase as a consequence. In this work, we evaluated the integrated production of biogas and yeast single-cell protein (SCP) for use as fish feed. On sterile-filtered substrate sourced from an operating biogas plant, yeast SCP (from Wickerhamomyces anomalus, Pichia kudriavzevii, and Blastobotrys adeninivorans) was produced in batch fermentations using CSTR bioreactors. Biomass concentration after 12–15 h fermentations were 7.0–14.8 g l-1. Amino acid (AA) analysis showed AA levels typical for yeast biomass, suitable for inclusion in fish feed formulations. A biomethanation potential assay showed no significant differences in methane production when combining whole and spent substrates, but there was a significant difference between spent substrates from two different yeast strains (20%, p=0.04). We conclude that the combination of biogas and yeast SCP production is feasible as there are no apparent negative effects on methane potential. Significant differences in methane potential yielded by the evaluated yeast strains are intriguing, and warrant further investigation.
Invited Oral Abstract
Removal of non-sugar compounds from energy cane bagasse enzymatic hydrolysate by flocculation and activated carbon for syrup production
Fang Deng, Louisiana State University, St. Gabriel, LA, USA and Giovanna Aita, Louisiana State University, St Gabriel, LA, USA
39th Symposium on Biotechnology for Fuels and Chemicals
Flocculants and activated carbon were used in the removal of non-sugar compounds (organic acids, furans and phenolics) from dilute ammonia pretreated energy cane bagasse enzymatic hydrolysate for syrup production. Energy cane bagasse (non-commercial variety Ho 02-114) was pretreated with ammonium hydroxide (28% v/v solution) and water at a ratio of 1:0.5:8 at 160 °C for 1h. Pretreated energy cane bagasse was then hydrolyzed by commercially available enzymes. Organic acids, furans and phenolic compounds are generated during pretreatment and can inhibit downstream processes such as fermentation and syrup production. Meanwhile, these non-sugar compounds can be recovered as building blocks for numerous value-added by-products. Flocculation and activated carbon adsorption methods were designed to maximize the removal and recovery of non-sugar compounds with minimum fermentable sugar (glucose and xylose) losses. Both flocculation and activated carbon adsorption methods significantly reduced the concentrations of non-sugar compounds without noticeable sugar losses. Activated carbon adsorption was more efficient in removing non-sugar compounds, while flocculation demonstrated a stronger performance in recyclability and non-sugar compounds recovery.
Invited Oral Abstract
Syrup production from lignocellulosic biomass
Dae-Yeol Cheong and Giovanna Aita, Louisiana State University, St. Gabriel, LA, USA
39th Symposium on Biotechnology for Fuels and Chemicals
Solutions produced from pretreatments and enzymatic hydrolysis of lignocellulosic biomass contain a mixture of sugars and a variety of non-sugar compounds which can affect downstream processes such as the quality and purity of lignocellulosic syrups. Some of these non-sugar compounds include organic acids, glycerol, lignin-derived phenolics, and furan derivatives. However, when separated from the sugar streams, the above mentioned non-sugar compounds can serve as building blocks for numerous value-added products (i.e., plasticizers, polymers, lubricating oils, animal feed, industrial solvents). A process was developed to separate and to recover these non-sugar compounds from dilute ammonia pretreated energy cane bagasse hydrolysates, primarily containing the sugars xylose and glucose, using activated carbon, polymeric adsorbent resins and ion exchange resins. The purified lignocellulosic sugar hydrolysate was concentrated to a high quality lignocellulosic sugar syrup of 60 Brix% with a final ash content of < 0.1% and a chemical composition comparable to that of refined corn syrup.
Invited Oral Abstract
Combined whole cell conversion processes for economic production of psicose and mannitol from fructose
Seong-Hee Jeong, Rachelle Canete, Dae-Yun Lee, Jae-Eun Kim and Prof. Seon-Won Kim, Gyeongsang National University, Jinju, Korea, Republic of (South)
39th Symposium on Biotechnology for Fuels and Chemicals
Rare sugars, defined by the International Society of Rare Sugars (ISRS), exist in nature as monosaccharides but are present in only limited quantities. Recently, rare sugars have received considerable attention because of its various clinical effects and specific biological functions. Likewise, D-psicose (D-ribo-2-hexulose or D-allulose), a C-3 epimer of D-fructose, also has many uses which include reducing intra-abdominal fat accumulation, protecting pancreas beta-islets and improving insulin sensitivity. Especially, D-psicose has only 0.3% calories compared to sucrose, while it has 70% relative sweetness. In 2012, D-psicose was approved as a food additive and designated as generally recognized as safe (GRAS) by Food and Drug Administration (FDA). Despite such abundant advantages, there is no economical way of mass production of D-psicose. Recently, biological production of D-psicose from D-fructose using D-psicose 3-epimerase (DPE) has been developed. However, the conversion yield is below 30%, which causes an increase of purification cost because of the similar solubility of psicose and fructose. Thus, we addressed the problem by converting the residual fructose of the psicose production reaction to D-mannitol which has a low solubility. This work was supported by a grant from the Next-Generation BioGreen 21 Program (SSAC, grant# : PJ01106201), RDA, Korea.
Invited Oral Abstract
Impact of nutrient in the fermentation of sweet sorghum syrup and sugar beet syrup to butanol
Thomas Klasson, USDA-ARS, New Orleans, LA, USA and Nasib Qureshi, USDA-ARS-NCAUR, Peoria, IL, USA
39th Symposium on Biotechnology for Fuels and Chemicals
Butanol production from sugars was once a commercial technology and it is once again being considered as a second generation liquid fuel after bioethanol. Sweet sorghum is a crop that when grown for sugar production generates a sugar syrup that is high in fructose, glucose, and sucrose, when compared to sugar beet syrup, which contains mainly sucrose. Sweet sorghum juice is also high in potential fermentation inhibitors (e.g., aconitic acid) but may have more available nutrients for bacterial growth than those in sugar beet syrup. In this study we will report on recent results with fermentation of synthetic sugar (glucose) as well as sugar crop syrups and the impact of added nutrients (yeast extract, salts, minerals, and vitamins).
Invited Oral Abstract
Effect of lignin and manganese peroxidases on biomethane production from lignin-rich residues
Dr. Thales Costa, Dr. Daniel Mulat and Prof. Svein Jarle Horn, Norwegian University of Life Sciences, s, Norway
39th Symposium on Biotechnology for Fuels and Chemicals
Biofuel generation from lignocellulosic materials has been extensively researched and optimized over the last decade. The focus has been to convert the carbohydrate fraction into fermentable sugars that can be converted to bioethanol. The emerging cellulosic bioethanol industry will inevitably generate large amounts of lignin-rich residues that typically will be burned for heat generation. However, these residues could be further valorized to improve the economy of biorefineries [1]. In order to add value to waste lignin, anaerobic digestion (AD) could be used for the conversion of lignin-rich materials to biomethane. However, lignin is very recalcitrant and its depolymerization under anaerobic conditions is very slow and needs to be increased for efficient conversion of lignin into biogas. It has been shown that addition of hydrogen peroxide, lignin- and manganese peroxidases (LiP and MnP) to anaerobic digesters can enhance the methane production from wastes rich in lignin [2, 3]. In this work, we evaluate the effect of LiP and MnP on biomethane production from birch hydrolysis-lignin (more than 80% lignin w/w) in batch bottles. The lignin rich fractions were prepared by enzymatic saccharification of steam exploded birch wood.
References:
1. Ragauskas, A.J., et al. Science, 2014. 344(6185).
2. Jayasinghe, P.A., et al. Bioresource Technology, 2011. 102(7): p. 4633-4637.
3. Hettiaratchi, J.P.A., et al. Bioresource Technology, 2014. 159: p. 433-436.
Invited Oral Abstract
One-pot process for hydrodeoxygenation of lignin to alkanes using ru-based bimetallic and bifunctional catalysts supported on Zeolite Y
Hongliang Wang1, Hao Ruan1, Maoqi Feng2, Yuling Qin1, Erik M. Kuhn3, Xiaowen Chen3, Melvin P. Tucker3 and Bin Yang1, (1)Washington State University, Richland, WA, USA, (2)Southwest Research Institute, San Antonio, TX, USA, (3)National Renewable Energy Laboratory, Golden, CO, USA
39th Symposium on Biotechnology for Fuels and Chemicals
The synthesis of highly efficient and low-cost catalysts for hydrodeoxygenation (HDO) of waste lignin into advanced biofuels is crucial for enhancing current biorefinery processes. Inexpensive transition metals, including Fe, Ni, Cu, and Zn, were separately co-loaded with Ru on HY zeolite to form bimetallic and bifunctional catalysts. These catalysts were subsequently tested for HDO conversion of softwood lignin and several lignin model compounds. Results indicated that the inexpensive earth abundant metals could modulate the hydrogenolysis activity of Ru and decrease the yield of low molecular weight gaseous products. Among these catalysts, Ru-Cu/HY showed the best HDO performance, giving the highest selectivity to hydrocarbon products. The catalytic performance of Ru-Cu/HY was improved probably due to the following three factors: (1) high total and strong acid sites, (2) good dispersion of metal species and limited segregation, (3) high adsorption capacity for polar fractions, including hydroxyl groups and ether bonds. Moreover, all the bifunctional catalysts were proven to be superior over the combination catalysts of Ru/Al2O3 and HY zeolite.
Invited Oral Abstract
Xylan extraction from pretreated sugarcane bagasse using alkaline and enzymatic approaches
Daniele Sporck, Andr Ferraz and Dr. Adriane M. F. Milagres, Escola de Engenharia de Lorena, Universidade de So Paulo, Lorena, Brazil
39th Symposium on Biotechnology for Fuels and Chemicals
Sugarcane bagasse was chemithermomechanically treated with an alkaline/sulfite solution followed by disk refining. The process caused considerable delignification, while the major part of xylan was retained in the solid residues. Residual xylan was extracted from pretreated bagasse by alkaline or enzymatic approaches. Alkaline methods employed high NaOH concentration (40% w/w) at moderate temperatures and reaction times, yielding 79% of xylan recover. The enzymatic approach provided a limited xylan yield of 22%. However, enzymatically recovered xylan showed more uniform chemical and structural characteristics. Prepared xylans were characterized by acid hydrolysis and gel permeation chromatography. All xylans presented xylose as major component (60-80%), followed by arabinosyl groups (7-12%), uronic acids (4-13%), hydroxycinnamic acids (0.3-1.2%) and residual lignin (3-10%). The xylose/arabinose ratio of xylans ranged from 7 to 10, while the xylose/uronic acids ratio showed a greater range (9-28). Varied degrees of substitution reflected in different xylan solubility in water. More substituted xylans presented higher solubility. Xylans isolated by the enzymatic approach exhibited two fractions with weight average molecular weights (Mw) of 3,700 g/mol and 800 g/mol. Higher Mw values were detected in the alkali-isolated xylans from the chlorite-delignified sample (24,450 g/mol) and from chemithermomecanically pretreated sample (24,780 g/mol). After xylan extraction, the residual solids presented increased cellulose digestibility by low celulases loadings.
Invited Oral Abstract
Production of platform chemical itaconic acid from pentose sugars
Badal C. Saha1, Gregory J. Kennedy2 and Nasib Qureshi1, (1)National Center for Agricultural Utilization Research, USDA-ARS, Peoria, IL, USA, (2)USDA-ARS-National Center for Agricultural Utilization Research, Peoria, IL, USA
39th Symposium on Biotechnology for Fuels and Chemicals
In recent years, itaconic acid (IA), an unsaturated five carbon dicarboxylic acid, has gained importance as a fully sustainable building block chemical (platform chemical) for wide range of applications in the manufacture of various synthetic resins, coatings and polymers. It is currently produced industrially from glucose by submerged fermentation using a filamentous fungus
Aspergillus terreus. Lignocellulosic biomass has the potential to serve as low cost feedstock for production of IA. However, only a limited research data is available on the use of other sugars for IA production rather than glucose and hydrolyzates of starchy materials. There is definitely a cost advantage associated with an
A. terreus strain that could use both pentose (xylose and arabinose) and hexose sugars (glucose, galactose and mannose) efficiently for IA production as typical lignocellulosic biomass hydrolyzates contain about 25-50% pentose sugars depending on the source. We have evaluated one hundred
A. terreus strains for production of IA from pentose sugars (xylose and arabinose). The results of IA production from glucose, xylose, arabinose and their mixture by some selected strains will be described. The problems of developing an efficient fermentation process for IA production from lignocellulosic biomass hydrolyzates and future directions of research will be highlighted.
Invited Oral Abstract
Lignin as a bio-adsorbent for the removal of heavy metals from water
Samira Fatemi and Abigail Engelberth, Purdue University, West Lafayette, IN, USA
39th Symposium on Biotechnology for Fuels and Chemicals
Metals such as lead (Pb) and cadmium (Cd) pose significant risks to human health and environmental well-being when present in water. Among various ailments are kidney dysfunction, developmental disabilities, and increased risk of cancer. Current methods to remove toxic metals include membrane filtration and activated carbon adsorption, but these methods are often capital-intensive. Here we investigate the utilization of lignin as a biorenewable approach to metal adsorption. Lignin was obtained from corn stover via acid hydrolysis. Metal solutions were prepared from Pb and Cd chloride salts. Solutions had initial concentrations ranging from 50 to 200 ppm, to represent typical contamination levels. Lignin was then added to the solution and left in an incubator shaker for 8 hours at 30°C. After lignin-metal association, the samples were filtered and centrifuged to separate solid and liquid fractions. The initial and final concentrations of the samples were analyzed via atomic absorption spectroscopy (AAS). Langmuir isotherms demonstrate the equilibrium adsorption of Pb and Cd onto lignin. Further work will investigate the effects of varying quantities of lignin, and interactions between lignin and non-metal substrates that may typically be found in water systems.
Invited Oral Abstract
Comparison of performances of commercial and in-house xylanase complexes for the hydrolysis of hemicellulosic biomass
Felipe A. S. Corradini1, Thais S. S. Milessi1, Thais O. Baldez1, Maria L. T. M. Polizeli2, Teresa C. Zangirolami1 and Raquel L. C. Giordano1, (1)Federal University of São Carlos, São Carlos, Brazil, (2)Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Universidade de São Paulo (USP), Ribeirão Preto, Brazil
39th Symposium on Biotechnology for Fuels and Chemicals
Lignocellulosic materials contain cellulose (glucose) and xylan (xylose) as the main sugar polymers in their compositions. Cellulose has been widely studied for several applications, mainly for fuel production. However, the economic viability of these processes would increase if the hemicellulosic fraction was also used for ethanol production and other valuable products, such as xylooligosaccharides (XOS). The hydrolysis of xylan requires the action of a complex of endo and exo-xylanases to obtain XOS. If ethanol is the aimed product, β-xylosidase is required to produce xylose from xylobiose. Accessory enzymes are also needed to remove the side chains from xylan. The product profile depends on the composition of the used xylanase complex. Therefore, substrate conversion and the xylanases complex selectivity are important variables to be considered. In this work, 10 different xylanases complexes (7 commercial and 3 in-house broth) were screened for xylan hydrolysis. The highest xylose productions were achieved using commercial Multifect CX XL A03139 and the in-house broth from Aspergillus niger, that presented after 24 hours of reaction, respectively, 79% and 76% of xylan conversion and similar product profiles (60% X1, 32% X2 and 8% X3). Considering XOS production, the commercial complexes NS22036 and NS50030 reached better results, with 64% and 58% of conversion, respectively. The products profile obtained were 16% X1, 81% X2 and 3% X3 with the NS22036 and 8% X1, 82% X2, 9% X3 and 1% X4 with NS50030. These results demonstrate the potential of the selected complexes for XOS and ethanol production.
Invited Oral Abstract
The potential of lipid production by growing oleaginous yeasts on steam pretreated biomass hydrolyzates
Seiji Nakagame1, Prof. Jack Saddler2, Hung Lee3 and Yuta Shimizu1, (1)Kanagawa Institute of Technology, Atsugi, Japan, (2)University of British Columbia, Vancouver, BC, Canada, (3)University of Guelph, Guelph, ON, Canada
39th Symposium on Biotechnology for Fuels and Chemicals
Three oleaginous yeasts (Lipomyces starkeyi, Trichosporon oleaginosus, Rhodotorula graminis) were selected after an initial screening of nine oleaginous yeasts (two L. starkeyi, T. oleaginosus, L. tetrasporus, L. spencer-martinsiae, R. glutinis, R. graminis, Rhodosporidium toruloides, Yarrowia lipolytica). The selection was based on growth and lipid production after growth on the hemicellulose-rich, water-soluble fraction of SO2 catalyzed steam pretreated biomass. After 6 days growth an OD660 of 9.9, 11.6, and 10.4 and a lipid production of 7.5×10-2, 3.8×10-1, and 1.8×10-1 g/L was reach respectively for L. starkeyi, T. oleaginosus, and R. graminis. Corn steep liquor (CSL) and urea were assessed as potential nitrogen sources instead of yeast extract and peptone, (cheaper nitrogen sources). Although urea decreased the growth rates of the three strains, CSL did not negatively affect the growth rates of L. starkeyi and R. graminis. The OD660 of L. starkeyi and R. graminis reached 6.8 and 9.3 after 6 days, respectively, while T. oleaginous did not grow well when CSL was used. The lipid produced by L. starkeyi and R. graminis was 0.59 g/L and 1.1 g/L, respectively. This was about eight and six times higher than the lipid obtained when using peptone and yeast extract as the nitrogen sources. Thus, R. graminis shows promise as a potential lipid producer when grown on the water soluble fraction of SO2 catalyzed steam pretreated biomass supplemented with CSL.
Invited Oral Abstract
Membrane bioreactors for hydrogen production by bacteria using waste products
Dr. Elvira Eivazova, Columbia State Community College, Columbia, TN, USA and Prof. Sergei Markov, Austin Peay State University, Clarksville, TN, USA
39th Symposium on Biotechnology for Fuels and Chemicals
Over the last several years, hollow-fiber bioreactor technology has been
actively pursued in wastewater treatment facilities, and therefore, industrial
scale bioreactors are available, and can be adopted for H2
production by bacteria using waste products. In
our experiments, carbon monoxide (waste product of thermochemical gasification of
biomass) or glycerol (waste product of biodiesel industry)
were used as substrates for bacterial growth in order to continuously make H2
by bacteria in hollow-fiber bioreactors. These bioreactors include thousands of hollow
fibers with immobilized bacterial cells.
The large surface-to-volume ratio of the hollow fibers allow us to grow
bacterial cells in high densities, to promote mass transport of carbon monoxide
or glycerol to cells, and to achieve higher rates of H2 production. We obtained higher H2 production rates, of up to 700 ml∙ g
cdw-1∙ h-1 using carbon monoxide or glycerol for
bacterial growth, compared to H2 rates from other microorganisms
reported in published papers. That was made
possible by increasing the mass transfer of carbon monoxide or glycerol into
bacterial cells using novel bioreactors.
Our
bioprocess for conversion for H2 production with
simultaneous waste utilization in hollow-fiber bioreactors is ready for
practical application. We were able to
inject H2 from our bioreactors directly into
fuel cells and generated enough electricity to power a motor.
Invited Oral Abstract
Self-regulated 1-butanol production in Escherichia coli based on the endogenous fermentative control
Mr. Rex Wen, National Tsing Hua University, Hsinchu, Taiwan, Taiwan and Prof. Claire Shen, National Tsing Hua University, Hsinchu, Taiwan
39th Symposium on Biotechnology for Fuels and Chemicals
In this work, we constructed a self-regulated 1-butanol production system in Escherichia coli by borrowing its endogenous fermentation regulatory elements (FRE) to automatically drive the 1-butanol biosynthetic genes in response to its natural fermentation need. Four different cassette of 5’ upstream transcription and translation regulatory regions controlling the expression of the major fermentative genes ldhA, frdABCD, adhE, and ackA were cloned individually to drive the 1-butanol pathway genes distributed among three plasmids, resulting in 64 combinations that were tested for 1-butanol production efficiency. Fermentation of 1-butanol was triggered by anaerobicity in all cases. In the growth-decoupled production screening, only combinations with formate dehydrogenase (Fdh) overexpressed under FREadhE demonstrated higher titer of 1-butanol anaerobically. In vitro assay revealed that 1-butanol productivity was directly correlated with Fdh activity under such condition. Switching cells to oxygen limiting condition prior to significant accumulation of biomass appeared to be crucial for the induction of enzyme synthesis and the efficiency of 1-butanol fermentation. With the selection pressure of anaerobic NADH balance, the engineered strain demonstrated stable production of 1-butanol anaerobically without the addition of inducer or antibiotics, reaching a titer of 10 g/L in 24 h and a yield of 0.25 g/g glucose under high density fermentation. Here, we successfully engineered a self-regulated 1-butanol fermentation system in E. coli based on the natural regulation of fermentation reactions. This work also demonstrated the effectiveness of selection pressure based on redox balance anaerobically.
Invited Oral Abstract
Selective production of vanillin derivatives from kraft lignin by peracetic acid depolymerization
Se-Yeong Park1, Jong-Chan Kim1, June-Ho Choi1, Seong-Min Cho1, Dr. Bonwook Koo2 and Prof. In-Gyu Choi1, (1)Seoul National University, Seoul, Korea, Republic of (South), (2)Korea Institute of Industrial Technology, Cheonan-si, Chungcheongnam-do, Korea, Republic of (South)
39th Symposium on Biotechnology for Fuels and Chemicals
Lignin is typically generated as a waste in paper industry and mostly used for power generation. Meanwhile, with the aim of producing value-added products from the lignin, depolymerization technology has been widely employed. Generally, studies on biorefinery lignins have been focused on selective production of low molecular lignin fragments. Among various methods for breaking the lignin structures, oxidative depolymerization using peracetic acid (PAA) has attracted for producing lignin fragments recently, since the peroxy acids are available for deconstruction of macromolecule lignin under low temperature condition. However, there has been little effort for applying PAA chemistry to converting lignin to valued products yet.
The purpose of this study is to generate low molecular weight lignin compounds by using PAA. Kraft lignin purchased from Sigma Aldrich was used as a starting material. PAA was prepared by reaction of acetic acid and hydrogen peroxide with different volume ratio. 1.5% sulfuric acid was added to above mixture as a catalyst. Depolymerization reaction of lignin was conducted under various conditions depending on PAA concentration, reaction time and temperature. After the reaction, solid and liquid fractions were separated by centrifugation. The liquid fraction was extracted with dichloromethane and ethyl acetate to obtain the lignin derived compounds, and then GC/MS analysis was carried out.
The main compounds detected by GC/MS were followed as; vanillin, vanillic acid, 3-vanilpropanol. As the reaction temperature increased, relative amount of vanillin derivatives increased in liquid fractions. The detailed quantitative analysis and the yield enhancement experiments will also carry out.
Invited Oral Abstract
Diesel fuel properties of essential oils extracted from four Korean domestic gymnosperms
Seong-Min Cho1, Jong-Hwa Kim1, Seon-Hong Kim1, Se-Yeong Park1, Soo-Kyeong Jang1, Dr. Bonwook Koo2 and Prof. In-Gyu Choi1, (1)Seoul National University, Seoul, Korea, Republic of (South), (2)Korea Institute of Industrial Technology, Cheonan-si, Chungcheongnam-do, Korea, Republic of (South)
39th Symposium on Biotechnology for Fuels and Chemicals
Petroleum diesel is a complex mixture of compounds which have mostly carbon numbers from 9 to 22. These components can be classified to the paraffinic, naphthenic or aromatic hydrocarbons. Therefore, the physical and chemical properties of diesel fuel performance thereof in a diesel engine are determined by the difference of relative proportions of these components. Terpenes, also known as constituents of essential oil (EO), are available from plant leaves by steam distillation. They are hydrocarbons or its derivatives from five-carbon isoprene units and, in particular, EO is composed of monoterpenes (C10), sesquiterpenes (C15) and a few kinds of diterpenes (C20).
In this study, the possibility for the replacement of petroleum diesel to EOs derived from woody plant was evaluated. Four Korean domestic gymnosperms (Cryptomeria japonica, Chamaecyparis obtusa, Pinus koriensis and Pinus densiflora) were selected to extract the EO and the calculated yields of four EOs were 2.32, 3.17, 0.53 and 1.01%, respectively. GC/MS results showed that C. japonica EO had 52.11% of sesquiterpenes and 19.90% of diterpene kaur-16-ene. However, 60.59% of monoterpenes was followed by 37.54% of sesquiterpenes in C. obtusa EO. This tendency was more obvious in the case of P. koriensis and P. densiflora which had 84.01% and 95.12% of monoterpenes, respectively. The physical and chemical properties including elemental composition, calorific value, density, viscosity and water content of four EOs will be assessed to compare with those of petroleum diesel.
Invited Oral Abstract
Synthesis and characterization of high purity lactide from cellulosic lactic acid
Hsien-Hsiu Chen and Wen-Hua Chen, Chemistry Division, Institute of Nuclear Energy Research, Taoyuan, Taiwan
39th Symposium on Biotechnology for Fuels and Chemicals
Lactide, a cyclic dimer of lactic acid, was an intermediate of Poly (Lactic acid) (PLA) polymerization. PLA is one of the promising biodegradable plastic with good mechanical properties, biocompatibility and environmentally friendly. It has a variety of application on packing industry, medical supplies and agriculture. Morever, ring-opening polymerization (ROP) of latides is the most general method that can obtain PLA with high molecular weight. Therefore, the chemical and optical purity of the lactide monomer is important to this reaction. Although the synthesis and the polymerization of PLA have been studied widely, reports of lactide synthesis are scarce. Therefore, in this study, a simply synthsis and purification apparatus to produce lactide from cellulosic lactic acid was established. Lactide was synthesized through following three steps: (i) remove free water from cellulosic lactic acid solution (ii) remove bound water and generate lactic acid oligomers (iii) depolymerization. The effects of vacuum degree, reaction temperature, and reaction time on purity and yield of lactide during the synthesis procedure were investigated. Then, the crude lactide was purified by crystallization method with ethyl acetate. The chemical properties and purity of the obtained lactide was characterized by FTIR, DSC and NMR. The results indicated that the proposed synthesis and purify processes can successfully producing high purity lactide from cellulosic lactic acid.
Invited Oral Abstract
Techno-economical and environmental analysis of a biorefinery for the production of ethanol and co-products from switchgrass in Uruguay
Valeria Larnaudie, Mario Daniel Ferrari and Claudia Lareo, Universidad de la Repblica, Montevideo, Uruguay
39th Symposium on Biotechnology for Fuels and Chemicals
The production of ethanol and co-products on a facility that processes 250 dry ton/day of switchgrass, a small scale compared with current studies, appropriate for feedstock supplies scenarios in locations like Uruguay, was studied. Environmental and economic metrics through life cycle assessment (LCA) and techno-economic analysis (TEA) were evaluated. The biofuel supply chain analyzed includes feedstock production and supply, liquid hot water pretreatment, hydrolysis, fermentation, purification, co-product generation, wastewater treatment and utilities. A process model in AspenPlus®, based on the NREL model for bioethanol from corn stover, was developed applying experimental and public data. SimaPro was used as tool for LCA. The resulting material and energy balances were used for the TEA and LCA, estimating minimum selling price, life cycle greenhouse gas (GHG) emissions and non-renewable energy consumption. The minimum selling price of ethanol ranged from 0.5 to 0.9 US dollars per liter depending on the use of xylose (ethanol or furfural production), process yields (60-90% hydrolysis and fermentation) and solids concentration in bioreactors (12.5-25%). This price range is consistent with values for advanced fuel alcohols. GHG emissions for the base case (xylose to ethanol, high conversion yields) were 29 gCO2eq/L, lower than 489 gCO2eq/L found for switchgrass (Spatari et al., 2005). This low value is due to low soil nutrient requirements in Uruguay, low chemicals requirement for pretreatment and credit from electricity. Energy return on investment (ethanol+electricity/fossil energy consumed) for the base case was 10 MJ/MJ, confirming the environmental potential of switchgrass for bioethanol production in Uruguay.
Invited Oral Abstract
Sugarcane agroindustry by-products for xylitol production: mixture of bagasse and straw as feedstock in a biorefinery
Mr. Andrs Felipe Hernndez1, Danielle Verde Nolasco1, Alejandra Camila Chaves-Villamil2 and Dr. Maria das Graas Almeida Felipe1, (1)Escola de Engenharia de Lorena, Universidade de So Paulo, Lorena, Brazil, (2)Universidad Distrital Francisco Jos Carlos Caldas, Bogot, Colombia
39th Symposium on Biotechnology for Fuels and Chemicals
The present work evaluated the use of hemicellulosic hydrolysate of the mixture of bagasse and straw for xylitol production, aiming to the integration of this bioprocess with the sugar-alcohol industry in the context of a biorefinery. The hemicellulosic hydrolysate was obtained from the diluted acid hydrolysis of the sugarcane biomass (straw and bagasse 1:1), concentrated six times at vacuum, and its composition was determined (g/L): xylose (74.59), glucose (7.55), arabinose (11.8), acetic acid (2.29), 5-hydroxymethilfurfural (0.43) and furfural (0.19). Two conditions were evaluated: detoxification with pH adjustment and activated charcoal, and without detoxification. Fermentations were conducted with the yeast Candida guilliermondii FTI 20037, cultivated in Erlenmeyer flasks (125 mL) with 50 ml of medium, initial pH 5.5, inoculum concentration 1.0 g/L, incubated for 72h at 200 rpm and 30°C. According to the results, 23.49 and 25.02 g/L of xylitol were produced corresponding to a consumption of 86% and 98% of xylose for detoxified and not detoxified hydrolysates, respectively. This demonstrates that detoxification of this hydrolysate in this bioprocess may be not necessary, contributing to reduction of time and costs. Based on these results, experiments are being conducted to establish important fermentative parameters in bench reactor, mainly oxygen availability, with the aim of contributing to develop the technology of xylitol production from a mixture of bagasse and straw. Results of this research can open alternatives to incorporate new bioproducts in sugar-alcohol industry as xylitol, which is a sugar-alcohol with applications in pharmaceutical, odontology and food industry.
Invited Oral Abstract
Comparison of butanol production from sugarcane-sweet sorghum juices by ABE and IBE fermentation-gas stripping integrated process
Eloísa Rochón, Florencia Cebreiros, Mario Daniel Ferrari and Claudia Lareo, Universidad de la República, Montevideo, Uruguay
39th Symposium on Biotechnology for Fuels and Chemicals
Butanol is an attractive alternative to ethanol biofuel due to its better performance in engines, can be produced by fermentation in the same industrial plant, using common raw materials and equipment. It can be produced by acetone–butanol–ethanol (ABE) or isopropanol-butanol-ethanol (IBE) fermentation. The production of isopropanol instead of acetone, which is corrosive, allows the produced mixture of solvents to be used as fuel. Both fermentations present butanol inhibition. The aim of this work was to compare ABE and IBE fermentations with product recovery by in situ gas stripping from a mixture of industrial juices of sugarcane (75%) and sweet sorghum (25%). Fermentation assays were done in a 2.5 L bioreactor using Clostridium acetobutylicum DSM 792 for ABE production and Clostridium beijerinckii DSM 6423 for IBE production. In batch fermentation without gas stripping C. acetobutylicum reached 10.5 g/L butanol (17.6 g/L total ABE solvents) at 100 h and C. beijerinckii reached 8.0 g/L of butanol (11.3 g/L total IBE solvents) at 44 h. Both microorganisms were not able to convert all the sugars because of butanol toxicity. When gas stripping was used, C. acetobutylicum achieved 100% of sugar conversion in 149 h. However, C. beijerinckii was not able to convert all the sugars (65% conversion) and the fermentation ceased at 48 h (8.4 g/L of butanol, 12.9 g/L total IBE solvents). Although the IBE mixture has better fuel properties and that C. beijerinckii presented a better butanol production kinetic, it showed problems facing the gas stripping process.
Invited Oral Abstract
Enhancement of butanol production and recovery in an integrated ABE fermentation-gas stripping process
Eloísa Rochón, Mario Daniel Ferrari and Claudia Lareo, Universidad de la República, Montevideo, Uruguay
39th Symposium on Biotechnology for Fuels and Chemicals
Butanol is an important bulk chemical with a large market which also presents good properties to be used as biofuel. It can be produced by acetone–butanol–ethanol (ABE) fermentation which presents severe product inhibition. The aim of this work was to evaluate butanol production in a fermentation coupled to an
in situ gas stripping that contributes both to mitigate the butanol inhibitory effect and to obtain a highly concentrated butanol condensate. The gas flowrate has to be selected in order to have a butanol concentration lower than its cell toxicity level (10 g/L) but higher enough (8 g/L) to obtain a concentrated butanol condensate with phase separation resulting in a more energy-efficient recovery process.
Kinetics models describing butanol formation by fermentation and butanol extraction by gas stripping were developed respectively. The effect of gas recycle rate on the performance of the integrated process was studied by integrating both models to define the most favorable operational condition.
Fermentation assays were done in 250 mL bottles using Clostridium acetobutylicum DSM 792. Gas stripping tests were conducted in a 2.5 L bioreactor. MATLAB® and Excel-MS® softwares were used to estimate parameters values. The models showed satisfactory agreement with the experimental data. A gas recycle flowrate in the range 0.4-0.6 vvm was obtained from models to maintain the butanol concentration in the broth at ~8 g/L. It was validated in a fed batch fermentation using sugarcane-sweet sorghum juices with in situ gas stripping where the butanol concentration was effectively maintained at 7-8 g/L.
Invited Oral Abstract
Biosurfactants produced by yeasts in sugarcane bagasse hemicellulosic hydrolyzate: new bio-based products for lignocellulosic biorefineries
Dr. Paulo R. F. Marcelino1, Dr. Silvio Silverio Silva1, Ms. Olvia Valrio Guiotti1, Mr. Guilherme Fernando Dias Peres1 and Dr. Jlio C. Santos2, (1)University of So Paulo, Lorena, Brazil, (2)University of So Paulo - School of Engineering of Lorena, Lorena, Brazil
39th Symposium on Biotechnology for Fuels and Chemicals
Biosurfactants (BS) are compounds of microbial origin with emulsifying and tensoactive properties, and have been target of agriculture studies, since these compounds can be applied as biopesticides in biological control. Furthermore, they can be used against mosquitoes vectors of tropical diseases such as dengue, zika and chikungunya. However, these compounds production is still a challenge, due to its high costs. In this context, this concern can be reduced by their production from industrial by-products, such as lignocellulosics from sugar and alcohol industry. In the present work, BS were produced by five xylose fermenting-yeasts in culture medium supplemented with sugarcane bagasse hemicellulosic hydrolyzate. Bioproducts were isolated and characterized as glycolipids, probably cellobiose-lipid or sophorolipid type, and high emulsifiers properties. Preliminary studies in our lab demonstrated the larvicide feasibility of these compounds against vectors of tropical diseases such as
Aedes aegypti. We highlight the potentiality of the sugarcane bagasse hydrolysate to be used as substrate for BS production in future refineries from lignocellulosics.
Acknowledgments: CAPES, CNPq and FAPESP-2015/06238-4.
Invited Oral Abstract
Towards sustainable production of lactic acid
Martin Altvater1, Sinisa Petrik2, Diethard Mattanovich2 and Michael Sauer2, (1)University of Natural Resources and Life Sciences (BOKU), Vienna, Austria, (2)Austrian Center of Industrial Biotechnology, Vienna, Austria
39th Symposium on Biotechnology for Fuels and Chemicals
Lactic acid is an organic acid that is used in food, cosmetic and pharmaceutical industries and that serves as building block for polylactic acid (PLA), a biodegradable and biocompatible polymer. The global lactic acid market is estimated to increase from 714.2 kilo tons in 2013 to 1,960.1 kilo tons by 2020 with a revenue of USD 4.3 billion [1]. Currently, optically pure lactic acid is mainly produced by sugar fermentation of lactic acid bacteria. However, this production process has some drawbacks. Particularly, lactic acid bacteria are sensitive to low pH. Therefore, neutralizing agents have to be added to the production medium making the separation and purification of lactic acid very costly. In the presented project, we use
S. cerevisiae as production organism for lactic acid. Budding yeast has many advantages over
Lactobacilli such as the simple nutritional requirements or the tolerance to low pH, making it an excellent cell factory for producing organic acids. Since
S. cerevisiae cells do not naturally produce lactic acid, metabolic engineering is required in the first place to develop a lactic acid producing strain. Initially, the genes coding for pyruvate decarboxylases involved in the reduction of pyruvate to ethanol were deleted. By overexpressing a heterologous lactate dehydrogenase, pyruvate can then be converted into lactic acid. Furthermore, a combinatorial approach of modifications of gene expression levels, cell sorting for high intracellular pH, and evolutionary engineering of the yeast production strain will improve the final lactic acid yield.
[1] Grand View Research (2015) 978-1-68038-126-9
Invited Oral Abstract
Optimization of extraction conditions of polyphenolic compounds from Marine microalge (Tetraselmis KCTC 12236BP) using statically-based optimization
JinWoo Kim, JaeMin Jo, JungHyun Jin and BoRa Min, Sunmoon University, Asan-si, Korea, Republic of (South)
39th Symposium on Biotechnology for Fuels and Chemicals
Microalgae constitute an important source for bioactive materials, especially polyphenolic compounds, with a wide range of applications that can be used for high value commercial additives. In our study, a statistically-based methodology is applied to optimize the extraction of total polyphenolic compounds (TPC) from marine microalgae (
Tetraselmis KCTC 12236BP). Key variables, including extraction temperature, ethanol concentration, extraction time and liquid to solid ratio (L/S ratio) were optimized in order to maximize TPC. The Response Surface Methodology (RSM) provided predicted values of extraction conditions and maximum polyphenols amounts similar to those obtained experimentally. All variables, except ethanol concentration, showed significant positive effects on the extraction of TPC and ethanol concentration of near 65% showed the maximum concentration of TPC. Statically-based optimization resulted that the maximum TPC of 11.2 mg gallic acid equivalent/g dry weigh), was obtained with extraction temperature of 117.3°C, ethanol concentration of 61.0%, extraction time of 75.0 min and L/S ratio of 50.
Tetraselmis is a good source of bioactive constituents that might be used for various applications, particularly functional food ingredients and nutraceuticals.
Invited Oral Abstract
Carbon fibers from purified and fractionated eucalyptus kraft lignin
Adilson Roberto Goncalves, UNESP, Rio Claro, Brazil and George Jackson de Moraes Rocha, EEL-USP, Lorena, Brazil
39th Symposium on Biotechnology for Fuels and Chemicals
Carbon fibers will be obtained from purified fractions of eucalyptus kraft lignin, by melt spinning, in polyacrylonitrile (PAN) blends containing 10-100% lignin. Fractionated materials will be characterized by spectroscopic, thermal and macromolecular properties to feed a mathematical model to predict and to analyze the best conditions for obtainment of extruded lignin monofilaments, with and without PAN-blends. Filaments will be cabonized and pyrolyzed to obtain carbon fibers which properties will be evaluated in comparison with commercial fibers.
Invited Oral Abstract
Life cycle greenhouse gas and land use impacts of bio-isoprene
Bahar Riazi1, Mukund Karanjikar2 and Sabrina Spatari1, (1)Drexel University, Philadelphia, PA, USA, (2)Technology Holding LLC, Salt Lake City, UT, USA
39th Symposium on Biotechnology for Fuels and Chemicals
Biomass holds great promise for synthesizing fuels, chemicals, and polymeric materials to address energy security and climate change. Poly-isoprene, the raw material used to produce rubber, can be produced from rubber trees (as poly cis 1,4 isoprene, latex) and synthesized from the monomer isoprene that is derived from both petrochemical feedstocks and fermentable sugars in biomass. We explore select life cycle environmental impacts of an alternative pathway for isoprene synthesis from corn stover co-produced with dimethyl cyclooctadiene, a high density jet fuel blend. Following pretreatment and enzymatic hydrolysis, the sugars from corn stover are fermented to methyl-butenol and then dehydrated to isoprene. Within the same dehydration reactor, using a dehydro-coupling catalyst, methyl-butenol is also converted to dimethyl cyclooctadiene. We use life cycle assessment (LCA) and system expansion rules to compare the GHG emissions of the isoprene co-product with isoprene produced from petroleum. Moreover, we compare the land use intensity of isoprene for rubber produced from US Midwest corn stover and rubber tree plantations in Southeast Asia. We include the expected land use change (LUC) from forest to rubber trees, which may have added climate change and biodiversity impacts relative to modifications to corn agroecosystems in the US Midwest, and find that 1.57 ha and 1 ha are needed to produce 1 metric ton of isoprene from rubber trees and corn stover, respectively. We posit that producing isoprene from corn stover would result in a lower land use and GHG intensity from direct LUC and also fewer changes to biodiversity.
Invited Oral Abstract
Molecular modeling of high-oleic soybean oil as a universal solvent for extraction of hydrogen sulfide from natural gas
Emma C. Brace and Abigail Engelberth, Purdue University, West Lafayette, IN, USA
39th Symposium on Biotechnology for Fuels and Chemicals
The rise of hydraulic fracturing (fracking) in the United States has increased interest in utilizing natural gas in transportation fuels, since combustion of natural gas releases less carbon dioxide into the atmosphere than conventional gasoline. However, natural gas extracted by fracking has high concentrations of hydrogen sulfide, a corrosive compound that can damage processing equipment, and is harmful for human health. The purpose of this research is to evaluate the feasibility of using high oleic soybean oil to remove hydrogen sulfide from natural gas. The high degree of saturation in high-oleic soybean oil offers several binding sites for sulfur, and a molecular modeling approach was used to determine the conditions under which high-oleic soybean oil can be used as an extraction solvent. The Conductor-like Screening Model for Real Solvents (COSMO-RS) was used to calculate the partition coefficient of hydrogen sulfide between methane and soybean oil phases. This statistical thermodynamics approach simulates the partitioning of the target molecule (hydrogen sulfide) between the liquid (soybean oil) and gas (methane) phases. The molecular modeling approach to predict the partition coefficient allowed for more rapid determination and reduction of experimental effort (time, resources) when choosing the right concentrations of solvents and co-solvents (such as oxidizing agents) which aided the ability of the high oleic soybean oil to extract the hydrogen sulfide. This work demonstrates a novel use of high-oleic soybean oil as a bio-based solvent for cleaning natural gas, and will potentially improve the viability and economics of the natural gas industry.
Invited Oral Abstract
Prospects of ethyl levulinate production from sugarcane bagasse in Brazil: a cheap biodiesel additive
Rubens Maciel Filho1, Jean Felipe Leal Silva1, Ms. Rebecca Grekin2 and Dr. Adriano Pinto Mariano1, (1)University of Campinas, Campinas, Brazil, (2)Massachusetts Institute of Technology, Cambridge, MA, USA
39th Symposium on Biotechnology for Fuels and Chemicals
Biodiesel, a solution for climate change widely spread across several countries for the fuel market of heavy vehicles, still facing obstacles because of the need of additives to make it more suitable for use in cold weather and common engines. Ethyl levulinate is a green chemical which can be obtained by esterification of levulinic acid with ethanol, being the former obtained via hydrolysis of biomass. Compatible for blending with diesel, ethyl levulinate reduces soot emission and improves cold flow properties, a fundamental performance parameter for biodiesel. Because of this great potential, this study assessed the routes to produce ethyl levulinate from sugarcane bagasse focusing in the Brazilian market. Several hydrolysis technologies were taken into account together with different esterification setups in process simulations using the software Aspen Plus. Data such as yield of products from biomass and energy consumption of each process were then used in a estimation of operational costs. The MSP (minimum selling price) of ethyl levulinate was determined in order to attain an EBITDA (earnings before interest, taxes, depreciation and amortization) margin of 30%. Results showed that the MSP of ethyl levulinate in Brazil could be in the range of 40%-92% of the local wholesale price of ultra-low-sulfur diesel on an energy basis. These results prove the potential of sugarcane bagasse for the production of ethyl levulinate, and indicate the need to develop suitable process design in order to bring the production of this green chemical to large scale in Brazil.
Invited Oral Abstract
Mild organic catalyst for the depolymerization of lignin
Gracielou Klinger1, Prof. James Jackson1 and Prof. Eric Hegg2, (1)Michigan State University, East Lansing, MI, USA, (2)Michigan State University, Great Lakes Bioenergy Research Center, East Lansing, MI, USA
39th Symposium on Biotechnology for Fuels and Chemicals
Lignin is an aromatic polymer that accounts for nearly half of the energy density in plant cell walls. Replacing petro-materials with products derived from lignocellulosic biomass requires the depolymerization of the lignin polymer into fragments for further valorization into bio-based chemicals and fuels. A simple, inexpensive, and green depolymerization technique is desired for this step in the replacement of petroleum products. Nature affords an excellent model for studying the types of cleavage required to break lignin into energy dense material. The glutathione dependent enzyme, Sphingobium sp strain SYK-6, uses a simple peptide to reductively cleave aryl ether bonds. This type of bond is significant as it is the key connection in the B-O-4 linkage, the most common covalent link in lignin. By mimicking this etherase chemistry with simple chemicals, we have found high levels of cleavage in lignin models and significant molecular weight reduction in real lignin. This work exemplifies the first biomimetic approach to reductive lignin deconstruction to small-molecule fragments by mimicking small molecule-mediated enzymatic ether cleavage.
Invited Oral Abstract
A life cycle GHG emissions intensity assessment of ionic liquid-based biofuel production
Dr. Binod Neupane1, Dr. N.V.S.N. Murthy Konda1, Seema Singh2, Blake A. Simmons3 and Corinne Scown4, (1)Joint BioEnergy Institue, Lawrence Berkeley National Laboratory, Emeryville, CA, USA, (2)Joint BioEnergy Institute / Sandia National Laboratories, Emeryville, CA, USA, (3)Joint BioEnergy Institute, Emeryville, CA, USA, (4)Joint BioEnergy Institute, Berkeley, CA, USA
39th Symposium on Biotechnology for Fuels and Chemicals
Efficient pretreatment of biomass is important for higher sugar yield and subsequent conversion into biofuels and renewable chemicals. Ionic liquids (ILs) are considered to be a potential alternative to conventional solvents because of their unique properties (e.g., negligible vapor pressure), and their ability to facilitate efficient hydrolysis of lignocellulose to fermentable sugars. However, their greenhouse gas (GHG) emissions-intensity and water footprint are not yet well understood. Therefore, a detailed life-cycle assessment (LCA) is needed to evaluate IL-based pretreatment processes as compared to other alternatives, and elucidate opportunities to improve the resulting environmental impacts associated with advanced biofuel production.
In this study, we developed a detailed life-cycle inventory to examine GHG emissions-intensity of IL production, specifically Cholinium Lysinate ([Ch][Lys]), and of its subsequent use in a corn stover-to-ethanol biorefinery as a pretreatment solvent. Our results suggest that, depending on the location of lysine production, GHG emissions for [Ch][Lys] production range from 6 to 8 kg CO2e per kg. Based on the integrated biorefinery design considered in this study, the IL- based biofuel is able to reduce GHG emissions per MJ of fuel output by around 80% compared to gasoline. These reductions meet the RFS2 definition of cellulosic biofuel category. Additionally, we found that the GHG emissions-intensity of current IL production methods necessitates near-100% IL recovery and development of more environmentally sustainable IL production. Given the uncertainty with the several process parameters, detailed sensitivity analysis is conducted to better understand potential impact on emission intensity.
Invited Oral Abstract
Enhancement of fatty alcohols production withmodified E. coli strains
Jianmin Xing, Institute of Process Engineering, Chinese Academy of Sciences, Beijing, China
39th Symposium on Biotechnology for Fuels and Chemicals
Microbial synthesis of fatty alcohols from renewable resources has attracted increasing attentions. We designed a novel strategy for fatty alcohol production based on fatty acid starvation. For the first time, the deletion of acyl-ACP thioesterases coupled with overexpression of exogenous fatty acyl-ACP reductase were employed to enhance fatty alcohol production. Fatty alcohol titer increased about 58% while the accumulation of fatty acids concentration decreased 73%. In order to explore the effects of acyl-ACP thioesterase deletion on the biosynthesis of fatty alcohol, we performed whole-genome transcriptional analysis. Deletions of ldhA, pta and ackA from KLCB coupled with over expression of FAR were performed and resulted in strain MGL2. The highest OD600 were increased from 5.2 to 7.8. The fatty alcohol tilter was increased from 756 mg/L to 2024 mg/L. Total fatty alcohol accumulation reached a maximum of 6.33 g/L after 50 h of fed-batch fermentations with MGL2. Two saturated fatty alcohols (C14:0 and C16:0) and two unsaturated ones (C16:1 and C18:1) are the major components. C14:0 (2.42 g/L) and C16:1 (1.81 g/L) are the two most abundant fatty alcohols, constituting 38.2% and 28.6% of total fatty alcohols, respectively. The percentage of unsaturated fatty alcohols was up to 36.5% of the total fatty alcohols. Notably, our best strain MGL2 produced 6.33 g fatty alcohols.
Invited Oral Abstract
An integrated approach to obtain cellulose nanocrystals and ethanol using eucalyptus cellulose pulp as feedstock
Thalita Bondancia1, Luiz Henrique Mattoso1, Jos Manoel Marconcini1 and Cristiane S. Farinas2, (1)Brazilian Agricultural Research Corporation, Embrapa Instrumentation, So Carlos, Brazil, (2)Chemical Engineering, Federal University of Sao Carlos, So Carlos, Brazil
39th Symposium on Biotechnology for Fuels and Chemicals
The biorefinery concept has been identified as one of the most promising routes to build the new industries of the future, as it allows the use of renewable biomass such as lignocellulosic residues for the production of biofuels including cellulosic ethanol, together with other chemicals and bioproducts. Among these bioproducts, high added-value materials such as nanocellulose can significantly contribute to the economic viability of the overall process. Here, the feasibility of integration of cellulosic ethanol production with the manufacture of cellulose nanofibers and cellulose nanocrystals was evaluated using eucalyptus cellulose pulp as feedstock and employing the biochemical route alone. For the enzymatic hydrolysis step, experimental central composite design (CCD) methodology was used as a tool to evaluate the effects of solids loading (SL) and enzymatic loading (EL) on glucose release and cellulose conversion. Validation of the statistical model was performed at SL of 20% and EL of 10 mg protein/g, which was defined by the desirability function as the optimum condition. The sugars released were used for the production of ethanol, resulting in 95.5% yield. For all the CCD conditions, the residual solids presented cellulose nanofiber (CNF) characteristics. Moreover, the use of a new strategy with temperature reduction enabled cellulose nanocrystals (CNC) to be obtained after 144 h. The CNC showed characteristics suitable for many applications, such as reinforcement in polymeric materials. The findings indicate the viability of obtaining ethanol and CNC from eucalyptus cellulose pulp using the biochemical route exclusively, potentially contributing to the future implementation of forest biorefineries.
Invited Oral Abstract
One-pot process for biotransformation of ricinoleic acid to (E)-11-(heptanoyloxy) undec-9-enoic acid in recombinant Escherichia coli
Yong-Han Cho1, Ji-Young Kim1, Jun-Seok Gwak1, Dr. Soo-Jung Kim2, Prof. Dae-Hyuk Kweon3 and Yong Cheol Park1, (1)Kookmin University, Seoul, Korea, Republic of (South), (2)Korea Research Institute of Bioscience & Biotechnology, Daejeon, Korea, Republic of (South), (3)Sungkyunkwan University, Suwon, Republic of Korea, Korea, Republic of (South)
39th Symposium on Biotechnology for Fuels and Chemicals
(E)-11-(Heptanoyloxy) undec-9-enoic acid (11-HOUA) is an intermediate of ω-hydroxyundec-9-enoic acid, a valuable medium-chain fatty acid with industrial potentials. In order to produce (E)-11-(heptanoyloxy) undec-9-enoic acid from ricinoleic acid, a renewable fatty acid, we developed one-pot processes with connection of cell biocatalyst preparation stage and biotransformation stage by recombinant E. coli overexpressing an alcohol dehydrogenase from Micrococcus luteus and a Baeyer-Villiger monooxygenase (BVMO) from Pseudomonas putida KT2440. In all processes, feeding of carbon sources facilitated the preparation of high-density cell biocatalyst and supplementation of cofactors required for enzymatic reaction. In one-pot process feeding glucose, 30 mM of 11-HOUA was produced with 1.6-fold higher productivity than the same process without glucose feeding. Through one-pot process with intermittently supplying glycerol and 32 g/L of cell biocatalyst, 34.5 mM of 11-HOUA and 77 % conversion yield were obtained with 16 % improved productivity, compared to that in the one-pot process with glucose-feeding strategy. These results suggested that one-pot process with supplying carbon sources would be a potent strategy to enhance other fatty acid biotransformation.
Invited Oral Abstract
Optimization and growth kinetic modelling of ethanol production from sweet sorghum
Elham Ebrahimiaqda, University of Arizona, Tucson, AZ, USA
39th Symposium on Biotechnology for Fuels and Chemicals
Development of our changing society and innovation of recent technologies are strongly reliant upon energy supplies. Limited fossil fuel resources and rapid growth rate of industries justifies the increasing demand for renewable energy. Biofuel, categorized as a renewable energy resource, has been subject of many studies in recent years. Bioethanol is a promising fuel source that can be utilized either in its pure form or mixed with gasoline. The primary advantage of bioethanol is its ability to reduce greenhouse gases. In addition, the non-food crops used in biofuel production can serve as sustainable sources of energy. Due to its compatibility with a wide variety of soil conditions as well as its rapid growth rate, sweet sorghum is regarded as a low-cost candidate for production of bioethanol. The focus of this study is on optimization of fermentation step in bioethanol production. A statistical model was applied to optimize ethanol yield and fermentation efficiency as a function of temperature, DO and pH. Experiments were designed to monitor the effect of each parameter independently and the interaction of a multitude of variables on ethanol yield efficiency. The results showed a maximum ethanol yield efficiency of 74%, achieved at 28 °C, pH=5.5 and a DO of 0%. Also, a graphical user interface was developed in MATLAB to study kinetics of fermentation systems. The developed application was tested for several bio-system processes. Results of the suggested simulation method were commensurate with the experimental data
Invited Oral Abstract
Overexpression of Bacillus subtilis arabinose symporter enhanced xylose transport efficiency in xylitol production by engineered Saccharomyces cerevisiae
Hyun-Su Lee, HyoJoo Kim, TaeYeob Kim, Hye-Jin Lee and Yong Cheol Park, Kookmin University, Seoul, Korea, Republic of (South)
39th Symposium on Biotechnology for Fuels and Chemicals
Xylitol, a natural sweetener, is able to be produced from xylose by engineered
Sacchromyces cerevisiae. Since efficient xylose transportation is a pre-requisite for xylitol production, in this study, we overexpressed the
araE gene coding for an arabinose:H
+ symporter (AraE) from
Bacillus subtilis in a
S. cerevisiae mutant lacking all hexose transporters along with expression of the
XYL1 gene encoding xylose reductase. The resulting strain (EMXR p405HXT_AraE) exhibited 4.2- and 5.5-folds higher xylose consumption rate and maximum xylitol productivity, respectively, compared to the control strain without overexpressing the
araE gene. Also, we overexpressed the
araE gene in
S. cerevisiae having hexose transporters. In batch fermentation, overexpression of the
araE gene increased xylose consumption rate and maximum xylitol productivity by 63 % and 22 %, respectively. These results indicated that AraE protein might be a potent xylose transporter and confer higher xylose consuming ability to
S. cerevisiae.
Key words: AraE protein, Saccharomyces cerevisiae, xylitol, xylose transporter, xylose reductase
Invited Oral Abstract
Novel viscoelastic amine polymer derived from biorefining lignin
Dylan Packard and Zhenglun "Glen" Li, Oregon State University, Corvallis, OR, USA
39th Symposium on Biotechnology for Fuels and Chemicals
Lignin, a renewable resource available from lignocellulosic biomass, has the potential to replace petrochemical feedstock in the production of aromatic polymers. Applications of lignin as a polymer material, however, is hindered by brittleness and thermal instability of the material. Unless blended with other polymers (e.g. PLA, PP, PVA, PET) or added as a filler in composites, technical lignins are not suited for material applications. The complexity in lignin structure and depolymerization chemistry also complicates the use of lignin as a source of polymer building blocks.
We present a new process that converts enzymatic lignin to a viscoelastic material that contains over 80% lignin by weight. In this process, lignin derived from a commercial cellulosic ethanol process is oxidatively functionalized and repolymerized using renewable chemicals (hydrogen peroxide, methanol, and DMSO) under mild conditions (ambient pressure, under 100 degrees C), yielding a homogeneous polymeric with Mw>10,000 Dalton. The synthesized material contains native beta-aryl ether linkages in lignin, while also exhibiting new viscoelastic properties distinct from the starting material. In an effort to enhance the viscoelasticity of the lignin-rich material, our studies on lignin functionalization chemistry revealed the association between polymerization parameters and mechanical properties. This new polymer material has broad potential applications as a component for coating, adhesives, and carbon fiber precursor.
Invited Oral Abstract
Enhanced production of 2,3-butanediol by engineered Saccharomyces cerevisiae
Jin-Woo Kim1, Hye-Won Oh1, Soe-Hee Park1, Jungyeon Kim2, Seung-Oh Seo3, Prof. Kyoung Heon Kim2, Prof. Yong-Su Jin3 and Prof. Jin-Ho Seo1, (1)Seoul National University, Seoul, Korea, Republic of (South), (2)Korea University, Seoul, Korea, Republic of (South), (3)University of Illinois at Urbana-Champaign, Urbana, IL, USA
39th Symposium on Biotechnology for Fuels and Chemicals
2,3-Butanediol (2,3-BD) is a promising compound for various applications. Pyruvate decarboxylase (Pdc)-deficient Saccharomyces cerevisiae is an attractive host strain for producing 2,3-BD because a large amount of pyruvate could be shunted to 2,3-BD production instead of ethanol synthesis. However, 2,3-BD productivity by engineered yeast was inferior to native bacterial producers. To overcome these problems, the Candida tropicalis PDC1 gene (CtPDC1) was used to minimize the production of ethanol but maximize cell growth and 2,3-BD productivity. As a result, productivity of the BD5_G1CtPDC1 strain expressing an optimal level of Pdc was 2.3 folds higher than that of the control strain in flask cultivation. Through a fed-batch fermentation, 121.8 g/L 2,3-BD was produced in 80 h. NADH oxidase from Lactococcus lactis (noxE) was additionally expressed in the engineered yeast with an optimal activity of Pdc. The fed-batch fermentation with the optimized 2-stage aeration control led to production of 154.3 g/L 2,3-BD in 78 h. The overall yield of 2,3-BD was 0.404 g 2,3-BD/g glucose. A massive metabolic shift in the BD5_G1CtPDC1_nox strain expressing NADH oxidase was observed, suggesting redox imbalance was a major bottleneck for efficient production of 2,3-BD. Maximum 2,3-BD titer in this study was close to the highest among the reported microbial production studies. The results demonstrate that resolving both C2-compound limitation and redox imbalance is critical to increase 2,3-BD production in the Pdc-deficient S. cerevisiae. Our strategy to express fine-tuned PDC and noxE could be applicable not only to 2,3-BD production, but also other chemical production systems.
Invited Oral Abstract
Production of bioethanol from immobilized Enterobacter aerogenes byanaerobic culture with optimized media
Mr. Ju Hun Lee, Mr. Dong Sup Kim, Mr. Soo Kweon Lee, Mr. Ji Hyun Yang and Prof. Seung Wook Kim, Korea University, Seoul, Korea, Republic of (South)
39th Symposium on Biotechnology for Fuels and Chemicals
In recent years, crude glycerol which is by-product of the biodiesel process was rapidly increased in quantity. Glycerol is a promising substrate for production of many valuable products such as bioethanol, 1, 3-propanediol and succinate. So, this study was intended to produce bioethanol using immobilized cell reactor (ICR) from glycerol and crude glycerol by
Enterobacter aerogenes ATCC 29007. To produce bioethanol stably and efficiently, media optimization was performed using statistical methods as well.
Ethanol production from pure and crude glycerol by
Enterobacter aerogenes ATCC29007 was carried out in anaerobic culture. Also, the effect of media optimization was investigated for improved ethanol production. Media components for bioethanol production from glycerol were optimized via analysis of variance (ANOVA) and response surface methodology (RSM). The optimal concentrations of each factors as determined by RSM were approximately 6.5 g/L for peptone, 3.8 g/L for ammonium sulfate, 2.1 g/L for citrate dehydrate and 12 g/L for glycerol, respectively. The continuous ethanol production was performed with pure glycerol and crude glycerol under the optimized conditions using ICR. On average, ethanol production and ethanol yield were respective 5.38 g/L and 0.96 mol-ethanol/ mol-glycerol with the pure glycerol, whereas ethanol production and ethanol yield were approximately 5.29 g/L and 0.91 mol-ethanol/ mol-glycerol with the crude glycerol during 192 h fermentation.
Invited Oral Abstract
Effect of GO/Co/chitosan mediator made by acetic acid solution on capacity of enzymatic fuel cell
Mr. Dong Sup Kim1, Mr. Ju Hun Lee1, Mr. Soo Kweon Lee1, Mr. Ji hyun Yang1, Prof. Jingyoung Lee2 and Prof. Seung Wook Kim1, (1)Korea University, Seoul, Korea, Republic of (South), (2)Sangmyung University, Chonan, Korea, Republic of (South)
39th Symposium on Biotechnology for Fuels and Chemicals
Biofuel cells(BFCs) have received significant attention in the last few decades due to its potential application as alternative fuel and advantages over conventional BFCs. The application of redox enzyme electrodes has been applied to enzymatic fuel cells (EFCs). EFCs have utilized redox enzymes to increase electron transfer rate from available substrates for high power generation. Chitosan has a role of electrodeposition from aqueous solution by using acetic acid as a solvent. The effect of acetic acid concentration on electron transfer of EFC system with chitosan mediator was identified using electrochemical techniques. This work shows that chitosan was soluble in acetic acid with the range from 1% to 7%. Cyclic voltammetry (CV) experiment involves the scan of potential voltages while measuring current, Fourier transform infrared spectroscopy (FT-IR) were supported structure and the surface morphology was observed. The EFCs using composites of solved chitosan composites obtained high power density in the study (1,198±8.2 μW/cm
2) at 0.342 V and the satisfied open circuit voltage obtain (0.567 V) in 5% acetic acid solution.
Invited Oral Abstract
Production of 3- hydroxypropionic acid in engineered Escherichia coli from glucose and xylose
In-Young Joung1, Do-Haeng Lee1, Byung-Yeon Kim1, Jong-Won Lee1, Won-Ki Min1, Yong Cheol Park2 and Prof. Jin-Ho Seo1, (1)Seoul National University, Seoul, Korea, Republic of (South), (2)Kookmin University, Seoul, Korea, Republic of (South)
39th Symposium on Biotechnology for Fuels and Chemicals
Escherichia coli expressing the Lactobacillus brevis dhaB1B2B3 and dhaR1R2 clusters and Pseudomonas aeruginosa aldhH was engineered to produce 3-HP from glucose and xylose via the glycerol biosynthetic pathway. Glycerol, a key precursor for 3-HP biosynthesis was produced by overexpression of the GPD1 and GPP2 genes from Saccharomyces cerevisiae. For relief of carbon catabolite repression, deletion of the chromosomal ptsG gene and overexpression of the endogenous xylR gene rendered engineered E. coli JHS01300/pCPaGGRm to utilize glucose and xylose simultaneously and to produce glycerol at 0.48 g/g yield and 0.35 g/L-h productivity. Finally, engineered E. coli HS01300/pELDRR+pCPaGGRm produced 29.4 g/L of 3-HP with 0.54 g/L-h productivity and 0.36 g/g yield in a sugar-limited fed-batch fermentation. It was concluded that dual modulation of sugar transport and glycerol biosynthesis is a promising strategy for efficient conversion of glucose and xylose to 3-HP.
Invited Oral Abstract
Evaluation of pre-processing options of sugar beet vinasse as feedstock for large scale bioproduction
Nurashikin Suhaili, Max Cárdenas-Fernández, John M. Ward and Gary J. Lye, University College London, London, United Kingdom
39th Symposium on Biotechnology for Fuels and Chemicals
Vinasse, a waste stream after ethanol distillation offers various potential benefits as feedstock for bioproduction as it is inexpensive and contains useful substrates such as glycerol. Our preliminary results have shown its feasibility for production of ω-Transaminase (ω-TAm), an industrial biocatalyst for chiral amines synthesis. We have also optimised the ω-TAm production using vinasse medium in 24-well controlled microbioreactor (MBR). In translating the optimal production from MBR to conventional reactor, an efficient and cost effective pre-processing strategy for the vinasse need is essential. Filtration, which has been applied in our previous work might not be ideal as it is expensive. In this work, several pre-processing options of vinasse were performed and the resulting media namely filtered dilute vinasse (FDV), autoclaved dilute vinasse (ADV) and pasteurised dilute vinasse (PDV) (no sterilisation) were then tested for ω-TAm production in MBR. The best medium was further used in scaling up the biocatalyst production from MBR to 7.5 L stirred tank reactor (STR). Our results showed that, the use of PDV was the most relevant due to its comparable cell growth and ω-TAm titre with that obtained using FDV. Furthermore, a good agreement between the two scales was achieved and the results also suggested the feasibility of PDV for large scale fermentation. Its simple pre-processing procedure with no additional sterilisation after ethanol distillation offers an economic advantage in terms of time and energy saving. In general, this work suggested the potential of PDV as an efficient and cost effective feedstock for large scale bioproduction.
Invited Oral Abstract
D-lactic acid production from hemicellulosic hydrolysate of sugar cane by Lactobacillus pentosus
Daiana Wischral1, Carolina Araujo Barcelos2 and Nei Pereira Jr.1, (1)Federal University of Rio de Janeiro, Rio de Janeiro, Brazil, (2)Joint BioEnergy Institute, Emeryville, CA, USA
39th Symposium on Biotechnology for Fuels and Chemicals
D-lactic acid, traditionally obtained by fermentation process, has numerous applications in the chemical industry, including the production of polymers, more specifically the biodegradable polylactic acid (PLA). However, higher production costs have hindering the large-scale application of PLA because of lactic acid price. The development of low cost substrate fermentation can improve the economic viability of bioprocesses, such as the bioproduction of lactic acid. Thus, the present work reports the investigation of hemicellulosic hydrolysate from sugar cane bagasse used as sole carbon source for lactic acid production by Lactobacillus pentosus ATCC 8041. Initially, the acid pretreatment of sugar cane bagasse was performed, with a solid:liquid ratio of 1:2.8 (1 g of bagasse: 2.8 mL of sulfuric acid solution 1 % v/v) and temperature of 121°C during 27 minutes. Then the concentration of hemicellulosic hydrolysate and yeast extract in MRS medium were optimized using the response surface methodology through STATISTICA 6.0 software. The optimal conditions (40 % of hemicellulosic hydrolysate and 5 g/L of yeast extract) were validated in shaker flasks and bioreactor. The fermentations were performed in anaerobic conditions at 37°C and 120 rpm. Finally, hemicellulosic hydrolysate consumption was increased in bioreactor, reaching 42.4 g/L of lactic acid (25.0 g/L of D-lactic acid) at 41 h of fermentation, which corresponds to a volumetric productivity of 1.02 g/L.h-1, yield of 0.70 g/g and 100 % of xylose consumption. The findings of this work demonstrated that hemicellulosic hydrolysate of sugar cane is a promising carbon source for lactic acid production.
Invited Oral Abstract
Extraction and application of Eucalyptus urograndis hemicellulose fraction as substrate for the single cell oil production by Rhodosporidium toruloides CCT7815
Prof. Everson Alves Miranda1, Helberth Junnior Santos Lopes1 and Nemailla Bonturi2, (1)School of Chemical Engineering, State University of Campinas, Campinas, Brazil, (2)Institute of Technology, Tartu, Estonia
39th Symposium on Biotechnology for Fuels and Chemicals
Single cell oil (SCO) is a promising substitute for the vegetable oils as raw material for biodiesel production
since it does not compete with food supplies. Also, oleaginous microorganisms can grow in a variety of substrates including agricultural residues. However, studies are still required to reduce the production costs, for example, by using low cost carbon sources for the fermentation step such as hemicellulosic hydrolysates. The objective of this work was to study the extraction of the hemicellulose of Eucalyptus urograndis (extensively cultivated in Brazil for papermaking) and to use its undetoxified hydrolyzate as carbon source for SCO production by the yeast Rhodosporidium toruloides CCT7815. The hemicellulose extraction studies were conducted in a laboratory shaking oven under different temperatures (145-160 ºC), solid to liquid ratios (S:L, 1:4-1:8), and reaction times (60-240 min). The extracts obtained were hydrolyzed and used for lipid production according to a 22 factorial design experiments to evaluate the effects of C/N and C/P ratios. Virtually 100% of the hemicellulose was extracted at 160 ‹C, S:L ratio of 1:8 at 195 min but the xylose content of this extract was relatively low, 12.2 g/L. SCO production by the yeast using the undetoxified hydrolyzate was only affected by the C/N ratio. The best results ̶ lipid concentration of 1.45 g/L and lipid yield of 26.4% ̶ were obtained at C/N molar ratio of 100. We expect to increase lipid production by using hydrolysate concentration or by supplementing it with raw glycerol and salts.
Invited Oral Abstract
High concentration of bioethanol production process with microalgae
Jingliang Xu, Guangzhou Institute of Energy Conversion, Chinese Academy of Sciences, Guangzhou, China
39th Symposium on Biotechnology for Fuels and Chemicals
As efficient photosynthetic microorganism, microalgae can convert solar energy and CO
2 into biomass, such as starch and cellulose, which is the desired feedstock for bioethanol production. In present work, a high carbohydrate content microalgae strain was identified as
Scenedesmus raciborskii WZKMT.
Simulated flue gas was used as carbon source to study the effect of flue gas on microalgae cell components. Microalgae growth and starch accumulation was inhibited with simulated flue gas, the maximum biomass yield and starch content were 2.14 g/L and 36.23%，while with 7% CO2, those were 3.25 g/L and 53.16%, respectively. Liquid hot water was applied in the microalgae pretreatment process to increase the enzyme hydrolysis efficiency. The optimization pretreatment conditions analyzed by response surface methodology were as follows: ratio of liquid to solid 13:1, temperature 147℃, heating time 41 min. Under the conditions, the glucose concentration was 14.223 g/L, the glucose recovery was 89.32%, which was up to 5-fold higher than the one of the sample without LHW pretreatment (17.91%).
Process parameters were studied for high concentration sugar accumulation, eg. enzyme composition, enzyme loading, optimum pH value, optimum and optimum solid loading. When microalgae hydrolysis with fed batch mode at 33% of solid loading, the highest concentration of glucose, xylose and cellobiose could attain to 58.33g/L，12.57g/L and 1.87g/L, respectively. The highest ethanol concentration and yield with simultaneous saccharification and fermentation (SSF) could attain to 79.38g/Land 89.2%, respectively, which is the highest report for bioethanol production with microalgae.
Invited Oral Abstract
Catalytic processing for the hybrid conversion of lignin into biodiesel
Marcus Foston, Washington University, Saint Louis, MO, USA
39th Symposium on Biotechnology for Fuels and Chemicals
Future commercial viability of second generation biofuel and biochemical production depends on the efficient utilization of not only carbohydrates but also non-carbohydrate fractions. To enhance the valorization of biomass, we have developed a hybrid (i.e., thermo-catalytic and biological) conversion platform to generate value-added products from lignin. This hybrid conversion platform consists of two sequential processes: a thermo-catalytic process that depolymerizes lignin into an aqueous-soluble lignin breakdown product and a biological process that converts and funnels compounds in the lignin breakdown product into a single bio-product, a triacylglycerol (TAG, a biodiesel precursor). I will present the results of a study that elucidates the effect that catalyst and reaction conditions have on the catalytic hydrogenolysis of lignin and the effect that different lignin breakdown product compositions have on its biological conversion with a bacterium,
Rhodococcus opacus.
Invited Oral Abstract
Novel co-polymers synthesized from biorefinery by-products
Dr. Kalavathy Rajan1, Dr. Stephen Chmely1, Dr. Nicole Labbé1 and Dr. Danielle Julie Carrier2, (1)The University of Tennessee, Knoxville, TN, USA, (2)The University of Tennessee Knoxville, Knoxville, TN, USA
39th Symposium on Biotechnology for Fuels and Chemicals
Novel co-polymers were synthesized from biorefinery by-products such as lignin and hemicellulose. The lignin and hemicellulose macromolecules were functionalized by modifying the aliphatic alcohol groups with a 2-hydroxyethylmethacrylate-imidazole (HEMA-Im) precursor, resulting in their substitution with HEMA (2-hydroxyethylmethacrylate). Thus functionalized, the lignin and hemicellulose macromolecules were used to synthesize copolymers with acrylate monomers, such as HEMA and methyl methacrylate. The generated co-polymers displayed a wide range of properties, from soft hydrogels to hard plastics. For example, the hydrogel synthesized by co-polymerizing 20% functionalized lignin and 80% HEMA resulted in increases in water retention capacity of 17%, storage modulus (G’) of 256% and thermal decomposition temperature of 50 °C, as compared to that of the control polymer, which was made with 100% HEMA. This method of functionalization is versatile and suitable for the modification of both lignin and hemicellulose extracted from different bioenergy crops, such as hybrid poplar, pine and switchgrass. The properties of these co-polymers can be adjusted by altering the ratio of monomer to modified lignin/hemicellulose and the degree of substitution of aliphatic alcohol groups by HEMA. Thus, this research provides suitable means for valorizing biorefinery streams.
Invited Oral Abstract
Enhancement of cellulolytic efficiency of fungal biofilms for consolidated bioprocessing of plant biomass
Dr. Charilaos Xiros and Michael Studer, Bern University of Applied Sciences, Zollikofen, Switzerland
39th Symposium on Biotechnology for Fuels and Chemicals
In the multispecies biofilm membrane (MBM) reactor for direct biomass conversion, the simultaneous achievement and separation of aerobic and anaerobic conditions in the reactor allows fungal cellulolytic enzyme production and anaerobic fermentation of the hydrolysis-derived sugars at the same time. During the direct bioconversion of plant biomass in the MBM system, enzymatic hydrolysis was identified as a major bottleneck of the process. We therefore investigated and evaluated the MBM bioconversion process, regarding its cellulolytic production and the system was optimized with regard to the hydrolytic efficiency of cellulosic substrates. By developing the multi-fungi biofilm by A. phoenicis and T. reesei, we demonstrated that the MBM process can be adjusted to host more than one fungus in order to boost the cellulolytic efficiency of the system. The biofilms were characterized regarding fungal growth, enzyme production, and enzymes localization. The multi-species biofilm produced three times more b-Glucosidase activity than the single T. reesei one. A biomass hydrolysis study on steam pretreated beech wood solids using enzymatic systems produced by single-fungus or multi-fungi biofilms showed the importance of certain enzyme activities for maximizing the hydrolysis yields. The final yield achieved, using the multi-fungal enzymatic system, at low temperatures (30°C), was 88% of the theoretical.
Invited Oral Abstract
Enzymatic hydrolysis optimization of modified sweet potato (Ipomoea batatas Lam. (L)) using statistical approach
Kallyana Moraes Carvalho Dominices1, Emilia Savioli Lopes2, Robson dos Santos Barbosa3, Wesley Rosa Santana3, Tarso da Costa Alvim3, Prof. Laura Plazas Tovar4 and Prof. Rubens Maciel Filho5, (1)Federal Institute of Tocantis, Paraiso, Brazil, (2)Chemical Processes, School of Chemical Engineering, State University of Campinas, Campinas, Brazil, (3)Federal University of Tocantins, Palmas, Brazil, Brazil, (4)University of Santa Maria, Santa Maria, Brazil, Brazil, (5)State University of Campinas - UNICAMP, Campinas, Brazil
39th Symposium on Biotechnology for Fuels and Chemicals
Sweet potato is rich in starch is hydrolyzed by acid catalysis or enzymatic process. The enzymatic process involved two-steps, liquefaction and saccharification. In the first step, the enzyme α-amylase cleaves 1,4-glycosidic bonds to yield shorter chains of soluble dextrins. In the second step, the enzyme glucoamylase attacks 1,4 terminal bonds in the degraded molecules to release one glucose unit at a time. The values of optimum time, temperature, α-amylase dose, glucoamylase dose and solid loading other operating conditions differ between the two steps. In this study, two-step enzymatic hydrolysis of modified sweet potato was optimized. The modified sweet potato used in this study was variety ‘Duda’. The effects of enzymatic hydrolysis were investigated using the response surface methodology. For the liquefaction step was applied with a total of 28 experimental runs, and an ANOVA test showed the quadratic model obtained to be significant (p<0.05). The statistical model predicted the maximum glucose concentration to be 25.93g/L at a temperature of 79°C, α-amylase dose 0.5%(v/v) and time 89 min. For the saccharification step was applied and a total of 18 experimental runs, and again a quadratic model obtained and verified by ANOVA test showed to be significant (p<0.05). The statistical model for the second step predicted the maximum glucose concentration to be 127.77g/L, established at the temperature of 50°C, glucoamylase dose 0.5%(v/v) and time 40 min. As the enzymatic steps play an important role in the whole process cost the optimization of the operational condition may be crucial for economic viability.
Invited Oral Abstract
Comparison between sugarcane bagasse and eucalyptus hydrolysates for fermentation: Promissing biomass for ethanol 2G production
Emilia Savioli Lopes1, Daniel de Castro Assumpo1, Prof. Laura Plazas Tovar2, Dr. Adriano Pinto Mariano1 and Rubens Maciel Filho3, (1)Chemical Processes, School of Chemical Engineering, State University of Campinas, Campinas, Brazil, (2)University of Santa Maria, Santa Maria, Brazil, Brazil, (3)School of Chemical Engineering, State University of Campinas, Campinas, Brazil
39th Symposium on Biotechnology for Fuels and Chemicals
The energy consumption matrix worldwide predominately relies on fossil fuels and its demand is increasing over the years. The oil, gas and coal dependency represents geopolitical instability and also is harmful to the environment in a global scale. International entities and local governments are seeking solutions to supply the increasing energy demand in a sustainable way. Sugarcane bagasse and eucalyptus have been studied and show increasing interest as promising sources for the production of 2G ethanol. In order to obtain the best hydrolysis for subsequent fermentation, a comparison was made between these two biomasses. In the current study, sugarcane bagasse and eucalyptus were submitted individually to a chemical pretreatment with dilute sulfuric acid (0.5, 1.0 and 1.5 % w/w H2SO4), at 121 °C, residence time of 60 min and with 10, 12,5 and 15 % w/w solids loads, using a full factorial design 22. After that, the samples were submitted to enzymatic hydrolyzes with 8,0 % w/w solids loadings (dry basis) at 50 °C, 200 rpm and considering 15 FPU and 33,0 CBU per gram of dry biomass, for 72 hours. The pretreatment mass yield, glucose, acetic acid and inhibitors (furfural and 5-hydroxymetilfurfural) percentage after enzymatic hydrolysis were evaluated, besides an analysis on the total reducing sugars in function of the elapsed enzymatic hydrolysis time. Sugarcane bagasse presents better hydrolysates results for ethanol 2G production, but eucalyptus presents potential to be used as co-feedstock to extend the operational of 1G plants when integrated 1G2G concept is considered.
Invited Oral Abstract
Deep surveys of biological modules: K-biclustering gene expression and phenotype data
Marcin Joachimiak1, Cathy Tuglus2, Mark van der Laan2 and Adam Arkin3, (1)Lawrence Berkeley National Laboratory, Berkeley, CA, USA, (2)University of California, Berkeley, CA, USA, (3)University of California, Berkeley, Berkeley, CA, USA
39th Symposium on Biotechnology for Fuels and Chemicals
New algorithms that statistically associate features of genomic data to predict phenotypic outcome are becoming increasingly important in synthetic biology. One of the first critical steps is the detection of patterns in data which group together features across measured molecules that are similar over a subset of conditions. Many such algorithms are based on a biclustering procedure wherein sets of molecular features such as genetic sequence, molecular abundance or molecular interactions co-occur in certain patterns across a set of conditions or outcomes. While current approaches have proven useful they also suffer from limitations that reduce their predictive power: inability to identify overlapping patterns, discovery of biclusters that are too small or too large, focus on single data types, and low coverage of the available signal in datasets. Moreover, it has proven difficult to compare the strengths and weakness of different algorithms due to a lack of gold-standard data sets and criteria for evaluation.
We have developed a new algorithm, Massive Associative K-biclustering (MAK, http://genomics.lbl.gov/MAK), to address some key limitations in other biclustering approaches. To scale for the growth of biological data MAK leverages high performance computing by breaking up the bicluster discovery problem into many smaller, independent searches. In a formal evaluation against new simulated data sets with more realistic properties MAK significantly improved recovery of biclusters. We applied MAK to a large gene expression compendium from Saccharomyces cerevisiae and demonstrate that MAK biclusters are more coherent and functionally enriched and reveal many data associations not captured by other methods.
Invited Oral Abstract
Strain development and screening using a parallel bioreactor system operating at elevated pressure
Jasbir Singh, HEL Ltd, Borehamwood, Herts, United Kingdom
39th Symposium on Biotechnology for Fuels and Chemicals
The commercial feasibility of many bio-processes can depend on how fast gas transfer takes place. This is especially true if gas solubility is poor, for example when working with gases such as hydrogen and methane in the context of gas fermentation for the production of fuels and chemicals from waste gas. The engineering solution to poor gas transfer is limited to kLa increase through changes in sparger and stirring arrangement. This offers very limited scope for improvement and therefore many potentially interesting processes can be rendered uneconomic. A much more effective alternative is to operate the bio-reactor at elevated pressure as this can in principle increase gas transfer rate several-fold without any changes to sparging or agitation.
This presentation will discuss data from a mini-bioreactor platform used to screen and then develop bacterial strains at elevated pressure, allowing process economics to be directly improved through higher production rate and better yield. Substantial increases is solubility and mass flux will be demonstrated while at the same time achieving fine control of dissolved oxygen profile to suit different bacterial strains. Though operating at under 100ml in each bio reactor, successful scale up to multi-litre volume will also be demonstrated.
Invited Oral Abstract
Deregulation of feedback control of ATP phosphoribosyltransferase (HisG) of Escherichia coli for the production of L-Histidine
Mr. Jun Ho Lee and Prof. Pyung Cheon Lee, Ajou University, Suwon, Korea, Republic of (South)
39th Symposium on Biotechnology for Fuels and Chemicals
L-Histidine is an essential amino acid present in the human body and other mammals, and its inclusion through diet is necessary for normal metabolic functions. L-histidine biosynthesis in
Escherichia coli is tightly regulated by the end-product L-histidine feedback inhibition of ATP phosphoribosyltransferase (HisG), which catalyzes the first step of the pathway. In this study,
E.coli was metabolically engineered to produce L-histidine through deregulation of the feedback control of hisG. Native promoter of the
hisG gene in the chromosome was replaced with a strong trc promoter and the C-terminal regulatory domain of hisG was deleted to deregulate feedback control of L-histidine. The engineered
E. coli was able to produced 3.1g/L of L-Histidine in flask scale fermentation.
Invited Oral Abstract
Producing consistent, high-quality feedstock from mixed biomass sources
Dr. Timothy Rials1, Dr. Nicole Labb1, Dr. Nicolas Andre2 and Warren Edmunds1, (1)The University of Tennessee, Knoxville, TN, USA, (2)The University of Tennessee Institute of Agriculture, Knoxville, TN, USA
39th Symposium on Biotechnology for Fuels and Chemicals
The transition from raw biomass to feedstock is a critical step in providing a reliable and sustainable supply of high-quality raw material that consistently meets the specifications for a given chemical conversion process. Given the inherent variability of biomass, improving the efficiency and effectiveness of this operation represents a challenging, but valuable, target for cost reduction while meeting other performance expectations. One approach to achieving feedstock requirements is through blending of biomass sources to produce engineered bioenergy feedstocks; however, successful implementation requires timely access to new information on biomass properties such that adjustments can be made to accommodate, and possibly exploit, property variation. Research has developed the near infrared (NIR) spectrum as a fingerprint that can be calibrated to provide information on chemical and physical properties that impact downstream processes. Recent work has advanced NIR as a sensor for on-line monitoring of key biomass characteristics like moisture and ash content, and explored multivariate and neural network models for quality and robustness. Additionally, preliminary experience incorporating information from this new process sensor into a statistical process control framework will illustrate the potential to optimize process efficiencies that ultimately reduce cost through the blending of different biomass types. This biomass blend concept can allow biorefineries to purchase and convert a much larger amount of biomass within their procurement radius (e.g. the southeast U.S., which has both pine and herbaceous biomass available). This capability will ultimately reduce operational risks from supply chain disruptions, while allowing larger-scale plants to be constructed and operated.
Invited Oral Abstract
Production of biocontrol agents using soybean processing wastes
Ulalo Chirwa, Enshi Liu and Jian Shi, University of Kentucky, Lexington, KY, USA
39th Symposium on Biotechnology for Fuels and Chemicals
The three elements of the food-energy-water nexus are interconnected and all tie to sustainable agriculture. Biocontrol agents are a group of naturally occurring organisms which can interrupt the lifespan and suppress the propagation of disease organisms. The use of biocontrol agents offers an environmentally friendly and sustainable solution to the synthetic agrochemicals. Currently, synthetic medium is commonly used in submerged fermentation for the production of biocontrol agents. However, the cost associated with the medium and the difficulties in obtaining viable spores for long shelf life and stability warrant the development of new bioprocesses. In this study, we explored the potential of using soybean processing wastes such as soybean hulls, stalks and soybean meal as substrates for spore production of two model biocontrol microorganisms, Bacillus pumilus and Streptomyces griseus by solid state cultivation (SSC). Besides lower production cost, SSC offers more suitable cultivation conditions by mimicking the natural habitant environment of the soil microorganisms. Using a Box-Behnken central composite design, cultivation temperature, C/N ratio and initial moisture content were optimized for microbial growth, sporulation and spore viability. A flow cytometry method was established to differentiate and enumerate viable spores using propidium iodide dye. The effectiveness of the biocontrol agent will be assessed using plate based anti-microbial assay and direct application on soybean plant. Results from this study demonstrate the potential of using soybean processing wastes as a low cost, effective and environmentally safe mean for the production of biocontrol agents.
Invited Oral Abstract
Contributions of developmental stage and anatomical fraction to the recalcitrance of switchgrass (Panicum virgatum) to deconstruction by alkaline pretreatment and enzymatic hydrolysis
Jacob Crowe1, Nicholas Feringa1, Sivakumar Pattathil2, Brian Merritt3, Cliff Foster4, Dayna Dines4, Rebecca Garlock Ong5 and David B. Hodge6, (1)Michigan State University, East Lansing, MI, USA, (2)Mascoma LLC, Lebanon, NH, USA, (3)University of Georgia, Athens, GA, USA, (4)Michigan State University, Lansing, MI, USA, (5)Michigan Technological University, Houghton, MI, USA, (6)Department of Chemical Engineering and Materials Science and DOE Great Lakes Bioenergy Research Center, Michigan State University, East Lansing, MI, USA
39th Symposium on Biotechnology for Fuels and Chemicals
Heterogeneity in herbaceous biomass feedstocks can contribute significant challenges to processing and conversion. As a consequence, utilizing a physical fractionation prior to deconstruction may offer the potential to generate more homogeneous fractions whereby pretreatment conditions can be tailored to the biomass fraction or potentially biomass fractions can be targeted to different applications. For this work, we investigate the relationship between cell wall composition and morphology and its response to deconstruction by alkaline pretreatment and enzymatic hydrolysis in maturing internodes of three switchgrass tissues within a single cultivar of switchgrass (Panicum virgatum). The stem, sheath, and leaf fractions were shown to have different key features governing tissue-specific recalcitrance following alkaline pretreatment, with lignin content as the primary contributor to recalcitrance in stem internodes only, while sheath and leaf internode recalcitrance were shown to be impacted significantly by hemicellulose content and substitution, as well as structural pectin content. Interestingly, lignin content was not correlated to hydrolysis yields in untreated samples and, as ferulate content was inversely correlated to lignin content in all fractions, demonstrates that ferulate cross-linking in low lignin tissues may be a key feature of cell wall recalcitrance overcome by alkaline pretreatments. Furthermore, we were able to identify significant differences in non-structural, extractable sugar content between fractions with extractable glucose, sucrose, and starch comprising as much as 20% of the total carbohydrates in some fractions, with important implications for a conversion process.