Comparative Secretome Analysis of Trichoderma reesei and Aspergillus niger during Growth on Sugarcane Biomass

Background

Our dependence on fossil fuel sources and concern about the environment has generated a worldwide interest in establishing new sources of fuel and energy. Thus, the use of ethanol as a fuel is advantageous because it is an inexhaustible energy source and has minimal environmental impact. Currently, Brazil is the world''s second largest producer of ethanol, which is produced from sugarcane juice fermentation. However, several studies suggest that Brazil could double its production per hectare by using sugarcane bagasse and straw, known as second-generation (2G) bioethanol. Nevertheless, the use of this biomass presents a challenge because the plant cell wall structure, which is composed of complex sugars (cellulose and hemicelluloses), must be broken down into fermentable sugar, such as glucose and xylose. To achieve this goal, several types of hydrolytic enzymes are necessary, and these enzymes represent the majority of the cost associated with 2G bioethanol processing. Reducing the cost of the saccharification process can be achieved via a comprehensive understanding of the hydrolytic mechanisms and enzyme secretion of polysaccharide-hydrolyzing microorganisms. In many natural habitats, several microorganisms degrade lignocellulosic biomass through a set of enzymes that act synergistically. In this study, two fungal species, Aspergillus niger and Trichoderma reesei, were grown on sugarcane biomass with two levels of cell wall complexity, culm in natura and pretreated bagasse. The production of enzymes related to biomass degradation was monitored using secretome analyses after 6, 12 and 24 hours. Concurrently, we analyzed the sugars in the supernatant.

Results

Analyzing the concentration of monosaccharides in the supernatant, we observed that both species are able to disassemble the polysaccharides of sugarcane cell walls since 6 hours post-inoculation. The sugars from the polysaccharides such as arabinoxylan and β-glucan (that compose the most external part of the cell wall in sugarcane) are likely the first to be released and assimilated by both species of fungi. At all time points tested, A. niger produced more enzymes (quantitatively and qualitatively) than T. reesei. However, the most important enzymes related to biomass degradation, including cellobiohydrolases, endoglucanases, β-glucosidases, β-xylosidases, endoxylanases, xyloglucanases, and α-arabinofuranosidases, were identified in both secretomes. We also noticed that the both fungi produce more enzymes when grown in culm as a single carbon source.

Conclusion

Our work provides a detailed qualitative and semi-quantitative secretome analysis of A. niger and T. reesei grown on sugarcane biomass. Our data indicate that a combination of enzymes from both fungi is an interesting option to increase saccharification efficiency. In other words, these two fungal species might be combined for their usage in industrial processes.     

Addendum to Issue 1 - ENZITEC 2012Simultaneous biosynthesis of biomass-degrading enzymes using co-cultivation of Aspergillus niger and Trichoderma reesei

Abstract

The biotransformation of lignocellulosic materials into biofuels and chemicals requires the simultaneous action of multiple enzymes. Since the cost of producing an efficient enzyme system maybe high, mixed cultures of microorganisms maybe an alternative to increase enzymatic production and consequently reduce costs. This study investigated the effects of different inoculum ratios and inoculation delays on the biosynthesis of cellulases and xylanases during co-cultivation of Aspergillus niger and Trichoderma reesei under solid-state fermentation (SSF). While the monoculture of T. reesei was more efficient for CMCase production than the co-cultivation of A. niger and T. reesei, a significant increase in β-glucosidase and xylanase production was achieved by co-cultivation of both species. The maximum CMCase activity of 153.91 IU/g was obtained with T. reesei after 48 h of cultivation, while the highest β-glucosidase activity of 119.71 IU/g (after 120 h) was obtained by co-cultivation of A. niger and T. reesei with a 3:1 inoculum ratio (A. niger: T. reesei). The greatest xylanase activity observed was 589.39 IU/g after 72 h of mixed culturing of A. niger and T. Reesei with a 1:1 inoculum ratio. This is the first study where the effects of inoculum ratio and inoculation delay in mixed culture of T. reesei and A. niger under SSF have been systematically assessed, and it indicates co-cultivation as a feasible alternative to increase enzymatic production.     

Enzymatic hydrolysis and simultaneous saccharification and fermentation of steam-pretreated spruce using crude Trichoderma reesei and Trichoderma atroviride enzymes

The aim of this study was to compare the performance of the enzymes produced by Trichoderma reesei Rut C30 and the good extracellular β-glucosidase-producing mutant Trichoderma atroviride TUB F-1663 to that of commercial preparations in the enzymatic hydrolysis and the simultaneous saccharification and fermentation (SSF) of steam-pretreated spruce (SPS).The concentrated TUB F-1663 enzyme was found to be the most efficient in the hydrolysis of washed SPS at 50 g/L water-insoluble solids (WIS) in terms of the glucose produced (18.5 g/L), even in comparison with commercial cellulases (14.1–16.7 g/L). The enzyme preparations were studied at low enzyme loadings (5 FPU/g WIS) in SSF to produce ethanol from SPS. The enzyme supernatant and whole fermentation broth of T. atroviride as well as the whole broth of T. reesei proved to be as efficient in SSF as the commercial cellulase mixtures (ethanol yields of 61–76% of the theoretical were achieved), while low ethanol yields (<40%) were obtained with the β-glucosidase-deficient T. reesei supernatant.Therefore, it seems, that instead of using commercial cellulases, the TUB F-1663 enzymes and the whole broth of Rut C30 may be produced on-site, using a process stream as carbon source, and employed directly in the biomass-to-bioethanol process.     

Enzyme production by the mixed fungal culture with nano‐shear pretreated biomass and lignocellulose hydrolysis

Cellulase, xylanase, and β‐glucosidase production was studied on novel nano‐shear pretreated corn stover by the mixed fungi culture. The high shear force from a modified Tayor‐Couette nano‐shear mixing reactor efficiently disintegrated corn stover, resulting in a homogeneous watery mash with particles in much reduced size. Scanning electron microscope study showed visible mini‐pores on the fiber cell wall surface, which could improve the accessibility of the pretreated corn stover to microorganisms. Mixed fungal culture of Trichoderma reesei RUT‐C30 and Aspergillus niger produced enzymes with higher cellulolytic and xylanolytic activities on corn stover pretreated with nano‐shear mixing reactor, in comparison with other pretreatment methods, including acid and ammonia fiber explosion (AFEX) pretreatment. The hydrolytic potential of the whole fermentation broth from the mixed fungi was studied, and the possibility of applying the whole cell saccharification concept was also investigated to further reduce the cost of lignocellulose hydrolysis. Biotechnol. Bioeng. 2013; 110: 2123–2130. © 2013 Wiley Periodicals, Inc.     

Functional assembly and characterization of a modular xylanosome for hemicellulose hydrolysis in yeast

Five trimeric xylanosomes were successfully assembled on the cell surface of Saccharomyces cerevisiae. Three dockerin‐tagged fungal enzymes, an endoxylanase (XynAc) from Thermomyces lanuginosus, a β‐xylosidase (XlnDt) from Aspergillus niger and an acetylxylan esterase (AwAXEf) from Aspergillus awamori, were displayed for the synergistic saccharification of birchwood xylan. The surface‐expression scaffoldins were modular constructs with or without carbohydrate binding modules from Thermotoga maritima (family 22) or Clostridium thermocellum (family 3). The synergy due to enzyme–enzyme and enzyme–substrate proximity, and the effects of binding domain choice and position on xylan hydrolysis were determined. The scaffoldin‐based enzymes (with no binding domain) showed a 1.6‐fold increase in hydrolytic activity over free enzymes; this can be attributed to enzyme–enzyme proximity within the scaffoldin. The addition of a xylan binding domain from T. maritima improved hydrolysis by 2.1‐fold relative to the scaffoldin without a binding domain (signifying enzyme–substrate synergy), and 3.3‐fold over free enzymes, with a xylose productivity of 105 mg g^?1 substrate after 72 h hydrolysis. This system was also superior to the xylanosome carrying the cellulose binding module from C. thermocellum by 1.4‐fold. Furthermore, swapping the xylan binding module position within the scaffoldin resulted in 1.5‐fold more hydrolysis when the binding domain was adjacent to the endoxylanase. These results demonstrate the applicability of designer xylanosomes toward hemicellulose saccharification in yeast, and the importance of the choice and position of the carbohydrate binding module for enhanced synergy. Biotechnol. Bioeng. 2013; 110: 275–285. © 2012 Wiley Periodicals, Inc.     

Saccharification of used paper with different cellulases

Various used paper materials have been exposed to the action of cellulases from Penicillium funiculosum, Trichoderma reesei, Trichoderma viride and Aspergillus niger. A 2 h incubation period showed cellulase from T. viride the most active except for office paper that was maximally degraded by A. niger cellulase. Cellulase mixtures increased saccharification while sequential treatment with cellulases from T. reesei and P. funiculosum increased biodegradation at values between 15% and 190%. The maximum increase of saccharification (190%) was obtained when T. reesei cellulase initiated the sequential treatment of newspaper relative to the sole action of P. funiculosum cellulase on this non-pretreated and pretreated material.     

Evaluation of cellulases produced from four fungi cultured on furfural residues and microcrystalline cellulose

Four fungal strains—Trichoderma viride, Aspergillus niger, Trichoderma koningii, and Trichoderma reesei—were selected for cellulase production using furfural residues and microcrystalline cellulose (MCC) as the substrates. The filter paper activity (FPA) of the supernatant from each fungus was measured, and the performance of the enzymes from different fungal strains was compared. Moreover, the individual activities of the three components of the cellulase system, i.e., β-glucosidase, endoglucanase, and exoglucanase were evaluated. T. koningii showed the highest activity (27.81 FPU/ml) on furfural residues, while T. viride showed an activity of 21.61 FPU/ml on MCC. The FPA of the crude enzyme supernatant from T. koningii was 30% higher on furfural residues than on MCC. T. koningii and T. viride exhibited high stability and productivity and were chosen for cellulases production. The crystallinity index (CrI) of the furfural residues varied after digested by the fungi. The results indicated differences in the functioning of the cellulase system from each fungus. In the case of T. koningii, T. reesei and T. viride, furfural residues supported a better environment for cellulase production than MCC. Moreover, the CrI of the furfural residues decreased, indicating that this material was largely digested by the fungi. Thus, our results suggest that it may be possible to use the cellulases produced from these fungi for the simultaneous saccharification and fermentation of lignocellulosic materials in ethanol production.     

Saccharification of biomass using whole solid‐state fermentation medium to avoid additional separation steps

The enzymatic hydrolysis of steam‐exploded sugarcane bagasse (SESB) was investigated using enzymatic extracts (EE) and whole fermentation media (WM), produced in‐house, from Aspergillus niger 3T5B8 and Trichoderma reesei Rut‐C30 cultivated on wheat bran under solid‐state fermentation (SSF). A detailed and quantitative comparison of the different hydrolysis conditions tested was carried out using the Chrastil approach for modeling enzymatic reactions by fitting the experimental data of total reducing sugar (TRS) released according to hydrolysis time. Conversion of SESB using A. niger enzymatic complex were up to 3.2‐fold higher (in terms of TRS) than T. reesei at similar enzyme loadings, which could be correlated to the higher β‐glucosidase levels (up to 35‐fold higher) of A. niger enzymatic complex. Conversion yields after 72 h exceeded 40% in terms of TRS when the WM was supplemented with a low dosage of a commercial enzyme preparation. When the combination of WM (from either T. reesei or A. niger) and commercial cellulase was used, the dosage of the commercial enzyme could be reduced by half, while still providing a hydrolysis that was up to 36% more efficient. Furthermore, SESB hydrolysis using either EE or WM resulted in similar yields, indicating that the enzyme extraction/filtration steps could be eliminated from the overall process. This procedure is highly advantageous in terms of reduced enzyme and process costs, and also avoids the generation of unnecessary effluent streams. Thus, the enzymatic conversion of SESB using the WM from SSF is cost‐effective and compatible with the biorefinery concept. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:1430–1440, 2013     

Production and characterization of a highly active cellobiase from Aspergillus niger grown in solid state fermentation

Summary Aspergillus niger produced extracellular cellobiase when grown on different lignocellulosic substrates in solid state fermentation. The enzyme activity and yield were variable according to the carbon source. In Vogel’s medium, the cellobiase productivity was significantly higher on wheat bran, followed by Leptochloa fusca (kallar grass) straw augmented with corn steep liquor. Maximum yield of cellobiase/g wheat bran was significantly higher than the values reported on other potent fungi, bacteria and recombinants, harboring heterologous gene for cellobiase. This enzyme in the presence and absence of Trichoderma reesei and celluloclast, saccharified the biomass and the percentage saccharification as well as glucose yield from lignocellulosic biomass was doubled in its presence. The partially purified enzyme was thermotolerant as evidenced by melting temperature, activation energy demand for active catalysis, enthalpy and entropy of activation for reversible or irreversible thermal inactivation.     

Enzymatic hydrolysis of cellulose: Evaluation of cellulase culture filtrates under use conditions

Culture filtrates from three mutant strains of Trichoderma reesei grown on lactose and on cellulose were compared under use conditions on four cellulose substrates. Cellulose culture filtrates contained five to six times as much cellulase as lactose culture filtrates. Unconcentrated cellulose culture filtrates produced up to 10% sugar solutions from 15% cellulose in 24 h. Specific activity in enzyme assays and efficiency in saccharification tests were low for enzymes from all the mutants. Over a wide range the percent saccharification of a substrate in a given times was directly proportional to the logarithm of the ratio of initial concentrations of enzyme and substrate. As a result of this, dilute enzyme is more efficient than concentrated enzyme, but if high sugar concentrations are desired, very large quantities of enzyme are required. Since the slopes of these plots varied, the relative activity of cellulase on different substrates may be affected by enzyme concentration.     

Mixed culture solid substrate fermentation for cellulolytic enzyme production

Summary Cellulolytic and hemicellulolytic enzymes were produced on extracted sweet sorghum silage by mixed culture solid substrate fermentation with Trichoderma reesei LM-1 (a Peruvian mutant) and Aspergillus niger ATCC 10864. Optimal cellulose and xylanase levels of 4 IU/g dry weight (DW) and 180 IU/g DW, respectively, were achieved in 120 h-fermentation when T. reesei, inoculated at 0 h, was followed by the inoculation of A. niger at 48 h.     

Deciphering the molecular mechanisms behind cellulase production in Trichoderma reesei,the hyper-cellulolytic filamentous fungus

The filamentous fungus Trichoderma reesei is a potent cellulase producer and the best-studied cellulolytic fungus. A lot of investigations not only on glycoside hydrolases produced by T. reesei, but also on the machinery controlling gene expression of these enzyme have made this fungus a model organism for cellulolytic fungi. We have investigated the T. reesei strain including mutants developed in Japan in detail to understand the molecular mechanisms that control the cellulase gene expression, the biochemical and morphological aspects that could favor this phenotype, and have attempted to generate novel strains that may be appropriate for industrial use. Subsequently, we developed recombinant strains by combination of these insights and the heterologous-efficient saccharifing enzymes. Resulting enzyme preparations were highly effective for saccharification of various biomass. In this review, we present some of the salient findings from the recent biochemical, morphological, and molecular analyses of this remarkable cellulase hyper-producing fungus.     

Sugarcane Bagasse Hydrothermal Pretreatment Liquors as Suitable Carbon Sources for Hemicellulase Production by <Emphasis Type="Italic">Aspergillus niger</Emphasis>

The aim of this study was to valorize the hemicellulose-rich liquid fraction (liquor) arising from hydrothermal pretreatment of sugarcane bagasse (SCB) through its utilization as an unconventional, soluble carbon source for the production of hemicellulases, namely xylanases and α-L-arabinofuranosidases (ABFases), by Aspergillus niger DCFS11. Through the use of factorial design, pretreatment conditions producing liquors optimized for either early- or late-phase enzyme production were identified. Subsequent deep characterization of liquor components using liquid chromatography and mass spectrometry was performed to identify compounds likely responsible for hemicellulase induction. SCB liquors arising from various pretreatment configurations induced up to 2- and 8.6-fold higher xylanase and ABFase production, respectively, by A. niger DCFS11 than raw SCB substrate owing to the strong inducing potential of arabinosylated xylooligosaccharides and free arabinose solubilized during pretreatment. Notably, unlike the severe pretreatment conditions required for maximum cellulose saccharification and ethanol yields during biomass conversion, low severity and low biomass loading are required if enzyme production from liquor is desired at early-phase growth with no additional detoxification steps. This suggests that for effective application in biorefineries, separate or multi-step processes would be required to optimize both hemicellulase production by A. niger DCFS11 and cellulose digestion. This work demonstrates the potential of hydrothermal pretreatment of lignocellulosic substrates as a tool to increase the production of enzymes by filamentous fungi.     

Simplification of the Biomass to Ethanol Conversion Process by Using the Whole Medium of Filamentous Fungi Cultivated Under Solid-State Fermentation

A novel simplified configuration is proposed for the conversion of biomass to ethanol using whole medium enzymatic cocktails (WM) and enzymatic extracts (EE) from different filamentous fungi (Trichoderma reesei, Aspergillus niger, and Aspergillus oryzae) cultivated under solid-state fermentation (SSF) for the hydrolysis of steam-exploded sugarcane bagasse (SESB). The hydrolyzed material derived from the saccharification of SESB using the combinations A. niger WM + T. reesei EE, A. oryzae WM + A. niger EE, and A. niger EE + T. reesei WM resulted in the best biomass conversion yields (66, 65, and 64 % of the theoretical reducing sugar yields, respectively). The best ethanol production (84 % of the theoretical yield) was obtained using the material hydrolyzed by a combination of A. oryzae WM + A. niger EE. The enzymatic conversion of SESB using on-site produced enzymes from the whole SSF cultivation medium, followed by an ethanol production step, is a potential configuration for the biomass to ethanol conversion process. This novel simplified configuration would enable the use of a single reactor system, avoiding the need for additional separation steps.     

Secretome analysis of Trichoderma reesei and Aspergillus niger cultivated by submerged and sequential fermentation processes: Enzyme production for sugarcane bagasse hydrolysis

Cellulases and hemicellulases from Trichoderma reesei and Aspergillus niger have been shown to be powerful enzymes for biomass conversion to sugars, but the production costs are still relatively high for commercial application. The choice of an effective microbial cultivation process employed for enzyme production is important, since it may affect titers and the profile of protein secretion. We used proteomic analysis to characterize the secretome of T. reesei and A. niger cultivated in submerged and sequential fermentation processes. The information gained was key to understand differences in hydrolysis of steam exploded sugarcane bagasse for enzyme cocktails obtained from two different cultivation processes. The sequential process for cultivating A. niger gave xylanase and β-glucosidase activities 3- and 8-fold higher, respectively, than corresponding activities from the submerged process. A greater protein diversity of critical cellulolytic and hemicellulolytic enzymes were also observed through secretome analyses. These results helped to explain the 3-fold higher yield for hydrolysis of non-washed pretreated bagasse when combined T. reesei and A. niger enzyme extracts from sequential fermentation were used in place of enzymes obtained from submerged fermentation. An enzyme loading of 0.7 FPU cellulase activity/g glucan was surprisingly effective when compared to the 5–15 times more enzyme loadings commonly reported for other cellulose hydrolysis studies. Analyses showed that more than 80% consisted of proteins other than cellulases whose role is important to the hydrolysis of a lignocellulose substrate. Our work combined proteomic analyses and enzymology studies to show that sequential and submerged cultivation methods differently influence both titers and secretion profile of key enzymes required for the hydrolysis of sugarcane bagasse. The higher diversity of feruloyl esterases, xylanases and other auxiliary hemicellulolytic enzymes observed in the enzyme mixtures from the sequential fermentation could be one major reason for the more efficient enzyme hydrolysis that results when using the combined secretomes from A. niger and T. reesei.     

13C-metabolic flux ratio and novel carbon path analyses confirmed that Trichoderma reesei uses primarily the respirative pathway also on the preferred carbon source glucose

Background  

The filamentous fungus Trichoderma reesei is an important host organism for industrial enzyme production. It is adapted to nutrient poor environments where it is capable of producing large amounts of hydrolytic enzymes. In its natural environment T. reesei is expected to benefit from high energy yield from utilization of respirative metabolic pathway. However, T. reesei lacks metabolic pathway reconstructions and the utilization of the respirative pathway has not been investigated on the level of in vivo fluxes.     

Recombinant expression, activity screening and functional characterization identifies three novel endo-1,4-β-glucanases that efficiently hydrolyse cellulosic substrates

The hydrolysis of cellulose into fermentable sugars is a costly and rate-limiting step in the production of biofuels from renewable feedstocks. Developing new cellulase systems capable of increased cellulose hydrolysis rates would reduce biofuel production costs. With this in mind, we screened 55 fungal endoglucanases for their abilities to be expressed at high levels by Aspergillus niger and to hydrolyze amorphous cellulose at rates significantly greater than that obtained with TrCel5A, one of the major endoglucanases in the Trichoderma reesei cellulase system. This screen identified three endoglucanases, Aureobasidium pullulans ApCel5A, Gloeophyllum trabeum GtCel12A and Sporotrichum thermophile StCel5A. We determined that A. niger expressed the three endoglucanases at relatively high levels (≥0.3 g/l) and that the hydrolysis rate of ApCel5A and StCel5A with carboxymethylcellulose 4M as substrate was five and two times greater than the T. reesei Cel5A. The ApCel5A, GtCel12A and StCel5A enzymes also demonstrated significant synergy with Cel7A/CbhI, the major exoglucanase in the T. reesei cellulase system. The three endoglucanases characterized in this study are, therefore, promising candidate endoglucanases for developing new cellulase systems with increased rates of cellulose saccharification.     

Production of cellulosic ethanol and enzyme from waste fiber sludge using SSF,recycling of hydrolytic enzymes and yeast,and recombinant cellulase-producing Aspergillus niger

Bioethanol and enzymes were produced from fiber sludges through sequential microbial cultivations. After a first simultaneous saccharification and fermentation (SSF) with yeast, the bioethanol concentrations of sulfate and sulfite fiber sludges were 45.6 and 64.7 g/L, respectively. The second SSF, which included fresh fiber sludges and recycled yeast and enzymes from the first SSF, resulted in ethanol concentrations of 38.3 g/L for sulfate fiber sludge and 24.4 g/L for sulfite fiber sludge. Aspergillus niger carrying the endoglucanase-encoding Cel7B gene of Trichoderma reesei was grown in the spent fiber sludge hydrolysates. The cellulase activities obtained with spent hydrolysates of sulfate and sulfite fiber sludges were 2,700 and 2,900 nkat/mL, respectively. The high cellulase activities produced by using stillage and the significant ethanol concentrations produced in the second SSF suggest that onsite enzyme production and recycling of enzyme are realistic concepts that warrant further attention.     

Enzymatic hydrolosis of isolated and fibre-bound galactoglucomannans from pine-wood and pine kraft pulp

Fibre-bound and isolated galactoglumanans from pine-wood and pine kraft pulp were hydrolysed with purified mannanases from Trichoderma reesei and Bacillus subtilis. The isolated galactoglucomannans from both wood and pulp could be hydrolysed fairly extensively with both enzymes. In addition to mixed oligomers, the fungal mannase produced mannobiose as the main hydrolysis product whereas the bacterial mannanase produced mannobiose, mannotriose and mannotetraose. Both enzymes hydrolysed the native galactoglucomannan in finely ground pinewood, whereas galactoglucomannan in pine kraft pulp was only hydrolysed by the T. ressei mannanase. Thus, mannanases exhibit different specificities on fibre-bound, modified substrates. In spite of the high enzyme loading, the degree of hydrolysis of fibre-bound substrates did not exceed 10% of the theoretical, probably due to poor accessibility of the substrates. Correspondence to: M. Rättö     

Development of a serial bioreactor system for direct ethanol production from starch using<Emphasis Type="Italic">Aspergillus niger</Emphasis> and<Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Aspergillus niger hyphae were found to grow with unliquefied potato starch under aerobic conditions, but did not grow under anaerobic conditions. The raw culture ofA. niger catalyzed saccharification of potato starch to glucose, producing approximately 12 g glucose/L/day/ The extracellular enzyme activity was decreased in proportion to incubation time, and approximately 64% of initial activity was maintained after 3 days. At 50°C,A. niger hyphae growth stopped, while the extracellular enzyme activity peaked. On the basis of theA. niger growth property and enzyme activity, we designed a serial bioreactor system composed of four different reactors. Fungal hyphae were cultivated in reactor I at 30°C, uniquefied starch was saccharified to glycose by a fungal hyphae culture in reactors II and III at 50°C, and glucose was fermented to ethanol bySaccharomyces cerevisiae in reactor IV. The total glucose produced by fungal hyphae in reactor I and saccharification in reactor II was about 42 g/L/day. Ethanol production in reactor IV was approximately 22 g/L/day, which corresponds to about 79% of the theoretical maximum produced from 55 g starch/L/day.