Zymomonas Mobilis 8b Research Articles

Cellulosic ethanol production by consortia of Scheffersomyces stipitis and engineered Zymomonas mobilis

BackgroundAs one of the clean and sustainable energies, lignocellulosic ethanol has achieved much attention around the world. The production of lignocellulosic ethanol does not compete with people for food, while the consumption of ethanol could contribute to the carbon dioxide emission reduction. However, the simultaneous transformation of glucose and xylose to ethanol is one of the key technologies for attaining cost-efficient lignocellulosic ethanol production at an industrial scale. Genetic modification of strains and constructing consortia were two approaches to resolve this issue. Compared with strain improvement, the synergistic interaction of consortia in metabolic pathways should be more useful than using each one separately.ResultsIn this study, the consortia consisting of suspended Scheffersomyces stipitis CICC1960 and Zymomonas mobilis 8b were cultivated to successfully depress carbon catabolite repression (CCR) in artificially simulated 80G40XRM. With this strategy, a 5.52% more xylose consumption and a 6.52% higher ethanol titer were achieved by the consortium, in which the inoculation ratio between S. stipitis and Z. mobilis was 1:3, compared with the Z. mobilis 8b mono-fermentation. Subsequently, one copy of the xylose metabolic genes was inserted into the Z. mobilis 8b genome to construct Z. mobilis FR2, leading to the xylose final-consumption amount and ethanol titer improvement by 15.36% and 6.81%, respectively. Finally, various corn stover hydrolysates with different sugar concentrations (glucose and xylose 60, 90, 120 g/L), were used to evaluate the fermentation performance of the consortium consisting of S. stipitis CICC1960 and Z. mobilis FR2. Fermentation results showed that a 1.56–4.59% higher ethanol titer was achieved by the consortium compared with the Z. mobilis FR2 mono-fermentation, and a 46.12–102.14% higher ethanol titer was observed in the consortium fermentation when compared with the S. stipitis CICC1960 mono-fermentation. Furthermore, qRT-PCR analysis of xylose/glucose transporter and other genes responsible for CCR explained the reason why the initial ratio inoculation of 1:3 in artificially simulated 80G40XRM had the best fermentation performance in the consortium.ConclusionsThe fermentation strategy used in this study, i.e., using a genetically modified consortium, had a superior performance in ethanol production, as compared with the S. stipitis CICC1960 mono-fermentation and the Z. mobilis FR2 mono-fermentation alone. This result showed that this strategy has potential for future lignocellulosic ethanol production.

Understanding the structural characteristics of water-soluble phenolic compounds from four pretreatments of corn stover and their inhibitory effects on enzymatic hydrolysis and fermentation

BackgroundFor bioethanol production from lignocellulosic biomass, phenolics derived from pretreatment have been generally considered as highly inhibitory towards enzymatic hydrolysis and fermentation. As phenolics are produced from lignin degradation during pretreatment, it is likely that the pretreatment will exert a strong impact on the structure of phenolics, resulting in varied levels of inhibition of the bioconversion process. Despite the extensive studies on pretreatment, it remains unclear how pretreatment process affects the properties of generated phenolics and how the inhibitory effect of phenolics from different pretreatment varies on enzymatic hydrolysis and fermentation.ResultsIn this study, the structural properties of phenolic compounds derived from four typical pretreatment [dilute acid (DA), liquid hot water pretreatment (LHW), ammonia fiber expansion (AFEX) and alkaline pretreatment (AL)] were characterized, and their effect on both enzymatic hydrolysis and fermentation were evaluated. The inhibitory effect of phenolics on enzymatic hydrolysis followed the order: AFEX > LHW > DA > AL, while the inhibitory effect of phenolics on Zymomonas mobilis 8b strain fermentation followed the order: AL > LHW > DA > AFEX. Interestingly, this study revealed that phenolics derived from AFEX showed more severe inhibitory effect on enzymatic hydrolysis than those from the other pretreatments at the same phenolics concentrations (note: AFEX produced much less amount of phenolics compared to AL and DA), while they exhibited the lowest inhibitory effect on fermentation. The composition of phenolics from different pretreatments was analyzed and model phenolics were applied to explore the reason for this difference. The results suggested that the amide group in phenolics might account for this difference.ConclusionsPretreatment process greatly affects the properties of generated phenolics and the inhibitory effects of phenolics on enzymatic hydrolysis and fermentation. This study provides new insight for further pretreatment modification and hydrolysate detoxification to minimize phenolics-caused inhibition and enhance the efficiency of enzymatic hydrolysis and fermentation.

Transcriptomic Profiles of Zymomonas mobilis 8b to Furfural Acute and Long-Term Stress in Both Glucose and Xylose Conditions.

Zymomonas mobilis 8b is an ethanologenic bacterium engineered to utilize both glucose and xylose. The impacts of lignocellulosic hydrolyzate inhibitors on the growth of Zymomonas mobilis 8b have been investigated. However, the molecular responses of these inhibitors have not been completely elucidated yet. In this study, molecular responses to furfural were investigated using transcriptomic approaches of both chip-based microarray and a directional mRNA-Seq. Furfural acute shock time-course experiment with 3 g/L furfural supplemented when cells reached exponential phase and stress response experiment in the presence of 2 g/L furfural from the beginning of fermentation were carried out to study the physiological and transcriptional profiles of short-term and long-term effects of furfural on 8b. Furfural negatively affected 8b growth in terms of final biomass and the fermentation time. Transcriptomic studies indicated that the response of 8b to furfural was dynamic and complex, and differences existed between short-term shock and long-term stress responses. However, the gene function categories were similar with most down-regulated genes related to translation and biosynthesis, while the furfural up-regulated genes were mostly related to general stress responses. Several gene candidates have been identified and genetic studies indicated that expression of ZMO0465 and cysteine synthase operon ZMO0003-0006 driven by its native promoter in a shuttle vector enhanced the furfural tolerance of 8b. In addition, the relationship between microarray and mRNA-Seq was compared with good correlations. The directional mRNA-Seq data not only provided the gene expression profiling, but also can be applied for transcriptional architecture improvement to identify and confirm operons, novel transcripts, hypothetical gene functions, transcriptional start sites, and promoters with different strength.

Boosting Ethanol Productivity of Zymomonas mobilis 8b in Enzymatic Hydrolysate of Dilute Acid and Ammonia Pretreated Corn Stover Through Medium Optimization, High Cell Density Fermentation and Cell Recycling.

The presence of toxic degradation products in lignocellulosic hydrolysate typically reduced fermentation rates and xylose consumption rate, resulting in a decreased ethanol productivity. In the present study, Zymomonas mobilis 8b was investigated for high cell density fermentation with cell recycling to improve the ethanol productivity in lignocellulosic hydrolysate. The fermentation performances of Z. mobilis 8b at various conditions were first studied in yeast extract-tryptone medium. It was found that nutrient level was essential for glucose and xylose co-fermentation by Z. mobilis 8b and high cell density fermentation with cell recycling worked well in yeast extract-tryptone medium for 6 rounds fermentation. Z. mobilis 8b was then studied in enzymatic hydrolysates derived from dilute acid (DA) pretreated corn stover (CS) and ammonia pretreated CS for high cell density fermentation with cell recycling. Ethanol productivity obtained was around three times higher compared to traditional fermentation. Ethanol titer and metabolic yield were also enhanced with high cell density fermentation. Z. mobilis 8b cells showed high recyclability in ammonia pretreated CS hydrolysate.

Mixing alkali pretreated and acid pretreated biomass for cellulosic ethanol production featuring reduced chemical use and decreased inhibitory effect

Alkali and acid pretreatment based processes for lignocellulosic biofuel production typically consume substantial amount of alkali and acid. In addition, alkali pretreatment generates compounds with high toxicity rendering washing/detoxification inevitable. In this work, alkali (AL) and dilute acid (DA) pretreatment based processes were compared side by side for ethanol production from corn stover (CS) using commercial enzymes and Zymomonas mobilis 8b strain. Next, AL and DA pretreated CS slurries (without removing liquid stream) were mixed to neutralize for enzymatic hydrolysis and ethanol fermentation. The digestibility and fermentability of the mixed pretreated biomass were systematically studied and compared to unmixed pretreated biomass. Results showed that mixed biomass process saved 40–50% alkali/acid use and enhanced fermentability compared to AL-CS. This work suggests that it could be beneficial to have both alkali and acid pretreatments operating in a biorefinery, which could accommodate various feedstocks.

Diverse lignocellulosic feedstocks can achieve high field‐scale ethanol yields while providing flexibility for the biorefinery and landscape‐level environmental benefits

AbstractIncreasing the diversity of lignocellulosic feedstocks accepted by a regional biorefinery has the potential to improve the environmental footprint of the facility; harvest, storage, and transportation logistics; and biorefinery economics. However, feedstocks can vary widely in terms of their biomass yields and quality characteristics (chemical composition, moisture content, etc.). To investigate how the diversity of potential biofuel cropping systems and feedstock supply might affect process and field‐scale ethanol yields, we processed and experimentally quantified ethanol production from five different herbaceous feedstocks: two annuals (corn stover and energy sorghum) and three perennials (switchgrass, miscanthus, and mixed prairie). The feedstocks were pretreated using ammonia fiber expansion (AFEX), hydrolyzed at high solid loading (~17%–20% solids, depending on the feedstock), and fermented separately using microbes engineered to utilize xylose: yeast (Saccharomyces cerevisiaeY128) or bacteria (Zymomonas mobilis8b). The field‐scale ethanol yield from each feedstock was dependent on biomass quality and cropping system productivity; however, biomass yield had a greater influence on the ethanol yield for low‐productivity crops, while biomass quality was the main driver for ethanol yields from high‐yielding crops. The process ethanol yield showed similar variability across years and feedstocks. A low process yield for corn stover was determined to result from inhibition of xylose utilization by unusually elevated levels of hydroxycinnamates (p‐coumaric and ferulic acids) in the untreated biomass and their acid and amide derivatives in the resulting hydrolyzate. This finding highlights the need to better understand factors that influence process ethanol yield and biomass quality. Ultimately we provide evidence that most feedstocks fall within a similar range of process ethanol yield, particularly for the more resistant strain Z. mobilis8b. This supports the claim that the refinery can successfully diversify its feedstock supply, enabling many social and environmental benefits that can accrue due to landscape diversification.

Accounting for all sugars produced during integrated production of ethanol from lignocellulosic biomass

Accurate mass balance and conversion data from integrated operation is needed to fully elucidate the economics of biofuel production processes. This study explored integrated conversion of corn stover to ethanol and highlights techniques for accurate yield calculations. Acid pretreated corn stover (PCS) produced in a pilot-scale reactor was enzymatically hydrolyzed and the resulting sugars were fermented to ethanol by the glucose–xylose fermenting bacteria, Zymomonas mobilis 8b. The calculations presented here account for high solids operation and oligomeric sugars produced during pretreatment, enzymatic hydrolysis, and fermentation, which, if not accounted for, leads to overestimating ethanol yields. The calculations are illustrated for enzymatic hydrolysis and fermentation of PCS at 17.5% and 20.0% total solids achieving 80.1% and 77.9% conversion of cellulose and xylan to ethanol and ethanol titers of 63g/L and 69g/L, respectively. These procedures will be employed in the future and the resulting information used for techno-economic analysis.

Scaling up and benchmarking of ethanol production from pelletized pilot scale AFEX treated corn stover using Zymomonas mobilis 8b

ABSTRACTThis report outlines the recent scale-up of AFEX pretreatment from the laboratory to pilot scale. Sugar yields were improved by 19 and 15% for glucose and xylose, respectively. Further improvement was achieved when scaling up the hydrolysis and fermentation of AFEX-treated corn stover to 2500 L working volume. Benchmarking was performed using CTec 3 and HTec 3 enzymes along with Zymomonas mobilis 8b. Subsequently, the seed train was modified to use a hydrolysate-based medium as a replacement for pure sugars and nutrients. Fermentation performance was comparable following this change. Economic analysis showed a 19% reduction in MESP for the new process over the previous benchmark process.

Insights into acetate toxicity in Zymomonas mobilis 8b using different substrates.

BackgroundLignocellulosic biomass is a promising renewable feedstock for biofuel production. Acetate is one of the major inhibitors liberated from hemicelluloses during hydrolysis. An understanding of the toxic effects of acetate on the fermentation microorganism and the efficient utilization of mixed sugars of glucose and xylose in the presence of hydrolysate inhibitors is crucial for economic biofuel production.ResultsA new microarray was designed including both coding sequences and intergenic regions to investigate the acetate stress responses of Zymomonas mobilis 8b when using single carbon sources of glucose or xylose, or mixed sugars of both glucose and xylose. With the supplementation of exogenous acetate, 8b can utilize all the glucose with a similar ethanol yield, although the growth, final biomass, and ethanol production rate were reduced. However, xylose utilization was inhibited in both media containing xylose or a mixed sugar of glucose and xylose, although the performance of 8b was better in mixed sugar than xylose-only media. The presence of acetate caused genes related to biosynthesis, the flagellar system, and glycolysis to be downregulated, and genes related to stress responses and energy metabolism to be upregulated. Unexpectedly, xylose seems to pose more stress on 8b, recruiting more genes for xylose utilization, than does acetate. Several gene candidates based on transcriptome results were selected for genetic manipulation, and a TonB-dependent receptor knockout mutant was confirmed to have a slight advantage regarding acetate tolerance.ConclusionsOur results indicate Z. mobilis utilized a different mechanism for xylose utilization, with an even more severe impact on Z. mobilis than that caused by acetate treatment. Our study also suggests redox imbalance caused by stressful conditions may trigger a metabolic reaction leading to the accumulation of toxic intermediates such as xylitol, but Z. mobilis manages its carbon and energy metabolism through the control of individual reactions to mitigate the stressful conditions. We have thus provided extensive transcriptomic datasets and gained insights into the molecular responses of Z. mobilis to the inhibitor acetate when grown in different sugar sources, which will facilitate future metabolic modeling studies and strain improvement efforts for better xylose utilization and acetate tolerance.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-014-0140-8) contains supplementary material, which is available to authorized users.

Studying the rapid bioconversion of lignocellulosic sugars into ethanol using high cell density fermentations with cell recycle.

BackgroundThe Rapid Bioconversion with Integrated recycle Technology (RaBIT) process reduces capital costs, processing times, and biocatalyst cost for biochemical conversion of cellulosic biomass to biofuels by reducing total bioprocessing time (enzymatic hydrolysis plus fermentation) to 48 h, increasing biofuel productivity (g/L/h) twofold, and recycling biocatalysts (enzymes and microbes) to the next cycle. To achieve these results, RaBIT utilizes 24-h high cell density fermentations along with cell recycling to solve the slow/incomplete xylose fermentation issue, which is critical for lignocellulosic biofuel fermentations. Previous studies utilizing similar fermentation conditions showed a decrease in xylose consumption when recycling cells into the next fermentation cycle. Eliminating this decrease is critical for RaBIT process effectiveness for high cycle counts.ResultsNine different engineered microbial strains (including Saccharomyces cerevisiae strains, Scheffersomyces (Pichia) stipitis strains, Zymomonas mobilis 8b, and Escherichia coli KO11) were tested under RaBIT platform fermentations to determine their suitability for this platform. Fermentation conditions were then optimized for S. cerevisiae GLBRCY128. Three different nutrient sources (corn steep liquor, yeast extract, and wheat germ) were evaluated to improve xylose consumption by recycled cells. Capacitance readings were used to accurately measure viable cell mass profiles over five cycles.ConclusionThe results showed that not all strains are capable of effectively performing the RaBIT process. Acceptable performance is largely correlated to the specific xylose consumption rate. Corn steep liquor was found to reduce the deleterious impacts of cell recycle and improve specific xylose consumption rates. The viable cell mass profiles indicated that reduction in specific xylose consumption rate, not a drop in viable cell mass, was the main cause for decreasing xylose consumption.

Saccharification and Fermentation of Waste Sweet Potato for Bioethanol Production

AbstractWe have investigated the relative performance of the two enzymes, α‐amylase and amyloglucosidase, and two microbial strains, Zymomonas mobilis AX101 and 8b, for saccharification and fermentation of sweet potato. Modified Michaelis–Menten and Monod‐type mathematical models were developed and verified with experimental results for saccharification using amyloglucosidase and fermentation using Z. mobilis 8b, respectively. The results showed that 91.5% of the starch and sucrose were converted to glucose and fructose using amyloglucosidase at pH 3.5 and 55C. The Z. mobilis 8b was able to convert more than 90% of the total sugars into ethanol within 18 h with 87.2% of the theoretical yield and 49.07 g/L final concentration of ethanol. A mass balance and cost analysis show that commercial production of ethanol from sweet potato is limited by the feedstock cost.Practical ApplicationsThis study shows that sweet potatoes that are unmarketable, small in size, bruised, cut or damaged otherwise from the harvesting process, supplied by a local farmer in the Central Texas area, can be used to produce bioethanol. Normally, farmers do not collect the damaged sweet potatoes because there is no demand for those in the market. This is supported by the fact that 20% of the total sweet potatoes cultivated are left in the ground due to damage while harvesting with plows. If collected and used for ethanol production, local farms can earn additional revenue.

Inhibition of growth of Zymomonas mobilis by model compounds found in lignocellulosic hydrolysates

BackgroundDuring the pretreatment of biomass feedstocks and subsequent conditioning prior to saccharification, many toxic compounds are produced or introduced which inhibit microbial growth and in many cases, production of ethanol. An understanding of the toxic effects of compounds found in hydrolysate is critical to improving sugar utilization and ethanol yields in the fermentation process. In this study, we established a useful tool for surveying hydrolysate toxicity by measuring growth rates in the presence of toxic compounds, and examined the effects of selected model inhibitors of aldehydes, organic and inorganic acids (along with various cations), and alcohols on growth of Zymomonas mobilis 8b (a ZM4 derivative) using glucose or xylose as the carbon source.ResultsToxicity strongly correlated to hydrophobicity in Z. mobilis, which has been observed in Escherichia coli and Saccharomyces cerevisiae for aldehydes and with some exceptions, organic acids. We observed Z. mobilis 8b to be more tolerant to organic acids than previously reported, although the carbon source and growth conditions play a role in tolerance. Growth in xylose was profoundly inhibited by monocarboxylic organic acids compared to growth in glucose, whereas dicarboxylic acids demonstrated little or no effects on growth rate in either substrate. Furthermore, cations can be ranked in order of their toxicity, Ca++ > > Na+ > NH4+ > K+. HMF (5-hydroxymethylfurfural), furfural and acetate, which were observed to contribute to inhibition of Z. mobilis growth in dilute acid pretreated corn stover hydrolysate, do not interact in a synergistic manner in combination. We provide further evidence that Z. mobilis 8b is capable of converting the aldehydes furfural, vanillin, 4-hydroxybenzaldehyde and to some extent syringaldehyde to their alcohol forms (furfuryl, vanillyl, 4-hydroxybenzyl and syringyl alcohol) during fermentation.ConclusionsSeveral key findings in this report provide a mechanism for predicting toxic contributions of inhibitory components of hydrolysate and provide guidance for potential process development, along with potential future strain improvement and tolerance strategies.

Fermentation of Reactive-Membrane-Extracted and Ammonium-Hydroxide-Conditioned Dilute-Acid-Pretreated Corn Stover

Acid-pretreated biomass contains various compounds (acetic acid, etc.) that are inhibitory to fermentative microorganisms. Removing or deactivating these compounds using detoxification methods such as overliming or ammonium hydroxide conditioning (AHC) improves sugar-to-ethanol yields. In this study, we treated the liquor fraction of dilute-acid-pretreated corn stover using AHC and a new reactive membrane extraction technique, both separately and in combination, and then the sugars in the treated liquors were fermented to ethanol with the glucose-xylose-fermenting bacterium, Zymomonas mobilis 8b. We performed reactive extraction with mixtures of octanol/Alamine 336 or oleyl alcohol/Alamine 336. The best ethanol yields and rates were achieved for oleyl alcohol-extracted hydrolysates followed by AHC hydrolysates, while octanol-extracted hydrolysates were unfermentable because highly toxic octanol was found in the hydrolysate. Adding olive oil significantly improved yields for octanol-extracted hydrolysate. Additional work is underway to determine if this technology is a cost-effective alternative to traditional hydrolysate conditioning processes.

Conditioning of dilute-acid pretreated corn stover hydrolysate liquors by treatment with lime or ammonium hydroxide to improve conversion of sugars to ethanol

Dilute-acid pretreatment of lignocellulosic biomass enhances the ability of enzymes to hydrolyze cellulose to glucose, but produces many toxic compounds that inhibit fermentation of sugars to ethanol. The objective of this study was to compare the effectiveness of treating hydrolysate liquor with Ca(OH)2 and NH4OH for improving ethanol yields. Corn stover was pretreated in a pilot-scale reactor and then the liquor fraction (hydrolysate) was extracted and treated with various amounts of Ca(OH)2 or NH4OH at several temperatures. Glucose and xylose in the treated liquor were fermented to ethanol using a glucose-xylose fermenting bacteria, Zymomonas mobilis 8b. Sugar losses up to 10% occurred during treatment with Ca(OH)2, but these losses were two to fourfold lower with NH4OH treatment. Ethanol yields for NH4OH-treated hydrolysate were 33% greater than those achieved in Ca(OH)2-treated hydrolysate and pH adjustment to either 6.0 or 8.5 with NH4OH prior to fermentation produced equivalent ethanol yields.

Ethanol production from paper sludge by simultaneous saccharification and co‐fermentation using recombinant xylose‐fermenting microorganisms

Simultaneous saccharification and co-fermentation (SSCF) of waste paper sludge to ethanol was investigated using two recombinant xylose-fermenting microbes: Zymomonas mobilis 8b and Saccharomyces cerevisiae RWB222. S. cerevisiae RWB222 produced over 40 g/L ethanol with a yield of 0.39 g ethanol/g carbohydrate on paper sludge at 37 degrees C, while similar titers and yields were achieved by Z. mobilis 8b at 30 degrees C. Both S. cerevisiae RWB222 and Z. mobilis 8b exhibited decreasing cell viability at 37 degrees C when producing over 40 g/L ethanol. A high ethanol concentration can account for S. cerevisiae RWB222 viability loss, but ethanol concentration was not the only factor influencing Z. mobilis 8b viability loss at 37 degrees C. Over 3 g/L residual glucose was observed at the end of paper sludge SSCF by Z. mobilis 8b, and a statistical analysis revealed that a high calcium concentration originating from paper sludge, a high ethanol concentration, and a high temperature were the key interactive factors resulting in glucose accumulation. The highest ethanol yields were achieved by SSCF of paper sludge with S. cerevisiae RWB222 at 37 degrees C and Z. mobilis 8b at 30 degrees C. With good sugar consumption at 37 degrees C, S. cerevisiae RWB222 was able to gain an improvement in the polysaccharide to sugar yield compared to that at 30 degrees C, whereas Z. mobilis 8b at 30 degrees C had a lower polysaccharide to sugar yield, but a higher sugar to ethanol yield than S. cerevisiae. Both organisms under optimal conditions achieved a 19% higher overall conversion of paper sludge to ethanol than the non-xylose utilizing S. cerevisiae D5A at its optimal process temperature of 37 degrees C.

Impact of recycling stillage on conversion of dilute sulfuric acid pretreated corn stover to ethanol

Both the current corn starch to ethanol industry and the emerging lignocellulosic biofuels industry view recycling of spent fermentation broth or stillage as a method to reduce fresh water use. The objective of this study was to understand the impact of recycling stillage on conversion of corn stover to ethanol. Sugars in a dilute-acid pretreated corn stover hydrolysate were fermented to ethanol by the glucose-xylose fermenting bacteria Zymomonas mobilis 8b. Three serial fermentations were performed at two different initial sugar concentrations using either 10% or 25% of the stillage as makeup water for the next fermentation in the series. Serial fermentations were performed to achieve near steady state concentration of inhibitors and other compounds in the corn stover hydrolysate. Little impact on ethanol yields was seen at sugar concentrations equivalent to pretreated corn stover slurry at 15% (w/w) with 10% recycle of the stillage. However, ethanol yields became progressively poorer as the sugar concentration increased and fraction of the stillage recycled increased. At an equivalent corn stover slurry concentration of 20% with 25% recycled stillage the ethanol yield was only 5%. For this microorganism with dilute-acid pretreated corn stover, recycling a large fraction of the stillage had a significant negative impact on fermentation performance. Although this finding is of concern for biochemical-based lignocellulose conversion processes, other microorganism/pretreatment technology combinations will likely perform differently.

Development of a high-throughput method to evaluate the impact of inhibitory compounds from lignocellulosic hydrolysates on the growth of Zymomonas mobilis

Overcoming the effects of hydrolysate toxicity towards ethanologens is a key technical barrier in the biochemical conversion process for biomass feedstocks to ethanol. Despite its importance, the complexity of the hydrolysate toxicity phenomena and the lack of systematic studies, analysis and tools surrounding this issue have blocked a full understanding of relationships involving toxic compounds in hydrolysates and their effects on ethanologen growth and fermentation. In this study, we developed a quantitative, high-throughput biological growth assay using an automated turbidometer to obtain detailed inhibitory kinetics for individual compounds present in lignocellulosic biomass hydrolysate. Information about prolonged lag time and final cell densities can also be obtained. The effects of furfural, hydroxymethylfurfural (HMF), acetate and ethanol on growth rate and final cell densities of Zymomonas mobilis 8b on glucose are presented. This method was also shown to be of value in toxicity studies of hydrolysate itself, despite the highly colored nature of this material. Using this approach, we can generate comprehensive inhibitory profiles with many individual compounds and develop models that predict and examine toxic effects in the complex mixture of hydrolysates, leading to the development of improved pretreatment and conditioning processes as well as fermentation organisms.

Formation of ethyl β-xylopyranoside during simultaneous saccharification and co-fermentation of paper sludge

An unexpected product was detected during simultaneous saccharification and co-fermentation (SSCF) of paper sludge using added commercial cellulase (Spezyme CP) by Saccharomyces cerevisiae RWB222, S. cerevisiae D5A, and Zymomonas mobilis 8b. Based on glycosyl composition analysis, linkage analysis and NMR analysis, the compound was identified as ethyl β-xylopyranoside (EXP). The carbon mass balance analysis showed up to 25% of xylan originally present in paper sludge was converted to EXP. EXP formation was found in simultaneous saccharification of beech wood xylan as well, and later proved to be produced by the Trichoderma reesei derived cellulase and hemicellulase mixture (Spezyme CP) during the course of xylan hydrolysis in the presence of ethanol, and its production increased with an increased concentration of ethanol, xylan, and T. reesei enzyme. Similar condensation reactions were also observed with other alcohols. These alcoholysis reactions were found to be reversible. Thermoanaerobacterium saccharolyticum was found to be able to degrade EXP.

Performance of a newly developed integrant of Zymomonas mobilis for ethanol production on corn stover hydrolysate.

Efficient conversion of lignocellulosic biomass requires biocatalysts able to tolerate inhibitors produced by many pretreatment processes. Recombinant Zymomonas mobilis 8b, a recently developed integrant of Zymomonas mobilis 31821(pZB5), tolerated acetic acid up to 16 g l(-1) and achieved 82%-87% (w/w) ethanol yields from pure glucose/xylose solutions at pH 6 and temperatures of 30 degrees C and 37 degrees C. An ethanol yield of 85% (w/w) was achieved on glucose/xylose from hydrolysate produced by dilute sulfuric acid pretreatment of corn stover after an overliming' process was used to improve hydrolysate fermentability.