Synergistic co-utilization of biomass-derived sugars enhances aromatic amino acid production by engineered Escherichia coli.

Efficient co-utilization of mixed sugar feedstocks remains a biomanufacturing challenge, thus motivating ongoing efforts to engineer microbes for improved conversion of glucose-xylose mixtures. This study focuses on enhancing phenylalanine production by engineering Escherichia coli to efficiently co-utilize glucose and xylose. Flux balance analysis identified E4P flux as a bottleneck which could be alleviated by increasing the xylose-to-glucose flux ratio. A mutant copy of the xylose-specific activator (XylR) was then introduced into the phenylalanine-overproducing E. coli NST74, which relieved carbon catabolite repression and enabled efficient glucose-xylose co-utilization. Carbon contribution analysis through 13 C-fingerprinting showed a higher preference for xylose in the engineered strain (NST74X), suggesting superior catabolism of xylose relative to glucose. As a result, NST74X produced 1.76 g/L phenylalanine from a model glucose-xylose mixture; a threefold increase over NST74. Then, using biomass-derived sugars, NST74X produced 1.2 g/L phenylalanine, representing a 1.9-fold increase over NST74. Notably, and consistent with the carbon contribution analysis, the xylR* mutation resulted in a fourfold greater maximum rate of xylose consumption without significantly impeding the maximum rate of total sugar consumption (0.87 vs. 0.70 g/L-h). This study presents a novel strategy for enhancing phenylalanine production through the co-utilization of glucose and xylose in aerobic E. coli cultures, and highlights the potential synergistic benefits associated with using substrate mixtures over single substrates when targeting specific products.

A Tempered Particle Filter to Enhance the Assimilation of SAR-Derived Flood Extent Maps Into Flood Forecasting Models.

Data assimilation (DA) is a powerful tool to optimally combine uncertain model simulations and observations. Among DA techniques, the particle filter (PF) has gained attention for its capacity to deal with nonlinear systems and for its relaxation of the Gaussian assumption. However, the PF may suffer from degeneracy and sample impoverishment. In this study, we propose an innovative approach, based on a tempered particle filter (TPF), aiming at mitigating PFs issues, thus extending over time the assimilation benefits. Probabilistic flood maps derived from synthetic aperture radar data are assimilated into a flood forecasting model through an iterative process including a particle mutation in order to keep diversity within the ensemble. Results show an improvement of the model forecasts accuracy, with respect to the Open Loop: on average the root mean square error (RMSE) of water levels decrease by 80% at the assimilation time and by 60% 2 days after the assimilation. A comparison with the Sequential Importance Sampling (SIS) is carried out showing that although SIS performances are generally comparable to the TPF ones at the assimilation time, they tend to decrease more quickly. For instance, on average TPF-based RMSE are 20% lower compared to the SIS-based ones 2 days after the assimilation. The application of the TPF determines higher critical success index values compared to the SIS. On average the increase in performances lasts for almost 3 days after the assimilation. Our study provides evidence that the application of the variant of the TPF enables more persistent benefits compared to the SIS.

Metabolic engineering of a stable haploid strain derived from lignocellulosic inhibitor tolerant Saccharomyces cerevisiae natural isolate YB-2625.

BACKGROUND: Significant genetic diversity exists across Saccharomyces strains. Natural isolates and domesticated brewery and industrial strains are typically more robust than laboratory strains when challenged with inhibitory lignocellulosic hydrolysates. These strains also contain genes that are not present in lab strains and likely contribute to their superior inhibitor tolerance. However, many of these strains have poor sporulation efficiencies and low spore viability making subsequent gene analysis, further metabolic engineering, and genomic analyses of the strains challenging. This work aimed to develop an inhibitor tolerant haploid with stable mating type from S. cerevisiae YB-2625, which was originally isolated from bagasse. RESULTS: Haploid spores isolated from four tetrads from strain YB-2625 were tested for tolerance to furfural and HMF. Due to natural mutations present in the HO-endonuclease, all haploid strains maintained a stable mating type. One of the haploids, YRH1946, did not flocculate and showed enhanced tolerance to furfural and HMF. The tolerant haploid strain was further engineered for xylose fermentation by integration of the genes for xylose metabolism at two separate genomic locations (ho∆ and pho13∆). In fermentations supplemented with inhibitors from acid hydrolyzed corn stover, the engineered haploid strain derived from YB-2625 was able to ferment all of the glucose and 19% of the xylose, whereas the engineered lab strains performed poorly in fermentations. CONCLUSIONS: Understanding the molecular mechanisms of inhibitor tolerance will aid in developing strains with improved growth and fermentation performance using biomass-derived sugars. The inhibitor tolerant, xylose fermenting, haploid strain described in this work has potential to serve as a platform strain for identifying pathways required for inhibitor tolerance, and for metabolic engineering to produce fuels and chemicals from undiluted lignocellulosic hydrolysates.

Chemotaxis to furan compounds by furan-degrading Pseudomonas strains.

Two Pseudomonas strains known to utilize furan derivatives were shown to respond chemotactically to furfural, 5-hydroxymethylfurfural, furfuryl alcohol, and 2-furoic acid. In addition, a LysR-family regulatory protein known to regulate furan metabolic genes was found to be involved in regulating the chemotactic response.

Hydrothermal pretreatment of sugarcane bagasse using response surface methodology improves digestibility and ethanol production by SSF.

Sugarcane bagasse was characterized as a feedstock for the production of ethanol using hydrothermal pretreatment. Reaction temperature and time were varied between 160 and 200°C and 5-20 min, respectively, using a response surface experimental design. The liquid fraction was analyzed for soluble carbohydrates and furan aldehydes. The solid fraction was analyzed for structural carbohydrates and Klason lignin. Pretreatment conditions were evaluated based on enzymatic extraction of glucose and xylose and conversion to ethanol using a simultaneous saccharification and fermentation scheme. SSF experiments were conducted with the washed pretreated biomass. The severity of the pretreatment should be sufficient to drive enzymatic digestion and ethanol yields, however, sugars losses and especially sugar conversion into furans needs to be minimized. As expected, furfural production increased with pretreatment severity and specifically xylose release. However, provided that the severity was kept below a general severity factor of 4.0, production of furfural was below an inhibitory concentration and carbohydrate contents were preserved in the pretreated whole hydrolysate. There were significant interactions between time and temperature for all the responses except cellulose digestion. The models were highly predictive for cellulose digestibility (R (2) = 0.8861) and for ethanol production (R (2) = 0.9581), but less so for xylose extraction. Both cellulose digestion and ethanol production increased with severity, however, high levels of furfural generated under more severe pretreatment conditions favor lower severity pretreatments. The optimal pretreatment condition that gave the highest conversion yield of ethanol, while minimizing furfural production, was judged to be 190°C and 17.2 min. The whole hydrolysate was also converted to ethanol using SSF. To reduce the concentration of inhibitors, the liquid fraction was conditioned prior to fermentation by removing inhibitory chemicals using the fungus Coniochaeta ligniaria.

Increased expression of the fluorescent reporter protein ymNeonGreen in Saccharomyces cerevisiae by reducing RNA secondary structure near the start codon.

Expression of a new fluorescent reporter protein called mNeonGreen, that is not based on the jellyfish green fluorescent protein (GFP) sequence, shows increased brightness and folding speed compared to enhanced GFP. However, in vivo brightness of mNeonGreen and its yeast-optimized variant ymNeonGreen in S. cerevisiae is lower than expected, limiting the use of this high quantum yield, fast-folding reporter in budding yeast. This study shows that secondary RNA structure near the start codon in the ymNeonGreen ORF inhibits expression in S. cerevisiae. Removing secondary structure, without altering the ymNeonGreen protein sequence, led to a 2 and 4-fold increase in fluorescence when expressed in S. cerevisiae and E. coli, respectively. In S. cerevisiae, increased fluorescence was seen with strong and weak promoters and led to higher transcript levels suggesting greater transcript stability and improved expression in the absence of stable secondary RNA structure near the start codon.

Transformation and electrophoretic karyotyping of Coniochaeta ligniaria NRRL30616.

Coniochaeta ligniaria NRRL30616 is an ascomycete that grows with yeast-like appearance in liquid culture. The strain has potential utility for conversion of fibrous biomass to fuels or chemicals. Furans and other inhibitory compounds in lignocellulosic biomass are metabolized by NRRL30616, facilitating subsequent microbial fermentation of biomass sugars. This study undertook initial characterization of the genetic system of C. ligniaria NRRL30616. Transformation using hygromycin as a dominant selectable marker was achieved using protoplasts generated by incubating cells in 1% (v/v) ß-mercaptoethanol, followed by cell wall-digesting enzymes. Thirteen chromosomes with an estimated total size of 30.1 Mb were detected in C. ligniaria. The GC content of chromosomal DNA and of coding regions from cDNA sequences were 49.2 and 51.9%, respectively. This study is the first report of genome size, electrophoretic karyotype, and transformation system for a member of the Coniochaetales.

Saccharomyces cerevisiae engineered for xylose metabolism requires gluconeogenesis and the oxidative branch of the pentose phosphate pathway for aerobic xylose assimilation.

Saccharomyces strains engineered to ferment xylose using Scheffersomyces stipitis xylose reductase (XR) and xylitol dehydrogenase (XDH) genes appear to be limited by metabolic imbalances, due to differing cofactor specificities of XR and XDH. The S. stipitis XR, which uses both NADH and NADPH, is hypothesized to reduce the cofactor imbalance, allowing xylose fermentation in this yeast. However, unadapted S. cerevisiae strains expressing this XR grow poorly on xylose, suggesting that metabolism is still imbalanced, even under aerobic conditions. In this study, we investigated the possible reasons for this imbalance by deleting genes required for NADPH production and gluconeogenesis in S. cerevisiae. S. cerevisiae cells expressing the XR-XDH, but not a xylose isomerase, pathway required the oxidative branch of the pentose phosphate pathway (PPP) and gluconeogenic production of glucose-6-P for xylose assimilation. The requirement for generating glucose-6-P from xylose was also shown for Kluyveromyces lactis. When grown in xylose medium, both K. lactis and S. stipitis showed increases in enzyme activity required for producing glucose-6-P. Thus, natural xylose-assimilating yeast respond to xylose, in part, by upregulating enzymes required for recycling xylose back to glucose-6-P for the production of NADPH via the oxidative branch of the PPP. Finally, we show that induction of these enzymes correlated with increased tolerance to the NADPH-depleting compound diamide and the fermentation inhibitors furfural and hydroxymethyl furfural; S. cerevisiae was not able to increase enzyme activity for glucose-6-P production when grown in xylose medium and was more sensitive to these inhibitors in xylose medium compared to glucose.

Comparison of separate hydrolysis and fermentation and simultaneous saccharification and fermentation processes for ethanol production from wheat straw by recombinant Escherichia coli strain FBR5.

Ethanol production by recombinant Escherichia coli strain FBR5 from dilute acid pretreated wheat straw (WS) by separate hydrolysis and fermentation (SHF) and simultaneous saccharification and fermentation (SSF) was studied. The yield of total sugars from dilute acid (0.5% H(2)SO(4)) pretreated (160 °C, 10 min) and enzymatically saccharified (pH 5.0, 45 °C, 72 h) WS (86 g/l) was 50.0 ± 1.4 g/l. The hydrolyzate contained 1,184 ± 19 mg furfural and 161 ± 1 mg hydroxymethyl furfural per liter. The recombinant E. coli FBR5 could not grow at all at pH controlled at 4.5 to 6.5 in the non-abated wheat straw hydrolyzate (WSH) at 35 °C. However, it produced 21.9 ± 0.3 g ethanol from non-abated WSH (total sugars, 44.1 ± 0.4 g/l) in 90 h including the lag time of 24 h at controlled pH 7.0 and 35 °C. The bioabatement of WS was performed by growing Coniochaeta ligniaria NRRL 30616 in the liquid portion of the pretreated WS aerobically at pH 6.5 and 30 °C for 15 h. The bacterium produced 21.6 ± 0.5 g ethanol per liter in 40 h from the bioabated enzymatically saccharified WSH (total sugars, 44.1 ± 0.4 g) at pH 6.0. It produced 24.9 ± 0.3 g ethanol in 96 h and 26.7 ± 0.0 g ethanol in 72 h per liter from bioabated WSH by batch SSF and fed-batch SSF, respectively. SSF offered a distinct advantage over SHF with respect to reducing total time required to produce ethanol from the bioabated WS. Also, fed-batch SSF performed better than the batch SSF with respect to shortening the time requirement and increase in ethanol yield.

Impact of stress-response related transcription factor overexpression on lignocellulosic inhibitor tolerance of Saccharomyces cerevisiae environmental isolates.

Numerous transcription factor genes associated with stress response are upregulated in Saccharomyces cerevisiae grown in the presence of inhibitors that result from pretreatment processes to unlock simple sugars from biomass. To determine if overexpression of transcription factors could improve inhibitor tolerance in robust S. cerevisiae environmental isolates as has been demonstrated in S. cerevisiae haploid laboratory strains, transcription factors were overexpressed at three different expression levels in three S. cerevisiae environmental isolates. Overexpression of the YAP1 transcription factor in these isolates did not lead to increased growth rate or reduced lag in growth, and in some cases was detrimental, when grown in the presence of either lignocellulosic hydrolysates or furfural and 5-hydroxymethyl furfural individually. The expressed Yap1p localized correctly and the expression construct improved inhibitor tolerance of a laboratory strain as previously reported, indicating that lack of improvement in the environmental isolates was due to factors other than nonfunctional expression constructs or mis-folded protein. Additional stress-related transcription factors, MSN2, MSN4, HSF1, PDR1, and RPN4, were also overexpressed at three different expression levels and all failed to improve inhibitor tolerance. Transcription factor overexpression alone is unlikely to be a viable route toward increased inhibitor tolerance of robust environmental S. cerevisiae strains.

Defining the eco-enzymological role of the fungal strain Coniochaeta sp. 2T2.1 in a tripartite lignocellulolytic microbial consortium.

Coniochaeta species are versatile ascomycetes that have great capacity to deconstruct lignocellulose. Here, we explore the transcriptome of Coniochaeta sp. strain 2T2.1 from wheat straw-driven cultures with the fungus growing alone or as a member of a synthetic microbial consortium with Sphingobacterium multivorum w15 and Citrobacter freundii so4. The differential expression profiles of carbohydrate-active enzymes indicated an onset of (hemi)cellulose degradation by 2T2.1 during the initial 24 hours of incubation. Within the tripartite consortium, 63 transcripts of strain 2T2.1 were differentially expressed at this time point. The presence of the two bacteria significantly upregulated the expression of one galactose oxidase, one GH79-like enzyme, one multidrug transporter, one laccase-like protein (AA1 family) and two bilirubin oxidases, suggesting that inter-kingdom interactions (e.g. amensalism) take place within this microbial consortium. Overexpression of multicopper oxidases indicated that strain 2T2.1 may be involved in lignin depolymerization (a trait of enzymatic synergism), while S. multivorum and C. freundii have the metabolic potential to deconstruct arabinoxylan. Under the conditions applied, 2T2.1 appears to be a better degrader of wheat straw when the two bacteria are absent. This conclusion is supported by the observed suppression of its (hemi)cellulolytic arsenal and lower degradation percentages within the microbial consortium.

c-Myb is required for neural progenitor cell proliferation and maintenance of the neural stem cell niche in adult brain.

Ongoing production of neurons in adult brain is restricted to specialized neurogenic niches. Deregulated expression of genes controlling homeostasis of neural progenitor cell division and/or their microenvironment underpins a spectrum of brain pathologies. Using conditional gene deletion, we show that the proto-oncogene c-myb regulates neural progenitor cell proliferation and maintains ependymal cell integrity in mice. These two cellular compartments constitute the neurogenic niche in the adult brain. Brains devoid of c-Myb showed enlarged ventricular spaces, ependymal cell abnormalities, and reduced neurogenesis. Neural progenitor cells lacking c-Myb showed a reduced intrinsic proliferative capacity and reduction of Sox-2 and Pax-6 expression. These data point to an important role for c-Myb in the neurogenic niche of the adult brain.

Genetic transformation of Coniochaeta sp. 2T2.1, key fungal member of a lignocellulose-degrading microbial consortium.

Coniochaeta sp. strain 2T2.1 is a key member of a microbial consortium that degrades lignocellulosic biomass. Due to its ecological niche and ability to also grow in pure culture on wheat straw, protocols for transformation and antibiotic selection of the strain were established. Hygromycin was found to be a reliable selectable transformation marker, and the mammalian codon-optimized green fluorescent protein was expressed and used to visualize fluorescence in transformed cells of strain 2T2.1.

Development and characterization of vectors for tunable expression of both xylose-regulated and constitutive gene expression in Saccharomyces yeasts.

Synthetic hybrid promoters for xylose-regulated gene expression in the yeast Saccharomyces cerevisiae have recently been developed. However, the narrow range of expression level from these new hybrid promoters limits their utility for pathway optimization in engineered strains. To expand the range of xylose-regulated gene expression, a series of expression vectors was created using a xylose derepressible promoter (PXYL) and varied termination regions from several S. cerevisiae genes. The new set of vectors showed a 26-fold range of gene expression under inducing conditions and a 13-fold average induction due to xylose. In the presence of the XylR repressor, gene expression was very sensitive to xylose concentration and full induction was observed with 0.10 g/L xylose. In the absence of XylR, gene expression from the vector set did not require xylose and was constitutive over a similar 26-fold range of expression. These results show that the vectors are extremely versatile for constitutive expression as well as for fine-tuning both the timing of gene expression and expression level using xylose as an inexpensive inducer.

Genome expansion by allopolyploidization in the fungal strain Coniochaeta 2T2.1 and its exceptional lignocellulolytic machinery.

BACKGROUND: Particular species of the genus Coniochaeta (Sordariomycetes) exhibit great potential for bioabatement of furanic compounds and have been identified as an underexplored source of novel lignocellulolytic enzymes, especially Coniochaeta ligniaria. However, there is a lack of information about their genomic features and metabolic capabilities. Here, we report the first in-depth genome/transcriptome survey of a Coniochaeta species (strain 2T2.1). RESULTS: The genome of Coniochaeta sp. strain 2T2.1 has a size of 74.53 Mbp and contains 24,735 protein-encoding genes. Interestingly, we detected a genome expansion event, resulting ~ 98% of the assembly being duplicated with 91.9% average nucleotide identity between the duplicated regions. The lack of gene loss, as well as the high divergence and strong genome-wide signatures of purifying selection between copies indicates that this is likely a recent duplication, which arose through hybridization between two related Coniochaeta-like species (allopolyploidization). Phylogenomic analysis revealed that 2T2.1 is related Coniochaeta sp. PMI546 and Lecythophora sp. AK0013, which both occur endophytically. Based on carbohydrate-active enzyme (CAZy) annotation, we observed that even after in silico removal of its duplicated content, the 2T2.1 genome contains exceptional lignocellulolytic machinery. Moreover, transcriptomic data reveal the overexpression of proteins affiliated to CAZy families GH11, GH10 (endoxylanases), CE5, CE1 (xylan esterases), GH62, GH51 (α-l-arabinofuranosidases), GH12, GH7 (cellulases), and AA9 (lytic polysaccharide monoxygenases) when the fungus was grown on wheat straw compared with glucose as the sole carbon source. CONCLUSIONS: We provide data that suggest that a recent hybridization between the genomes of related species may have given rise to Coniochaeta sp. 2T2.1. Moreover, our results reveal that the degradation of arabinoxylan, xyloglucan and cellulose are key metabolic processes in strain 2T2.1 growing on wheat straw. Different genes for key lignocellulolytic enzymes were identified, which can be starting points for production, characterization and/or supplementation of enzyme cocktails used in saccharification of agricultural residues. Our findings represent first steps that enable a better understanding of the reticulate evolution and "eco-enzymology" of lignocellulolytic Coniochaeta species.

Combined neonatal stress and young-adult glucocorticoid stimulation in rats reduce BDNF expression in hippocampus: effects on learning and memory.

Epidemiological studies suggest that multiple developmental disruptions are involved in the etiology of psychiatric illnesses including schizophrenia. In addition, altered expression of brain-derived neurotrophic factor (BDNF) has been implicated in these illnesses. In the present study, we examined the combined long-term effect of an early stress, in the form of maternal deprivation, and a later stress, simulated by chronic young-adult treatment with the stress hormone, corticosterone, on BDNF expression in the hippocampus of rats. To assess whether there were behavioral effects, which may correlate with the BDNF changes, learning and memory was tested in the Y-maze test for short term spatial memory, the Morris water maze for long-term spatial memory, and the T-maze test for working memory. Four groups of rats received either no stress, maternal deprivation, corticosterone treatment, or both. Dorsal hippocampus sections obtained from parallel groups were used for BDNF mRNA in situ hybridization. Rats which had undergone both maternal deprivation and corticosterone treatment displayed a unique and significant 25-35% reduction of BDNF expression in the dentate gyrus (DG), and similar trends in the CA1 and CA3 regions of the hippocampus. These "two-hit" animals exhibited a learning delay in the Morris water maze test, a marked deficit in the Y-maze, but little change in the T-maze test. However, some aspects of cognition were also altered in rats with either maternal deprivation or corticosterone treatment. This study demonstrates a persistent effect of two developmental disruptions on BDNF expression in the hippocampus, with parallel, but not completely correlative changes in learning and memory.

Identification and transcriptional profiling of Pseudomonas putida genes involved in furoic acid metabolism.

Pseudomonas putida Fu1 metabolizes furfural through a pathway involving conversion to 2-oxoglutarate, via 2-furoic acid (FA) and coenzyme A intermediates. Two P. putida transposon mutants were isolated that had impaired growth on furfural and FA, and DNA flanking the transposon insertion site was cloned from both mutants. The transposons disrupted psfB, a LysR-family regulatory gene in mutant PSF2 and psfF, a GcvR-type regulatory gene in PSF9. Disruption of two genes adjacent to psfB demonstrated that both are important for growth on FA, and ORFs in the proximity of psfB and psfF were transcriptionally activated during growth of P. putida on FA. Transcript levels increased in response to FA by 10-fold (a putative permease gene) to >1000-fold (a putative decarboxylase gene). The LysR-family gene appears to act positively, and the GcvR-family gene negatively, in regulating expression of neighboring genes in response to FA.

Use of green fluorescent protein to monitor fungal growth in biomass hydrolysate.

A reporter gene encoding green fluorescent protein (GFP) was introduced into the ascomycete Coniochaeta ligniaria NRRL30616, and fluorescence of cultures was monitored as a measure of cell growth. Fluorescence in the GFP-expressing strain was measured during growth of cells in defined and complex media as well as in the liquor derived from pretreatment of corn stover, an agricultural residue. Fluorescence mirrored growth of cultures, as measured by optical density and counts of colony forming units. Because traditional methods to monitor growth cannot be used in biomass liquors due to its fibrous, dark-colored nature, the speed and convenience of using GFP to monitor growth is advantageous. Fluorescence of cultures in biomass hydrolysate also correlated with the concentration of furfural in hydrolysate. Furfural and other compounds, present in hydrolysate due to physico-chemical pretreatment of biomass, are inhibitory to fermenting microbes. Therefore, measurement of fluorescence in GFP-expressing C. ligniaria is a proxy for measures of microbial growth and furfural consumption, and serves as a convenient indicator of metabolism of fermentation inhibitors in biomass hydrolysate.

Coexpression of pyruvate decarboxylase and alcohol dehydrogenase genes in Lactobacillus brevis.

Lactobacillus brevis ATCC367 was engineered to express pyruvate decarboxylase (PDC) and alcohol dehydrogenase (ADH) genes in order to increase ethanol fermentation from biomass-derived residues. First, a Gram-positive Sarcina ventriculi PDC gene (Svpdc) was introduced into L. brevis ATCC 367 to obtain L. brevis bbc03. The SvPDC was detected by immunoblot using an SvPDC oligo peptide antiserum, but no increased ethanol was detected in L. brevis bbc03. Then, an ADH gene from L. brevis (Bradh) was cloned behind the Svpdc gene that generated a pdc/adh-coupled ethanol cassette pBBC04. The pBBC04 restored anaerobic growth and conferred ethanol production of Escheirichia coli NZN111 (a fermentative defective strain incapable of growing anaerobically). Approximately 58 kDa (SvPDC) and 28 kDa (BrADH) recombinant proteins were observed in L. brevis bbc04. These results indicated that the Gram-positive ethanol production genes can be expressed in L. brevis using a Gram-positive promoter and pTRKH2 shuttle vector. This work provides evidence that expressing Gram-positive ethanol genes in pentose utilizing L. brevis will further aid manipulation of this microbe toward biomass to ethanol production.

Biofilm formation by bacterial contaminants of fuel ethanol production.

Commercial fuel ethanol production facilities were previously shown to have characteristic populations of bacterial contaminants which reduce product yield and are difficult to eradicate. Bacterial contaminants were found, for the first time, to form biofilms under laboratory conditions. Fermentor samples from a commercial fuel ethanol production facility were used to inoculate a biofilm reactor and purified bacterial isolates were identified. Biofilms were composed of many of the same species present in production samples, with lactic acid bacteria predominating.