The progressive depletion of non-renewable energy sources worldwide, together with the fact that their use has resulted in environmental deterioration and public health problems, has led to development of renewable energy harvesting technologies. Current bioethanol research focusses mainly on waste lignocellulosic feedstocks like agricultural, industrial and municipal solid wastes due to their abundance and renewability. However, the current technologies in use suffer from various drawbacks including low ethanol yield and productivity, co-fermentation of pentose and hexose sugars, low tolerance to product and inhibitors. The present study was carried out using pretreated paddy straw under 0.5% NaOH treatment with 20% solid loading at 15 psi for 10 min. The pretreated straw was treated with Cellic Ctec 2 with enzyme loading of 60 mg enzyme protein g^-1 dry biomass at 20% biomass loading and thermotolerant isolate Kluyveromyces marxianus NIRE-K3 cells at 45 ^oC in simultaneous saccharification and fermentation (SSF). SSF configuration was supplemented with medium components with composition (in g l^-1) yeast extract, 2.93; K₂HPO₄, 1.99; NaH₂PO₄, 0.24; MgSO₄, 0.42; (NH₄)₂SO₄, 1.34, pH 5.5. This resulted in maximum ethanol concentration of 25.34 g l^-1 after 24 h which corresponds to 182.22 g ethanol/kg of raw paddy straw. Thus, it can be concluded that the thermotolerant isolate K. marxianus could be exploited for commercial scale bioethanol production from alkali pretreated paddy straw in SSF configuration.

Recently, there has been much progress within discovery and engineering of thermostable cellulolytic enzymes. This is promising from an industrial perspective as saccharification of lignocellulosic biomass is best conducted at high temperatures. High temperatures are advantageous for different reasons including the repression of bacterial growth and reduction of viscosity, but the most important advantage is an increased reaction rate, which may lead to shorter process time and lower enzyme dosages. However, systematic investigations of temperature–activity relationships for cellulases are rare, and the potential activity gain of an increased process temperature remains uncertain.

We have studied temperature activation of the cellobiohydrolase Cel7A, and some other cellulases. In the range where the enzyme was stable, we found that the activity against a soluble substrate-analog approximately doubled for every 10°C temperature increment (Q₁₀≈2), as it is typical for hydrolases. On insoluble cellulose, however, the degree of temperature activation was much lower. This was shown to rely on a temperature dependent dissociation of enzyme from the substrate surface, which counteracted the inherent acceleration by higher temperature. Interestingly, enzyme variants that were engineered to have higher affinity for the substrate were less prone to dissociation and hence more activated by temperature.

The depolymerization of complex plant biomass represents a major pillar in the Earth´s carbon cycle and relies on a network of enzymatic and chemical reactions that is still full of mysteries. For decades, the degradation of recalcitrant polysaccharides was thought to be mainly achieved by an arsenal of hydrolytic enzymes called glycoside hydrolases (GHs). This paradigm changed in 2010 with the discovery of a new class of enzymes today known as Lytic Polysaccharide Monooxygenases (LPMOs) (Vaaje-Kolstad et al., 2010, Science). LPMOs are mono-copper enzymes that have the unique ability to carry out oxidative cleavage of crystalline polysaccharides, thus making glycosidic chains more accessible for canonical GHs. Inspired by Nature, where LPMOs are notably abundant in wood-rotting fungal secretomes, these enzymes also have become instrumental for the development of economically sustainable lignocellulose biorefineries (Johansen et al., 2016, Biochem.Soc.Trans.). However, the mode of action of LPMOs remains largely enigmatic, which potentially hampers their optimal industrial harnessing.

We have recently been considering the possibility that the classification of LPMOs as “classical” monooxygenases may need to be revised. Here, we provide compelling evidence that H₂O₂, and not O₂, is the co-substrate of LPMOs during the reaction of polysaccharide oxidation (Bissaro et al., 2016, bioRxiv 097022). These findings are shattering established dogmas and offer new perspectives regarding the mode of action of copper enzymes and the enzymatic conversion of biomass in Nature. We will also illustrate how the design of industrial lignocellulose biorefining processes may be improved based on our recent findings.

Lytic polysaccharide monooxygenases (LPMOs) are industrially important enzymes that are found widespread in the microbial world. LPMOs carry out oxidative cleavage of glycosidic bonds in recalcitrant polysaccharides such as cellulose and thereby boost the activity of glycosyl hydrolases. LPMOs have evolved to become substrate controlled but in the absence of substrate and in the presence of oxygen and a reducing agent, LPMOs release reactive oxygen species as a product of each redox cycle. Liberation of reactive oxygen species may cause chemical chain reactions that are detrimental to enzymes and to the microorganism. Oxidative inactivation of commercial cellulase mixtures has been shown to be a significant factor influencing the overall saccharification efficiency and the addition of catalase can successfully be used to protect cellulases mixtures from inactivation. In contrast, addition of hydrogen peroxide to the saccharification slurry after liquefaction had a clear negative effect on glucose yield. It is thus important to be able to understand the details of oxygen activation at the mononuclear copper active site of LPMOs. We have studied the interaction between two closely related members of the AA9 enzyme family and several polysaccharides. Although both enzymes acts on a range of polysaccharides, there are distinct differences which will be discussed.

Gen 1.5 describes the process of converting corn kernel fiber into ethanol. This process can give up to 10% more ethanol gallons, improve corn oil recovery by up to 70% and yields DDGs with improved digestibility. We will provide a review of current Gen 1.5 technologies in the market, and the prerequisites for this technology. We will discuss the science behind mechanical, chemical and enzymatic corn fiber treatment. We will present results on corn fiber saccharification and fermentation using enzymes and describe how different cellulolytic enzymes act on corn fiber. We will also provide an overview of the value of Gen 1.5 technology.

One of the technical barriers for biofuels production from biomass is the costly cellulases required to break down lignocellulosic feedstocks to sugars due to the general recalcitrance of plant cell walls. To overcome this hurdle, a “one pot” or “consolidated bioprocessing (CBP)” biomass processing scheme has been proposed, which includes cellulase production, cell wall polymer hydrolysis, and sugar fermentation in a single step. Simultaneous production of cellulases in fermentation host organisms for ethanol production especially in Saccharomyces cerevisiae has been explored. We are investigating the potential of the oleaginous yeasts as CBP host for production of long chain hydrocarbon biofuels. Cellobiohydrolase I (CBH I) is a key cellulase required for effectively break down cellulose and is found difficult to be functionally expressed in fermentation organisms at high level. While the most active Trichoderma reesei CBH I failed to be expressed successfully, recently researchers discovered the Talaromyces emersonii-Trichoderma reesei chimeric cellobiohydrolases I (Te-Tr chimeric CBH I) can be functionally expressed in S. cerevisiae. We showed here that the chimeric CBH I as well as its newly constructed fusion proteins can be expressed in oleaginous yeasts, Lipomyces starkeyi and Yarrowia lipolytica. All parent chimeric CBH I and fusion proteins are highly active and converted pretreated corn stover substrates. In addition, we will also discuss the co-expression of fungal cellulases including CBH I, CBHII and EGII in the oleaginous yeast and their impact on lipid accumulation.

Pyrolysis is one means of creating bio-oils, bio-chars, and syngas useful as combustible biofuels from lignocellulosic materials. The pyrolysis process also generates high yields of the anhydrosugar levoglucosan. Because this sugar is not readily metabolized by typical production organisms, there has been an interest in expressing enzymes which catabolize levoglucosan in production organisms.

Levoglucosan dehydrogenase (LGDH) is the only known bacterial enzyme to act on levoglucosan, and previously only one species of Arthrobacter has been observed to express LGDH. It was proposed that intracellularly this enzyme oxidizes levoglucosan to 3-keto-levoglucosan, which then chemically hydrates to 3-keto-glucose before reducing this substrate to glucose. Is this enzyme and its associated metabolic pathway specific to a particular taxonomic group of bacteria? What residues or structural moieties make LGDH specific to levoglucosan conversion?

Four bacterial strains were isolated showing growth on levoglucosan and LGDH activity. Through Illumina sequencing these isolates were determined to be Microbacterium sp., Paenibacillus sp., Shinella sp., and a strain of Klebsiella pneumoniae. The gene lgdh has been identified in all isolates, and we are presently looking at neighboring genes for other enzymes critical to levoglucosan conversion.

To understand how LGDH interacts with levoglucosan, the LGDH from Arthrobacter phenanthrenivorans Sphe3 was crystallized. X-ray diffraction data were used to assemble enzyme structures, revealing that while major structural features including the Rossman-fold for NADH binding and the carbohydrate active site are conserved relative to known dehydrogenases, LGDH has several different residues in the carbohydrate-binding site likely responsible for its preferential activity on levoglucosan.