Invited Oral Abstract Presentation
DNA from cell lysis drives increased viscosity of E. coli fermentation broth
Robert S. Kuczenski1, Jordan Baker1 and Dorothea Reilly2, (1)Genentech, South San Francisco, CA, USA, (2)Genentech Inc., South San Francisco, CA, USA
Recent Advances in Fermentation Technology (RAFT tm)
Increased broth viscosity can challenge bioprocessing by reducing mixing and the oxygen transfer rate as well as complicating downstream recovery operations. Here we characterize the rheology of E. coli fermentation broth. The data supports increased broth viscosity being driven by two mechanisms. First, the increased cell density over the growth phase is coincident with an initial rise in broth viscosity. Second, an increase in broth viscosity and shear thinning can be attributed to an increase in supernatant DNA. The rheological data is fit to a composite model that includes contributions from both cell density and supernatant DNA. This model captures the contributions of cell density and supernatant DNA to viscosity but is challenged to predict the end-of-run rheology when the cell density drops.
Invited Oral Abstract Presentation
Dynamics of phenotypical adaptation of yeast to main inhibitors found in lignocellulocidhydrolysate: a single cell analysis approach
Mr. Pau Cabaneros1, Mr. Chuan Tao Peng2, Anna Eliasson Lantz3, Prof. Nils Arneborg2 and Prof. Krist V. Gernaey3, (1)Technical University of Denmark, Lyngby, Denmark, (2)University of Copenhagen, Copenhagen, Denmark, (3)Technical University of Denmark, Kgs. Lyngby, Denmark
Recent Advances in Fermentation Technology (RAFT tm)
The inhibitors generated during the pretreatment of lignocellulocic biomass reduce the performance
of yeast to produce 2G bioethanol. In this context, several studies have reported that phenotypical
adaptation of the cells during their propagation step results in signicant improvements of the ethanol
production.
In this study, single cell analysis was used to elucidate the mechanisms of inhibition and the dynamics
of phenotypical adaptation of Saccharomyces cerevisiae to inhibitors commonly found in biomass
hydrolysate (acetic acid, furfural and vanillin). Their effects were studied using a circumscribed central
composite design. The phenotype of the yeast was assessed every 2 hours using multi-parametric ow
cytometry based on three criteria: cell membrane integrity (relates to viability), membrane potential and
cytosolic ROS concentration (both indicating metabolic stress). The results revealed that treatments
with acetic acid (7.5g/L) and furfural (3.5g/L) entail a quick drop of the membrane potential, and an
increase of the ROS concentration, indicating high metabolic stress. After 10 hours, the ratio of dead
cells increased notoriously. Whilst cells treated with acetic acid never recovered from the shock, the cells
treated with furfural recovered normal metabolic stress levels after 15 hours, indicating detoxication
of furfural. The cells treated with vanillin (1.5g/L) also showed increased levels of metabolic stress, but
were able to recover after 5 hours of fermentation.
This study shows the dynamics of phenotypical adaptation of yeast to inhibitors at a single cell level,
and will be used to optimize the propagation of yeast to improve the production of 2G bioethanol.
Invited Oral Abstract Presentation
High cell density fed-batch fermentation in micro-scale bioreactors â feed rate optimisation and strain selection for the production of HMO in Escherichia coli
Dr. Peter Becker1, Dr. Cristina Bernal Martinez2, Carolina Santos Fernandes2 and Nicolas Fierfort1, (1)Glycom, Hørsholm DK, Denmark, (2)Applikon Biotechnology, Delft, Netherlands
Recent Advances in Fermentation Technology (RAFT tm)
Human Milk Oligosaccharides (HMO) are a diverse group of natural oligosaccharides which are present in high concentrations in human milk. They are the 3rd most abundant component of human milk and contribute to many of the health benefits associated with breastfeeding. With the introduction of fermentation-based processes for the production of HMOs, it has become feasible to develop improved infant formula and to look at other applications outside the infant-space. At the core of the fermentation process is an E. coli strain which has undergone extensive metabolic engineering to be able to synthesize HMOs. The fermentation strategy is based on a classical fed-batch culture with glucose as the only carbon and energy source and lactose as an acceptor for the production of activated sugars such as GDP-fucose. This work focusses on the application of micro-scale bioreactors in the development of a fermentation process for the production of HMOs. Applikonâs micro-Matrix platform, operating 24 microbioreactors at a working volume of 2-5 mL with individual control of pH, dO2 and temperature, was evaluated against the use of benchtop fermenters and a conventional deep-well assay. A feeding strategy based on an automated pH-triggered feed start was applied as part of a high-cell density process using a defined medium. Four different feed regimes/flow rates were tested to establish conditions that give an overall productivity similar to that of a benchtop fermenter. The results demonstrate that the micro-Matrix is useful for the screening of different flow rates and evaluation of new production strain candidates.
Invited Oral Abstract Presentation
Mass transfer improvements in industrial fermentors
Zachary Baumer, Keith Alsaker, Seth Woodard, Dan Hester and Tiffany Rau, Evonik Corporation, Lafayette, IN, USA
Recent Advances in Fermentation Technology (RAFT tm)
The oxygen transfer properties of fermentation processes are
critical for optimizing productivity. Enhanced mass transfer can facilitate
other process improvements through media reformulation, higher cell densities,
and improved regulation of oxygen-sensitive pathways. We analyzed kLa
at production scale (60,000 L) for a viscous fermentation process utilizing multiple
flights of Rushton and pitched blade impellers and examined the effects of
altering (a) sparger design, (b) blade count and diameter, and (c) agitator
replacement with low-shear A340 hydrofoils. The modification results varied
from kLa reductions to significant, 20-35% improvements. The
enhancement in oxygen transfer can be used as a path forward toward implementing
other productivity improvement strategies at multiple scales.
Invited Oral Abstract Presentation
Production of agarobiose by acid hydrolysis of agarose from red macroalgae
Dong Hyun Kim, Dr. Sang Hyun Lee and Prof. Kyoung Heon Kim, Korea University, Seoul, Korea, Republic of (South)
Recent Advances in Fermentation Technology (RAFT tm)
Macroalgae contain large amounts of carbohydrates. Therefore, they are considered as renewable resources of carbohydrates. Among various polysaccharides in macroalgae, agarose is the major component of red macroalgae. Oligosaccharides of agarose are being revealed to exhibit various physiological functions. To predominantly produce agarobiose among various agarooligosaccharides from agarose, we have developed an acid hydrolysis process using phosphoric acid in this study. To effectively produce agarobiose, prehydrolysis conditions were optimized. The optimal conditions were found to be the solids loading of 30.7% (w/v), the operating temperature of 110°C, the operating time length of 10 min, and the phosphoric acid concentration of 2% (w/v). At these optimal conditions, the agarobiose yield of 70.0% based on the input mass of agarose was obtained.
Invited Oral Abstract Presentation
Enhanced fatty acids production by Saccharomyces cerevisiae engineered by CRISPR-Cas9
Mr. Dohyoung Kim and Prof. Kyoung Heon Kim, Korea University, Seoul, Korea, Republic of (South)
Recent Advances in Fermentation Technology (RAFT tm)
Saccharomyces cerevisiae is a Generally Recognized As a Safe (GRAS) strain. Therefore, S. cerevisiae is widely used as a fermenting starter for the food and beverage industries. To fully meet the industrial needs, it is sometimes necessary to genetically modify S. cecerevisiae. For genetically modified organisms (GMO), always legal and consumersâ concerns are followed. To possibly avoid the GMO issue, in this study, we exploited the CRISPR/Cas9 system that is the marker- and scar-free genome editing tool. Using the CRISPR/Cas9 system, we engineered S. cerevisiae to increase fatty acid production. More specifically, isocitrate dehydrogenase genes (idh1 and idh2) in the TCA cycle was disrupted to accumulate citrate and then ATP-citrate lyase genes (ylacl1 and ylacl2) were overexpressed to convert citrate into Acetyl-CoA that is the essential precursor of fatty acids biosynthesis. These engineering resulted in 37.1% increase of total fatty acids compared with that of WT strain. This marker- and scar-free engineered S. cerevisiae could be used as an alternative starter with high fatty acid production capability.
Invited Oral Abstract Presentation
Production of high titer 3,6-anhydro-L-galatose from agar by chemical liquefaction and enzymatic saccharification
Kyungmun Cho, Dong Hyun Kim, Najung PARK and Prof. Kyoung Heon Kim, Korea University, Seoul, Korea, Republic of (South)
Recent Advances in Fermentation Technology (RAFT tm)
Red macroalage are regarded as renewable resources due to their high contents of carbohydrates. The main carbohydrate in red algae is agarose that is composed of 3,6-anhydro-L-galactose (AHG) and D-galactose. In particular, AHG is found to possess diverse biological properties such as anticancer and skin-whitening, moisturizing activities. To produce AHG from agar, the process combining chemical liquefaction and enzymatic sacchrification was developed by our group. In this study, to produce AHG from agar at high titers, we have optimized the three steps of AHG production process: chemical liquefaction of agar into agarooligosaccharides using TrisâHCl buffer; enzymatic hydrolysis of agarooligosacchrides into agarotriose and neoagarobiose using crude cell free extract of exo-β-agarase (Aga50D); and enzymatic hydrolysis of agarotriose and neoagarobiose into AHG and D-galactose using crude cell free-extract of both agarolytic β-galactosidase (ABG) and α-neoagarobiose hydrolase (NABH). The optimal chemical liquefaction conditions were 150°C and 40 min using 20 mM TrisâHCl buffer at a 20% (w/w) solids loading. The optimal loadings of the three crude enzymes, Aga50D, ABG, and NABH, were 60, 58, and 60 U/g agar, respectively. The final titer and yield of AHG achieved using the optimal conditions were 39.0 g/L and 28.2% (w/w), respectively. These results can be used as basic information for the industrial production of AHG from agar.
Invited Oral Abstract Presentation
Lacto-vinegar production by Zymomonas mobilis
Recent Advances in Fermentation Technology (RAFT tm)
Invited Oral Abstract Presentation
A three-zone predictive scale up method for system biology
Duen G. Mou, Moubio Knowledge Co., Taipei, Taiwan
Recent Advances in Fermentation Technology (RAFT tm)
Shake flask culture is known for limited surface aeration and atmospheric gas exchange. Ultimate enhancement of oxygen transfer in shaker culture relies on atmospheric oxygen enrichment or culture volume reduction or both. They made high throughput studies of micro-reactor cultures a success as soon as micro titer plates and their automation became available. However, problem remains in the low shear and low mixing surface aeration which cannot be resolved simply by a matching kLa â mass transfer coefficient kL, multiplied by specific gas-liquid interfacial area, a. On the other hand, impeller dispersed line gas sparging in production scale stirred tank reactor (STR) is not easily scale down to or reproduced in mini and micro scale vessels like shake flask or micro titer plate without compromising their advantage in simplicity and economy. As success of combinatorial genomic DNA reconstruction, editing and subsequent high volume phenotype screen and validation spread in the new system and synthetic biology, predictive scale up conditions, like realistic impeller stirring, gas-liquid mixing and gas phase oxygen and carbon dioxide partial pressures, in mini or micro scale stirred reaction vessels have driven the high throughput fermentation device market ever since. This presentation will go over pros and cons of mini jar (in 10-100s mL) and micro well (in 100s µL) reactors in gas-liquid mixing design, and how and why a 3-zone mixing model and reactor vessel innovation (US Patent 8,162,295), made without engineering jargon, can benefit high throughput fermentation in phenotype validation and predictive scale up.
Invited Oral Abstract Presentation
Yeast extracts & peptones: optimal nutritional sources for culturing lactic acid bacteria
Lisha Jacob, Sensient Technologies, Hoffman Estates, IL, USA and Doug Antibus, SENSIENT TECHNOLOGIES, Hoffman Estates, IL, USA
Recent Advances in Fermentation Technology (RAFT tm)
The increasing demand for the growth of dairy & probiotic cultures has led to a strong interest in understanding microbial physiology and establishing optimal fermentation media for these lactic acid bacteria . This study highlights how yeast extracts serve as highly effective complex nitrogen sources and have strong influence on both cell biomass quantity and quality. They are abundant in peptides, amino acids and various growth factors that meet the high nutritional demands of fastidious bacteria. The supplementation of yeast extracts with peptones resulted in further increase in biomass production. M17 & de Man Rogosa Sharpe (MRS) culture media were each modified using animal-free and allergen-free yeast extracts and peptones to enhance & optimize the growth of various lactic acid bacteria.
Invited Oral Abstract Presentation
Use of bio-capacitance probes as an advanced process analytical tool at the manufacturing scale
Dr. John Carvell, Aber Instruments Ltd, Aberystwyth, United Kingdom
Recent Advances in Fermentation Technology (RAFT tm)
Real-time bioprocess monitoring is fundamental for maximizing yield, improving efficiency and process reproducibility, minimizing costs, and optimizing product quality. . The FDAâs Process Analytical Technology initiative (PAT) encourages bioprocess workflows to operate under systems that provide timely, in-process results.The detection of biomass is one of the most requested parameters in industrial cell cultivation. The knowledge of the biomass progress during a fermentation process gives deeper process knowledge and control and helps to define harvest or infection points.
The radio frequency impedance (RFI) or bio-capacitance method for online in-situ detection of viable biomass has become well established in biopharmaceutical and the technology has an added appeal that it can now be applied to rocking motion and stirred disposable bioreactors.
Many of the modern cell culture processes are often operating at very high cell densities and in these cases the product titer and quality can be very sensitive to the amount of nutrient feed added. This paper will focus on how the industry is now using bio-capacitance probes for controlling nutrient addition. It will provide examples of how integral bio-capacitance is used in manufacturing processes up to 15,000L and will also discuss the merits of using uncorrected capacitance values rather than attempting to convert this into a figure that matched the offline VCD.
This paper will also focus on how several groups have used RFI scanning, from 100KHz to 20 MHz, to comparatively profile multiple bioreactor runs and elucidate fine details concerning cell viability and mechanism of cell death
Invited Oral Abstract Presentation
Dissolved carbon dioxide (dco2) as a critical process parameter in upstream bioprocessing
Joseph Lattari, Mettler Toledo, Billerica, MA, USA
Recent Advances in Fermentation Technology (RAFT tm)
This presentation would be a general review on the importance of measuring CO2 in bioprocessing.
DCO2 is a critical parameter from a quality and performance perspective, impacting productivity, growth rates, and product quality.
DCO2 concentration directly impacts key metabolic pathways and intracellular and extracellular pH
Inline DCO2 measurement can be critical for process control and optimization, scale-up and scale-down models, and process understanding.
Invited Oral Abstract Presentation
Development of a new fast fermentation screening system for cell and process optimization
Dr. Jens Rupprecht1, Dr. Barney Zoro2, Alison Rees-Manley2 and Dr. Thorsten Adams1, (1)Sartorius Stedim Biotech, Goettingen, Germany, (2)Sartorius Stedim Biotech, Royston, United Kingdom
Recent Advances in Fermentation Technology (RAFT tm)
The development of biopharmaceuticals or biotechnological products derived from microbial fermentation is a financially risky endeavors and time consuming process, requiring technical upstream solutions which help to speed up this route and increase likelihood of success.
We have identified in particular the early steps of strain and process development offering best prospects to speed up the entire process significantly by using a reliable screening system.
Based on the well-proven ambr® principle we designed with ambr 15 fermentation an instrument perfectly matching the demand of early steps in the development of microbial fermentation products. The multi-fermentation unit allows, with a working volume just large enough to resemble larger scale processes, the screening for suitable clones, strains or growth conditions.
In two case studies with industrial partners using E. coli and P. pastoris, consistent and efficient control of fermentations across a variety culture conditions (e.g. feed, temperature, duration, pH) could be demonstrated.
A comparison of multiple replicates proved the reproducibility of ambr 15 fermentation and reliability of the system for screening processes.
In particular two procedures are of high relevance in microbial fermentations. High cell densities could be achieved for both strains in concentrations typical for bench-top scale systems. Furthermore the system allows with fed-batch cultivation one of the most commonly used procedures already at milli-scale, opening up the opportunity to scale-up such a process as shown for a 1L bench-top and 30L stainless steel system.
Invited Oral Abstract Presentation
Engineer Aspergillus niger by CRISPR/Cas9 for industrial bioreactor
Ms. Laure Leynaud-Kieffer, Lawrence Berkeley National Laboratory, Emeryville, CA, USA
Recent Advances in Fermentation Technology (RAFT tm)
The conversion of the biomass into advanced biofuels faces many challenges, one of which is finding the right organism for the job. The filamentous fungus
Aspergillus niger has been chosen as a biocatalyst for cellulose, hemicellulose, and lignin degradation because it can secrete numerous hydrolytic enzymes, such as lignin modifying enzymes (LMEs) and its genome sequence is available.
However, we currently lack efficient tools for editing and augmenting the A. niger genome. While genome editing techniques such as CRISPR/Cas9 editing function in A. niger, we are limited by the difficulty of making multiple mutations, restricted selection of markers, and inefficient, expensive and time-consuming methodologies for genome engineering.
Here I present progress towards developing a method for efficiently making multiple genomic mutations via Cas9/gRNAs without the use of selective markers. This technique utilizes several approaches; 1) pyrG positive and negative selection for transient plasmid maintenance, 2) a self-targeting plasmid for selection of Cas9 activity. Once complete, this strategy should remove the need for screening of colonies to identify mutants. Our objective is to first establish this method for genome engineering, and build a library of Aspergillus niger strains. Then we will design two type of bioreactor, a submerged fermentation and solid state fermentation. The objective is to define the best strains and conditions for the productivity of LMEs in bioreactors at a pilot scale for industries.
Invited Oral Abstract Presentation
Boosting bioprocess performance by using the right yeast extract
Ms. Katelijne M. Bekers and Mrs. Mariët J. van der Werf, Ohly GmbH, Hamburg, Germany
Recent Advances in Fermentation Technology (RAFT tm)
Yeast extract is widely used in industry to boost cell growth and enhance productivity, titer and product yields in fermentation processes. Opposed to chemically defined media, it has the benefits of providing ready to use complete building blocks for cells, in addition to a range of cofactors, vitamins and growth factors. However, being such a complex mixture of nutrients, generally little is known on which components in the yeast extract are responsible for its beneficial results. Moreover, it is generally not known to biotechnologists that yeast extract can be highly different in composition. As a result, process optimization by adding yeast extract is generally only reviewed on a basic level.To review the impact yeast extract can have on fermentation processes, a study was conducted in which the effect of three highly different yeast extract (combination)s on the performance of industrial relevant organisms was evaluated. In particular, (combinations of) Ohly® KAT (yeast extract high in free amino acids), Ohly® CTT-R (yeast extract containing relatively high nucleotide concentrations) and Ohly® PTU (yeast extract high in peptides) were tested in this study. They were reviewed with a 10-point experimental design to find an optimal blend.
The results show that there is clearly a potential for process optimization, if the right combination of yeast extracts is supplied. The experimental approach of testing ten blends as applied in this research shows to be a simple pragmatic approach to improve industrial production processes without the need for indepth knowledge about the exact composition of the yeast extract.
Invited Oral Abstract Presentation
Evaluation of industrial-grade commercial cellulases for the enzymatic hydrolysis of hydrothermally-pretreated empty fruit bunches
Jae Kyun Kim, Dr. Jungwoo Yang and Prof. Kyoung Heon Kim, Korea University, Seoul, Korea, Republic of (South)
Recent Advances in Fermentation Technology (RAFT tm)
Performance of cellulase in the enzymatic hydrolysis of lignocellulosic biomass largely depends on the characteristics of biomass feedstocks. Pretreatment methods are known to significantly affect the characteristics of lignocellulose. To obtain high sugar yields from pretreated lignocellulose, wise selection of effective cellulase for specifically pretreated biomass, which is based on the characteristics of cellulase and pretreated biomass, is important. In this study, we have evaluated industrial-grade commercial cellulases from major enzyme companies, such as Accellerase 1000, Accellerase 1500, and Spezyme CP from DuPont and Cellic CTec2 from Novozymes, for their hydrolysis efficiency with hydrothermally-pretreated empty fruit bunches (EFBs). Among the four cellulases tested, Cellic CTec2, which indicated the highest cellobiohydrolase, xylanase, and β-glucosidase activities, showed the highest glucose yield. The highest glucose yields of 91.3% and 84.7% (both based on the theoretical maximum glucose) were attained with 30 FPU of Cellic CTec2/g glucan with and without Cellic HTec2, respectively. These results would be valuable information for the selection of enzymes for the industrial-level enzymatic hydrolysis of hydrothermally-pretreated EFBs.
Invited Oral Abstract Presentation
Use of osmotic and temperature shock to improve viability of spray-dried microbial fungicides
Srujana Koganti and Alex Schlesinger, AgBiome, Durham, NC, USA
Recent Advances in Fermentation Technology (RAFT tm)
Despite high-quality agronomic practices,
chemistries, and germplasm, growers continue to experience crop yield losses of
approximately 30% due to pests and diseases. At AgBiome,
we discover solutions to combat these problems based on a large and expanding
core collection of fully-sequenced microbes from the plant-soil microbiome. It
is well-known that production cost is a key factor in the commercial success of
biological pesticides, particularly for use in agronomic crops such as soy and
maize. Our lead biological product
candidate, HowlerTM,
is a wettable powder, and is efficacious against
multiple fungal pathogens in field testing across multiple locations. Manufacturing process development has focused
on low-cost drying options, including spray drying. The microbial active in Howler, Pseudomonas chlororaphis
AFS009, is a novel Gram-negative isolate which has been formulated to be
shelf-stable for 24 months and counting when blended with formulants and
tray-dried. Spray drying is a much more
attractive drying technology due to its lower cost and higher capacity;
however, the use of high temperature and high shear can negatively impact the
viability of biopesticides. The viability of this isolate in formulations
which are spray-dried has been improved by 100 fold by
the use of osmotic shock in the fermentation stage by the use of NaCl. Temperature
shock in stationary phase produced improvements of 5-10 fold. Taken together these process changes
represent a significant improvement in the ability to dry this product via
lower-cost technologies and reduce the overall production cost of the biopesticide.
Invited Oral Abstract Presentation
E. coli culture and kLa comparison of single-use and steel tank fermenters
Jason Brown, Christopher Brau and Nephi Jones, Thermo Fisher Scientific, Logan, UT, USA
Recent Advances in Fermentation Technology (RAFT tm)
Recent advancement has led to the development of single-use reactors for use in microbial fermentation. The Thermo Fisher Scientific Hyperforma Single-Use Fermentor (SUF) is the first specifically designed fermentor to deliver equivalent performance to stainless steel SIP/CIP reactors for research and pilot scale microbial bioproduction at 30L and 300L liquid working volume. Due to the single-use film wall, sparge, and agitation implementation; there can be limitations when moving aggressive dense cultures from steel tank to single-use reactors. To characterize the limitations of available single-use bioreactors a rigorous comparison was undertaken in collaboration with multiple vendors. For kLa evaluation the same procedure, salt solution formula, and single-use dissolved oxygen probes were used to obtain comparable results. The various reactors were tested at optimal preset RPM and air flow rates without any oxygen supplementation allowed. Results were supplied to reactor vendors without disclosing the supplier names. Here we present the performance of Thermo Fisher Scientific Hyperforma Single-Use Fermentor (SUF) 30L and 300L in comparison to other unnamed vendor options. In addition we compare growth and protein production in the SUF to traditional stainless steel fermentors standard procedures, which show equivalent growth of E.coli to 200 OD600 with product yield of 8 g/L as were seen in SIP/CIP fermenters.
Invited Oral Abstract Presentation
The effect of design and scale on the mixing and mass transfer in U-loop bioreactors
Mr. Leander Petersen1, Prof. John Villadsen1, Prof. Sten Bay Jørgensen1, Mr. Ib Christensen2, Anna Eliasson Lantz1 and Prof. Krist V. Gernaey1, (1)Technical University of Denmark, Kgs. Lyngby, Denmark, (2)Unibio A/S, Odense M, Denmark
Recent Advances in Fermentation Technology (RAFT tm)
A system capable of handling a large volumetric gas fraction while providing a high gas to liquid mass transfer is a necessity if the metanotrophic bacterium Methylococcus capsulatus is to be used in single cell protein (SCP) production.
Previous studies have proven that a U-loop fermenter, a novel vertical forced flow loop reactor where gas and liquid are driven through a series of static mixers in a U-shaped pipe, is quite capable of coping with these challenges in pilot scale. The critical question remains; what happens when the scale undergoes a more than 10 fold increase and the geometry is altered?
In this study we have investigated the mixing time and mass transfer capabilities of U-loop reactors of different geometries (high vs. diameter ratio) in pilot (0.15m3) and semi-industrial scales (2.2m3). A new expression for the mechanical power input into the system is also proposed, which indicates that an even more favorable relationship between power input and mass transfer rate (compared to previous literature) applies to U-loop fermenters.
Invited Oral Abstract Presentation
Imaging for monitoring downstream processing of fermentation broths
Dr. Rayisa Moiseyenko1, Simon Glanville2, Camilla Nørskov Laursen3, Dr. Andreas Baum1, Seyed Soheil Mansouri4, Prof. Krist V. Gernaey5 and Dr. Thomas Martini Jørgensen1, (1)Technical University of Denmark, Lyngby, Denmark, (2)Novozymes A/S, kalundborg, Denmark, (3)ParticleTech, Farum, Denmark, (4)Technical University of Denmark, kongens lyngby, Denmark, (5)Technical University of Denmark, Kgs. Lyngby, Denmark
Recent Advances in Fermentation Technology (RAFT tm)
In relation to downstream processing of a fermentation broth coagulation/flocculation is a typical pre-treatment method for separating undesirable particles/impurities from the wanted product. In the coagulation process the negatively charged impurities are destabilized by adding of a clarifying agent thereby neutralizing the charges on the particles. Particles thus agglomerate. Larger agglomerates are formed in the flocculation process by adding a polymer, which forms bridges between the particles. The operation of coagulators, flocculators and clarifiers requires trained operators implying the human factor to play a major risk with regard to performance. Better process monitoring will provide the means for improved control giving higher yield, better quality, and minimize the consumption of water. In particular, the optimal separation of biomass from a soluble enzyme phase is often dependent on an initial coagulation of the biomass and a final flocculation of the solids just prior to separation. We investigate flocculation processes at Novozymes facilities so that the response time and risk of error is minimized. We use oCelloScope [1], an automated microscope, for imaging samples from the flocculation process and subsequently we extract image features for qualitative and quantitative image characterization. The processing include image morphology, image segmentation and image quantification. The aim is to correlate image information to âqualityâ of the separation process. Here we report our initial finding.
[1] M.Fredborg et al. Journal of Clinical Microbiology Vol 51 Number 7 p. 2047â2053 (2013); http://www.biosensesolutions.dk
Invited Oral Abstract Presentation
Bioprocess optimization achieved two-fold increase in terpene and ketone titers in two non-canonical hosts
Mona Mirsiaghi, Fabrice Masson, Deepti Tanjore, Todd Pray and Eric Sundstrom, Lawrence Berkeley National Laboratory, Berkeley, CA, USA
Recent Advances in Fermentation Technology (RAFT tm)
The primary objective of this study was to develop industrially relevant processes with two non-canonical hosts,
Rhodosporidium toruloides and
Streptomyces albus, for conversion of cellulosic hydrolysate into renewable fuels. Collaborators at the Joint Bioenergy Institute (JBEI) engineered
R. toruloides and
S. albus for terpene and short-chain ketone production, respectively. At the Advanced Biofuels Process Development Unit (ABPDU), we scaled each process from shake flask to 2L fermentor and optimized production of target molecules by varying carbon sources, pH, dissolved oxygen concentration, and feeding regimes.
R. toruloides is capable of producing fatty acids along with terpenes, both of which can be used as biofuel, and is known to ferment glucose, xylose, and aromatic byproducts of lignin degradation. High gravity batch fermentation doubled terpene (bisabolene) titer as compared to fed-batch fermentation with corn stover hydrolysate. Two engineered Streptomyces albus strains were tested for production of a mixture of C5-C7 ketones. Streptomyces species generally require complex media for production of secondary metabolites; we were able to replace complex media components with cellulosic hydrolysates from corn stover, poplar, and bagasse. The optimized process eliminated several high-cost media components while doubling ketone titers as compared to the control medium. Alkali-pretreated corn stover hydrolysate led to the highest ketone titers reported to date (>500 mg/L in shake flasks); this feedstock was chosen for scale-up to 2L fed-batch fermentation. Full volatilization of C5 and C6 ketones was observed during fermentation process optimization at 2L-scale, highlighting the potential for low-cost recovery of gas-phase fuel molecules.
Invited Oral Abstract Presentation
Application of a model-based soft sensor to monitor lactic acid bacteria fermentations at pilot scale
Robert Spann1, Christophe Roca2, Anna Eliasson Lantz1, Prof. Krist V. Gernaey1 and Gürkan Sin1, (1)Technical University of Denmark, Kgs. Lyngby, Denmark, (2)Chr. Hansen Holding A/S, Hoersholm, Denmark
Recent Advances in Fermentation Technology (RAFT tm)
A model-based soft sensor was applied to monitor a 700 L
Streptococcus thermophilus fermentation. The soft sensor was based on a data reconciliation module and a first principles mechanistic model. The data reconciliation module used a general process stoichiometry model to update some of the mechanistic model parameters with 5 minutes intervals using the very limited available on-line measurements, which were ammonia addition and pH. The updated parameters were used as input to the mechanistic model that combined biological (biomass growth and lactic acid production) and chemical (pH) mechanisms of the fermentation process. The model was then used to predict unmeasured, important process parameters, such as biomass, lactose, and lactic acid, and the measured pH. This process analytical technology (PAT) monitoring system was applied in MATLAB® (The MathWorks®, Natick, MA) to a historical data set of a 700 L fermentation where the on-line data was used as available on-line. A good prediction accuracy was obtained with an error less than 10 % of the biomass concentration. Uncertainty analysis was also performed using the Monte Carlo technique to quantify model prediction uncertainty when making predictions for the fermentation batches. The presented monitoring system is a promising tool to improve the operation of LAB fermentations as it provides the operators with additional information of the process in real-time.
Acknowledgement
This project has received funding from the European Unionâs Horizon 2020 research and innovation program under the Marie SkÅodowska-Curie grant agreement No 643056. We are grateful to Chr. Hansen A/S for the experimental support.
Invited Oral Abstract Presentation
Lean process validation
Ms. Jane Gunson, Ms. Nitya Krishnan and Dr. Ryan Hamilton, Genentech, Inc, South San Francisco, CA, USA
Recent Advances in Fermentation Technology (RAFT tm)
A process validation (PV) effort was performed for an antibody fragment (Fab) produced in Escherichia coli. Using a Quality by Design (QbD) approach, the impact of eight production culture process parameters on eight potential critical quality attributes (pCQAs) was evaluated in a single PV study. The study consisted of a one statistical design of experiment (DOE), resolution V fractional factorial, in addition to several univariate assessments. No PV studies were performed for the inoculum culture steps in the fermentation process. The reduced number of studies along with the smaller number of process parameters and pCQAs evaluated in the single production culture study was the outcome of process parameter and pCQA risk ranking and filtering (RRF) exercises. These assessments leveraged existing process data and relevant stress model results to inform the lean PV effort.
Invited Oral Abstract Presentation
Raman spectroscopy for in situ, real-time fermentation monitoring
Dr. Karen Esmonde-White1, Maryann Cuellar1, Dr. Sean Gilliam1, Dr. Carsten Uerpmann2, Dr. Bruno Lenain2 and Dr. Ian Lewis1, (1)Kaiser Optical Systems Inc., Ann Arbor, MI, USA, (2)Kaiser Optical Systems SARL, Saint Priest, MI, France
Recent Advances in Fermentation Technology (RAFT tm)
In situ Raman during bioprocesses enables simultaneous measurement of multiple cellular biochemistry and bioreactor parameters and allows for in-process monitoring and control. Raman is well-suited for many upstream and downstream bioprocess applications because of its ability to directly measure aqueous systems, strong history as a process analytical technology in small molecule pharmaceutical manufacturing, excellent model transferability and sampling versatility.
Raman spectroscopy can be used to measure gases, liquids, solids and turbid media. We describe technologic and ergonomic considerations in sampling probe design and how these factors affect implementation of in situ Raman spectroscopy for bioprocessing applications. Immersion probes are compatible with the bioreactor environment and sterilization protocols, provide simultaneous in situ measurements of multiple bioprocess parameters, and enable real-time process monitoring and control. Examples of immersion Raman probes will be shown in bioreactor and fermentation biogas production. New sampling systems compatible with single-use bioreactors (SUB) will be introduced and data comparing SUB-compatible optics with immersion probes an analytical standards model will be discussed. Through representative examples, we show Raman spectroscopy as a robust and reliable analytical technology for in situ fermentation application.
Invited Oral Abstract Presentation
The narrow balance of feeding charged nutrients
Charles Sellers, Heliae Development, LLC, Gilbert, AZ, USA and Dr. Eneko Ganuza, Heliae Development LLC., Gilbert, AZ, USA
Recent Advances in Fermentation Technology (RAFT tm)
A pH-auxostat is a self-titrated fed-batch system that provides a nutrient (i.e acetic acid) on demand to the culture. As the name implies, this system is designed to maintain the residual nutrient (auxo-) concentration constant (-stat). However, along with the acetate oxidation there are other biochemical processes would impact the medium alkalinity, resulting in the buildup of toxic acetate concentrations or conversely the displacement of all the acetate from the system. Our goal was to study the impact of the nitrogen source on the residual acetate concentration and ultimately design a process that could operate according to the auxostat principles. We used an oleaginous microalgae strain,
Aurantiochytrium sp. HS399, that uses acetic acid as building block for lipid synthesis and energy. Real time monitoring of residual nutrients was achieved thanks to the Cedex Bio Analyzer (Roche Diagnostics (Schweiz, Switzerland), which conveniently produced results in less than 20 min. When glutamate was used as a nitrogen source residual acetate built up to potentially toxic concentrations. In turn, when ammonium chloride was used as a nitrogen source residual acetate was displaced from the media resulting in the arrest of the pH-auxostat system. Finally, when ammonia was provided as ammonium acetate, residual acetate was balanced to minimize toxicity while avoiding the auxostat interruption. Feeding charged nutrients such as ammonia, acetic acid and managing their toxicities requires a deep understanding of the metabolic processes that go beyond the pH-auxostat control of one particular nutrient.
[CS1]Average test is 12-18 minutes
Invited Oral Abstract Presentation
Development of generic Raman models for process monitoring
Brian Hadley and Thaddaeus Webster, Lonza Biologics, Portsmouth, NH, USA
Recent Advances in Fermentation Technology (RAFT tm)
Manufacturing safe and consistent bio-therapeutic products requires robust process monitoring and control of production bioreactors. This historically required sample removal, expensive and sometimes toxic reagents and skilled operators. The implementation of novel process analytical technologies for continuous monitoring has been proposed by regulatory agencies as a means of improving process control. Inline Raman spectroscopy coupled with multivariate software to develop predictive models for critical process parameters was used to create, generic, cell line-independent models to continuously monitor critical process parameters in bioreactors operating Lonzaâs platform processes.
Generic Raman models were developed by culturing two different GS-CHO cell lines using a platform process in 12 five-liter bioreactors. Raman spectra and offline measurements were collected twice daily during the course of cell culture in order to construct predictive models. Metabolites, VCC & TCC, and titer were measured offline using a Nova Bioprofile 400, Vi-Cell XR Analyzer, and Protein-A HPLC respectively. Projections on latent structure models for each parameter of interest were created in SIMCA v13.0.3 by regressing Raman spectra with their corresponding offline measurements.
Validation against a third independent GS-CHO cell line provided predictive error of the models. Raman was capable of monitoring changes in the concentration of glucose, lactate, ammonium, viable cell concentration, and total cell concentration with prediction errors of 0.44 g/L, 0.24 g/L, 0.028 g/L, 1.92 x 106 cells/mL, and 1.89 x 106 cells/mL respectively with Lonzaâs platform process. With online continuous monitoring, process improvements or realtime feedback control could be used for Lonzaâs platform processes in the future.
Invited Oral Abstract Presentation
Application of novel free-floating sensor device: Flow characteristics in stirred vessels
Mr. Jonas Bisgaard1, Dr. Ole Skyggebjerg2, Prof. Jakob K. Huusom1, Mr. Lars V. Petersen2, Dr. Sjef Cornelissen3, Prof. Niels K. Poulsen1 and Prof. Krist V. Gernaey1, (1)Technical University of Denmark, Kgs. Lyngby, Denmark, (2)Freesense ApS, Copenhagen N, Denmark, (3)Novozymes A/S, Bagsvaerd, Denmark
Recent Advances in Fermentation Technology (RAFT tm)
In this initial study, a novel process analytical technology (PAT) tool for online monitoring of industrial bioreactors is presented. The technology is a free-floating sensor device which is robust, steam sterilizable and capable of measuring critical culture parameters. The sensors are designed for high sample rates and fast response times, while a patented positioning system gives information on the position at which the parameters are measured. Consequently, this novel sensor can also be used for analysis of flow characteristics and gradients in industrial scale bioreactors.
The free-floating sensors have been tested in a large-scale bioreactor (>100 m3) filled with water, at different agitation speeds. Pulses of sodium hydroxide were added to compare the pH response of the free-floating sensor with the response of commercial pH-sensors fixed to the tank wall. Circulation times and spatial distributions were determined based on the collected pressure measurements. It could be seen from the distributions and axial velocities that mixing was limited in the top and bottom of the reactor. As expected, a noticeable decrease in the circulation time was found when increasing the impeller speed. Using free-floating sensors rather than fixed sensors can provide unexplored data which has not previously been available. This data can link measurements of culture parameters with positions and flow characteristics within the reactors. This can serve as an important tool in validation, control and regulation of industrial processes, as well as a tool for process optimization where it should be possible to reduce development cycle time during process scale-up.
Invited Oral Abstract Presentation
Biology's role in energy storage: A unique 2-step process to turn renewable electricity into renewable methane
Kevin W Harrison and Nancy Dowe, National Renewable Energy Laboratory, Golden, CO, USA
Recent Advances in Fermentation Technology (RAFT tm)
The cost of wind- and solar-generated electricity has decreased significantly over the past few decades and is now competitive with fossil-based generation. Low-cost and otherwise curtailed renewable electricity is beginning to open up new markets in the areas of gas fermentation for long-duration energy storage. The National Renewable Energy Laboratory (NREL) and Southern California Gas Company (SoCal Gas) are collaborating on a first-of-its-kind in the United States Power-to-Gas project to shift energy and enable even higher penetrations of renewable electricity generation on the utility grid. The two-step process first involves splitting water electrochemically via low-temperature water electrolysis to produce renewable hydrogen. Secondly, a biologically-based anaerobic methanation process converts the hydrogen and carbon dioxide in to methane, heat, and water. The methanogen, Methanothermobacter thermautotrophicus, is at the heart of the NREL/SoCal Gas collaboration and will be run in a new 700 Liter bioreactor at pressures up to 18 bar in Golden, Colorado. The team also includes Electrochaea GmbH, the company commercializing the technology, and Burns & McDonnell, the engineering firm that oversaw the design and fabrication of the system. NREL will characterize the system performance under steady-state and highly variable (i.e., wind and solar) power profiles to understand the microorganisms performance over the pressure, temperature and agitation range that the system will operate in. The NREL/SoCal Gas pilot project will be used to determine the commercial viability of this power-to-gas approach to energy storage and provide insights into megawatt-scale system designs.
Invited Oral Abstract Presentation
Real-time optimization of fermentations and cell cultures profitability - from R&D up to biomanufacturing
Dr. Joel Sirois, BioIntelligence Technologies inc, Sherbrooke, QC, Canada
Recent Advances in Fermentation Technology (RAFT tm)
Biomanufacturing and bioprocess development suffer from a lack of probes to perform a valuable monitoring of product biosynthesis and its optimization. Many samples have to be taken and analyzed with instruments such as HPLC to have an idea of the kinetics and yields during fermentations and cell cultures. This constrain induces delays and losses. The BioAnalyst is a new instrument collecting data from any source, being probes on a bioreactor, datafile from a HPLC or user entries from manual analysis, and performing real-time calculations to unveil hidden information such as biokinetics. The embedded algorithms push information on personal sets of dashboards for each user enabling them to accelerate decision making and implement real-time optimization. A layer of algorithms is dedicated to translate scientific information into financial information. This interconnectivity hub reduces time wasted on data capture, data management and data analysis, increases profit margin by reducing costs and losses, and increases throughput with no change on actual bioprocesses.
Paper
Bio Base Europe Pilot Plant: âclosing the gap in the innovation chainâ
Dr. Hendrik Waegeman, Bio Base Europe Pilot Plant, Ghent, Belgium
Recent Advances in Fermentation Technology (RAFT tm)
Invited Oral Abstract Presentation
The scale up and scale down of bioprocesses for efficient fermentation development
Dr. Alison Arnold, Ingenza Ltd, Midlothian, United Kingdom
Recent Advances in Fermentation Technology (RAFT tm)
This presentation will highlight how Ingenza Ltd â a leading UK Industrial Biotechnology company â has successfully scaled up a number of different bioprocess. These high cell density fermentations were carried out using a number of different microorganisms applied to various processes across a number of industry sectors such as chemical manufacturing or biologics production. Predictability in scale up is key. Ingenza Ltd are now augmenting their current upstream processing capabilities with the Sartoriusâ ambr® 250 modular bioreactor system. Ingenza Ltd has taken established bioprocesses and replicated these from large scale to small scale quickly and efficiently. Other technologies are being investigated such as real time biomass monitoring systems. All of these things are key to aid the ease and throughput of fermentation development at smaller scale, to lead to confidence that the processes can be scaled up.