Production of a Bacteriocin Like Protein PEG 446 from Clostridium tyrobutyricum NRRL B-67062.

Clostridium tyrobutyricum strain NRRL B-67062 was previously isolated from an ethanol production facility and shown to produce high yields of butyric acid. In addition, the cell-free supernatant of the fermentation broth from NRRL B-67062 contained antibacterial activity against certain Gram-positive bacteria. To determine the source of this antibacterial activity, we report the genome and genome mining of this strain. The complete genome of NRRL B-67062 showed one circular chromosome of 3,242,608 nucleotides, 3114 predicted coding sequences, 79 RNA genes, and a G+C content of 31.0%. Analyses of the genome data for genes potentially associated with antimicrobial features were sought after by using BAGEL-4 and anti-SMASH databases. Among the leads, a polypeptide of 66 amino acids (PEG 446) contains the DUF4177 domain, which is an uncharacterized highly conserved domain (pfam13783). The cloning and expression of the peg446 gene in Escherichia coli and Bacillus subtilis confirmed the antibacterial property against Lactococcus lactis LM 0230, Limosilactobacillus fermentum 0315-25, and Listeria innocua NRRL B-33088 by gel overlay and well diffusion assays. Molecular modeling suggested that PEG 446 contains one alpha-helix and three anti-parallel short beta-sheets. These results will aid further functional studies and facilitate simultaneously fermentative production of both butyric acid and a putative bacteriocin from agricultural waste and lignocellulosic biomass materials.

Antibacterial Property and Metagenomic Analysis of Milk Kefir.

Milk kefir fermentation has been used in households for generations. Consumption of milk kefir has been associated with various health benefits, presumably from the probiotics of yeast and bacteria that make up the kefir grains. In addition, many of the microbes are known to produce novel antimicrobial compounds that can be used for other applications. The microbes living inside kefir grains differ significantly depending on geographical location and production methods. In this study, we aimed to use metagenomic analysis of fermented milk by using three different kefir grains (kefir 1, kefir 2, and kefir 3) from different US sources. We analyzed the microbial compositions of the three milk fermentation samples. This study revealed that each sample contains unique and distinct groups of microbes, kefir 1 showed the least diversity, and kefir 3 showed the highest diversity. Kefir 3 is rich in Proteobacteria while kefir 2 is dominated by the Firmicutes. Using bacterial indicator growth analyses carried out by continuous readings from microplate-based bioreactor assays suggested that kefir 2 fermentation filtrate has higher antibacterial property. We have screened 30 purified cultures of kefir 2 sample and isolated two lactic acid bacteria strains with higher antibacterial activities; the two strains were identified as Leuconostoc mesenteroides 28-1 and Lentilactobacillus kefiri 25-2 by 16S genomic PCR with confirmed antibacterial activities of fermentation filtrate after growing under both aerobic and anaerobic conditions.

Proteomic Analysis Identifies Dysregulated Proteins in Butanol-Tolerant Gram-Positive Lactobacillus mucosae BR0713-33.

Butanol can be produced biologically through fermentation of lignocellulosic biomass-derived sugars by Gram-positive Clostridium species. For cost-effective production, increased butanol fermentation titers are desired. However, the currently available butanol-fermenting microbes do not tolerate sufficiently high butanol concentrations; thus, new butanol-tolerant strains are desired. One promising strategy is to genetically modify Clostridium species by introducing stress tolerance-associated genes. This study was aimed to seek butanol tolerance genes from other Gram-positive species, which might be better suited than those from Gram-negative E. coli or eukaryotic Saccharomyces cerevisiae. Several butanol-tolerant lactobacilli were reported previously, and Lactobacillus mucosae BR0713-33, which showed the most robust anaerobic growth in 4% butanol, was used here for proteomics analyses. Cellular proteins that responded to 2, 3, and 4% butanol were characterized. Twenty-nine proteins that were identified were dysregulated in response to increased concentrations of butanol in L. mucosae . Seventeen genes involved in coding for stress-tolerant proteins GroES, GroEL, and DnaK and genes involved in substrate utilization, fatty acid metabolism, and nucleotide synthesis were induced by increased butanol, and 12 genes involving energy production (F0F1ATP synthases) and redox balance preservation were repressed by increased butanol. These results can help guide targeted engineering strategies to improve tolerance and production of biobutanol.

Ethanol tolerance assessment in recombinant E. coli of ethanol responsive genes from Lactobacillus buchneri NRRL B-30929.

We previously identified specific proteins associated with ethanol stress response in a Lactobacillus buchneri strain capable of growing in 10% ethanol. In the current study, the exceptional roles of ethanol responsive genes are examined to determine if they can increase ethanol tolerance in E. coli host cells. The recombinant strains carrying ethanol responsive genes were subjected to growth analyses in media with and without 4% ethanol. Among the expression of these genes and growth analyses of the recombinant strains in ethanol, six genes Lbuc_0522 (NADPH-dependent methylglyoxal reductase), Lbuc_0354 (succinate semialdehyde dehydrogenase), Lbuc_1211(threonyl_tRNA synthetase), Lbuc_2051 (nitroreductase), Lbuc_0707 (branched chain amino acid aminotransferase) and Lbuc_1852 (proline-specific peptidase) conferred host cells tolerance to 4% ethanol. Six genes Lbuc_1523 (phage major capsid protein, HK 97 family), Lbuc_1319 (phosphoglycerate kinase), Lbuc_0787 encoding fumarylacetoacetate hydrolase, Lbuc_1219 encoding UDP-N-acetylmuramate-L-alanine ligase, Lbuc_0466 encoding ornithine carbamoyltransferase and Lbuc_0858 encoding glycine hydroxymethyltransferase showed no impact on growth in media with 4% ethanol with IPTG induction when compared with E. coli carrying control pET28b plasmid. The expression of two genes Lbuc_1557 (S-layer glycoprotein) and Lbuc_2157 (6-phosphogluconate dehydrogenase) resulted ethanol sensitivity phenotype.

Increased ethanol tolerance associated with the pntAB locus of Oenococcus oeni and Lactobacillus buchneri.

Lactobacillus buchneri and Oenococcus oeni are two unique ethanol-tolerant Gram-positive bacteria species. Genome comparison analyses revealed that L. buchneri and O. oeni possess a pntAB locus that was absent in almost all other lactic acid bacteria (LAB) genomes. Our hypothesis is that the pntAB locus contributes to the ethanol tolerance trait of these two distinct ethanol-tolerant organisms. The pntAB locus, consisting of the pntA and pntB genes, codes for NADP(H) transhydrogenase subunits. This membrane-bound transhydrogenase catalyzes the reduction of NADP+ and is known as an important enzyme in maintaining cellular redox balance. In this study, the transhydrogenase operon from L. buchneri NRRL B-30929 and O. oeni PSU-1 were cloned and analyzed. The LbpntB shared 71.0% identity with the O. oeni (OopntB). The entire pntAB locus was expressed in Lactococcus lactis ssp. lactis IL1403 resulting in an increased tolerance to ethanol (6%), butanol (1.8%) and isopropanol (1.8%) when compared to the control strain. However, the recombinant E. coli cells carrying the entire pntAB locus did not show any improved ethanol tolerance. Independent expression of OopntB and LbpntB in recombinant E. coli BL21(DE3)pLysS host demonstrated higher tolerance to ethanol when compared with a control E. coli BL21(DE3)pLysS strain carrying pET28b vector. Ethanol tolerance comparison of E. coli strains carrying LbpntB and OopntB showed that LbpntB conferred higher ethanol tolerance (4.5%) and resulted in greater biomass, while the OopntB conferred lower ethanol tolerance (4.0%) resulted lower biomass. Therefore, the pntB gene from L. buchneri is a better choice in generating higher ethanol tolerance. This is the first study to uncover the role of pntAB locus on ethanol tolerance.

Factors Affecting Production of Itaconic Acid from Mixed Sugars by Aspergillus terreus.

Itaconic acid (IA; a building block platform chemical) is currently produced industrially from glucose by fermentation with Aspergillus terreus. In order to expand the use of IA, its production cost must be lowered. Lignocellulosic biomass has the potential to serve as low-cost source of sugars for IA production. It was found that the fungus cannot produce IA from dilute acid pretreated and enzymatically saccharified wheat straw hydrolysate even at 100-fold dilution. The effects of typical compounds (acetic acid, furfural, HMF and Mn2+, enzymes, CaSO4), culture conditions (initial pH, temperature, aeration), and medium components (KH2PO4, NH4NO3, CaCl2·2H2O, FeCl3·6H2O) on growth and IA production by A. terreus NRRL 1972 using mixed sugar substrate containing glucose, xylose, and arabinose (4:3:1, 80 g L-1) mimicking the wheat straw hydrolysate were investigated. Acetic acid, furfural, Mn2+, and enzymes were strong inhibitors to both growth and IA production from mixed sugars. Optimum culture conditions (pH 3.1, 33 °C, 200 rpm) and medium components (0.8 g KH2PO4, 3 g NH4NO3, 2.0 g CaCl2·2H2O, 0.83-3.33 mg FeCl3·6H2O per L) as well as tolerable levels of inhibitors (0.4 g acetic acid, < 0.1 g furfural, 100 mg HMF, < 5.0 ppb Mn2+, 24 mg CaSO4 per L) for mixed sugar utilization were established. The results will be highly useful for developing a bioprocess technology for IA production from lignocellulosic feedstocks.

Yellow top (Physaria fendleri) presscake: A novel substrate for butanol production and reduction in environmental pollution.

Yellow Top (Physaria fendleri) is a plant that belongs to the mustard family. This plant is used to produce seeds that are rich in hydroxy oil. After extraction of oil, the presscake is land filled. The seedcake is rich in polymeric sugars and can be used for various bioconversions. For the present case, the seedcake or presscake was hydrolyzed with dilute (0.50% [v/v]) H2 SO4 and enzymes to release sugars including glucose, xylose, galactose, arabinose, and mannose. Then, the hydrolyzate was used to produce acetone-butanol-ethanol (ABE). Using 100 gL-1 presscake (prior to pretreatment), 19.22 gL-1 of ABE was successfully produced of which butanol was the major product. In this process, an ABE productivity of 0.48 gL-1 h-1 was obtained. These results are superior to glucose fermentation to produce ABE in which an ABE productivity of 0.42 gL-1 h-1 was obtained. Use of Yellow Top to produce butanol has the following advantages: (i) it is an economic feedstock and is expected to produce butanol economically; (ii) it avoids pollution concerns when not land filled; and (iii) rate of ABE production is not inhibited when fermented this substrate. It is suggested that the potential of this feedstock be further explored by optimizing process parameters for this valuable fermentation. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2767, 2019.

Butanol production from sweet sorghum bagasse with high solids content: Part I-comparison of liquid hot water pretreatment with dilute sulfuric acid.

In these studies, we pretreated sweet sorghum bagasse (SSB) using liquid hot water (LHW) or dilute H2 SO4 (2 g L-1 ) at 190°C for zero min (as soon as temperature reached 190°C, cooling was started) to reduce generation of sugar degradation fermentation inhibiting products such as furfural and hydroxymethyl furfural (HMF). The solids loading were 250-300 g L-1 . This was followed by enzymatic hydrolysis. After hydrolysis, 89.0 g L-1 sugars, 7.60 g L-1 acetic acid, 0.33 g L-1 furfural, and 0.07 g L-1 HMF were released. This pretreatment and hydrolysis resulted in the release of 57.9% sugars. This was followed by second hydrolysis of the fibrous biomass which resulted in the release of 43.64 g L-1 additional sugars, 2.40 g L-1 acetic acid, zero g L-1 furfural, and zero g L-1 HMF. In both the hydrolyzates, 86.3% sugars present in SSB were released. Fermentation of the hydrolyzate I resulted in poor acetone-butanol-ethanol (ABE) fermentation. However, fermentation of the hydrolyzate II was successful and produced 13.43 g L-1 ABE of which butanol was the main product. Use of 2 g L-1 H2 SO4 as a pretreatment medium followed by enzymatic hydrolysis resulted in the release of 100.6-93.8% (w/w) sugars from 250 to 300 g L-1 SSB, respectively. LHW or dilute H2 SO4 were used to economize production of cellulosic sugars from SSB. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:960-966, 2018.

High solid fed-batch butanol fermentation with simultaneous product recovery: Part II-process integration.

In these studies, liquid hot water (LHW) pretreated and enzymatically hydrolyzed Sweet Sorghum Bagasse (SSB) hydrolyzates were fermented in a fed-batch reactor. As reported in the preceding paper, the culture was not able to ferment the hydrolyzate I in a batch process due to presence of high level of toxic chemicals, in particular acetic acid released from SSB during the hydrolytic process. To be able to ferment the hydrolyzate I obtained from 250 g L-1 SSB hydrolysis, a fed-batch reactor with in situ butanol recovery was devised. The process was started with the hydrolyzate II and when good cell growth and vigorous fermentation were observed, the hydrolyzate I was slowly fed to the reactor. In this manner the culture was able to ferment all the sugars present in both the hydrolyzates to acetone butanol ethanol (ABE). In a control batch reactor in which ABE was produced from glucose, ABE productivity and yield of 0.42 g L-1 h-1 and 0.36 were obtained, respectively. In the fed-batch reactor fed with SSB hydrolyzates, these productivity and yield values were 0.44 g L-1 h-1 and 0.45, respectively. ABE yield in the integrated system was high due to utilization of acetic acid to convert to ABE. In summary we were able to utilize both the hydrolyzates obtained from LHW pretreated and enzymatically hydrolyzed SSB (250 g L-1 ) and convert them to ABE. Complete fermentation was possible due to simultaneous recovery of ABE by vacuum. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:967-972, 2018.

Production of itaconic acid from pentose sugars by Aspergillus terreus.

Itaconic acid (IA), an unsaturated 5-carbon dicarboxylic acid, is a building block platform chemical that is currently produced industrially from glucose by fermentation with Aspergillus terreus. However, lignocellulosic biomass has potential to serve as low-cost source of sugars for production of IA. Research needs to be performed to find a suitable A. terreus strain that can use lignocellulose-derived pentose sugars and produce IA. One hundred A. terreus strains were evaluated for the first time for production of IA from xylose and arabinose. Twenty strains showed good production of IA from the sugars. Among these, six strains (NRRL strains 1960, 1961, 1962, 1972, 66125, and DSM 23081) were selected for further study. One of these strains NRRL 1961 produced 49.8 ± 0.3, 38.9 ± 0.8, 34.8 ± 0.9, and 33.2 ± 2.4 g IA from 80 g glucose, xylose, arabinose and their mixture (1:1:1), respectively, per L at initial pH 3.1 and 33°C. This is the first report on the production of IA from arabinose and mixed sugar of glucose, xylose, and arabinose by A. terreus. The results presented in the article will be very useful in developing a process technology for production of IA from lignocellulosic feedstocks. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1059-1067, 2017.

Utilization of inulin-containing waste in industrial fermentations to produce biofuels and bio-based chemicals.

Inulins are polysaccharides that belong to an important class of carbohydrates known as fructans and are used by many plants as a means of storing energy. Inulins contain 20 to several thousand fructose units joined by ß-2,1 glycosidic bonds, typically with a terminal glucose unit. Plants with high concentrations of inulin include: agave, asparagus, coffee, chicory, dahlia, dandelion, garlic, globe artichoke, Jerusalem artichoke, jicama, onion, wild yam, and yacón. To utilize inulin as its carbon and energy source directly, a microorganism requires an extracellular inulinase to hydrolyze the glycosidic bonds to release fermentable monosaccharides. Inulinase is produced by many microorganisms, including species of Aspergillus, Kluyveromyces, Penicillium, and Pseudomonas. We review various inulinase-producing microorganisms and inulin feedstocks with potential for industrial application as well as biotechnological efforts underway to develop sustainable practices for the disposal of residues from processing inulin-containing crops. A multi-stage biorefinery concept is proposed to convert cellulosic and inulin-containing waste produced at crop processing operations to valuable biofuels and bioproducts using Kluyveromyces marxianus, Yarrowia lipolytica, Rhodotorula glutinis, and Saccharomyces cerevisiae as well as thermochemical treatments.

Progress and perspectives on improving butanol tolerance.

Fermentative production of butanol for use as a biofuel or chemical feedstock is regarded as a promising renewable technology that reduces greenhouse gas emissions and has the potential to become a substitute for non-sustainable chemical production route. However, butanol toxicity to the producing microbes remains a barrier to achieving sufficiently high titers for cost-effective butanol fermentation and recovery. Investigations of the external stress of high butanol concentration on butanol-producing microbial strains will aid in developing improved microbes with increased tolerance to butanol. With currently available molecular tool boxes, researchers have aimed to address and understand how butanol affects different microbes. This review will cover the individual organism's inherent responses to surrounding butanol levels, and the collective efforts by researchers to improve production and tolerance. The specific microorganisms discussed here include the native butanol producer Clostridium species, the fermentation industrial model Saccharomyces cerevisiae and the photosynthetic cyanobacteria, the genetic engineering workhorse Escherichia coli, and also the butanol-tolerant lactic acid bacteria that utilize diverse substrates. The discussion will help to understand the physiology of butanol resistance and to identify specific butanol tolerance genes that will lead to informed genetic engineering strategies for new strain development.

Biological pretreatment of corn stover with Phlebia brevispora NRRL-13108 for enhanced enzymatic hydrolysis and efficient ethanol production.

Biological pretreatment of lignocellulosic biomass by white-rot fungus can represent a low-cost and eco-friendly alternative to harsh physical, chemical, or physico-chemical pretreatment methods to facilitate enzymatic hydrolysis. In this work, solid-state cultivation of corn stover with Phlebia brevispora NRRL-13018 was optimized with respect to duration, moisture content and inoculum size. Changes in composition of pretreated corn stover and its susceptibility to enzymatic hydrolysis were analyzed. About 84% moisture and 42 days incubation at 28°C were found to be optimal for pretreatment with respect to enzymatic saccharification. Inoculum size had little effect compared to moisture level. Ergosterol data shows continued growth of the fungus studied up to 57 days. No furfural and hydroxymethyl furfural were produced. The total sugar yield was 442 ± 5 mg/g of pretreated corn stover. About 36 ± 0.6 g ethanol was produced from 150 g pretreated stover per L by fed-batch simultaneous saccharification and fermentation (SSF) using mixed sugar utilizing ethanologenic recombinant Eschericia coli FBR5 strain. The ethanol yields were 32.0 ± 0.2 and 38.0 ± 0.2 g from 200 g pretreated corn stover per L by fed-batch SSF using Saccharomyces cerevisiae D5A and xylose utilizing recombinant S. cerevisiae YRH400 strain, respectively. This research demonstrates that P. brevispora NRRL-13018 has potential to be used for biological pretreatment of lignocellulosic biomass. This is the first report on the production of ethanol from P. brevispora pretreated corn stover. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:365-374, 2017.

Growth, ethanol production, and inulinase activity on various inulin substrates by mutant Kluyveromyces marxianus strains NRRL Y-50798 and NRRL Y-50799.

Economically important plants contain large amounts of inulin. Disposal of waste resulting from their processing presents environmental issues. Finding microorganisms capable of converting inulin waste to biofuel and valuable co-products at the processing site would have significant economic and environmental impact. We evaluated the ability of two mutant strains of Kluyveromyces marxianus (Km7 and Km8) to utilize inulin for ethanol production. In glucose medium, both strains consumed all glucose and produced 0.40 g ethanol/g glucose at 24 h. In inulin medium, Km7 exhibited maximum colony forming units (CFU)/mL and produced 0.35 g ethanol/g inulin at 24 h, while Km8 showed maximum CFU/mL and produced 0.02 g ethanol/g inulin at 96 h. At 24 h in inulin + glucose medium, Km7 produced 0.40 g ethanol/g (inulin + glucose) and Km8 produced 0.20 g ethanol/g (inulin + glucose) with maximum CFU/mL for Km8 at 72 h, 40 % of that for Km7 at 36 h. Extracellular inulinase activity at 6 h for both Km7 and Km8 was 3.7 International Units (IU)/mL.

The yajC gene from Lactobacillus buchneri and Escherichia coli and its role in ethanol tolerance.

The yajC gene (Lbuc_0921) from Lactobacillus buchneri NRRL B-30929 was identified from previous proteomics analyses in response to ethanol treatment. The YajC protein expression was increased by 15-fold in response to 10 % ethanol vs 0 % ethanol. The yajC gene encodes the smaller subunit of the preprotein translocase complex, which interacts with membrane protein SecD and SecF to coordinate protein transport and secretion across cytoplasmic membrane in Escherichia coli. The YajC protein was linked to sensitivity to growth temperatures in E. coli, involved in translocation of virulence factors during Listeria infection, and stimulating a T cell-mediated response of Brucella abortus. In this study, the L. buchneri yajC gene was over-expressed in E. coli. The strain carrying pET28byajC that produces YajC after isopropyl ß-D-1-thiogalactopyranoside induction showed tolerance to 4 % ethanol in growth media, compared to the control carrying pET28b. This is the first report linking YajC to ethanol stress and tolerance.

Recovery of Butanol by Counter-Current Carbon Dioxide Fractionation with its Potential Application to Butanol Fermentation.

A counter-current CO2 fractionation method was applied as a mean to recover n-butanol and other compounds that are typically obtained from biobutanol fermentation broth from aqueous solutions. The influence of operating variables, such as solvent-to-feed ratio, temperature, pressure and feed solution composition was experimentally studied in terms of separation efficiency, butanol removal rate, total removal and butanol concentration in the extract at the end of the continuous cycle. With respect to the temperature and pressure conditions investigated, results show that the highest separation efficiency was obtained at 35 °C and 10.34 MPa. At these operating conditions, 92.3% of the butanol present in the feed solution was extracted, and a concentration of 787.5 g·L-1 of butanol in the extract was obtained, starting from a feed solution of 20 g·L-1. Selectivity was calculated from experimental data, concluding that our column performs much better than a single equilibrium stage. When adding ethanol and acetone to the feed solution, ethanol was detected in the water-rich fraction (raffinate), whereas the highest concentration of acetone was found in the butanol rich fraction (extract).

Butanol production from food waste: a novel process for producing sustainable energy and reducing environmental pollution.

BACKGROUND: Waste is currently a major problem in the world, both in the developing and the developed countries. Efficient utilization of food waste for fuel and chemical production can positively influence both the energy and environmental sustainability. This study investigated using food waste to produce acetone, butanol, and ethanol (ABE) by Clostridium beijerinckii P260. RESULTS: In control fermentation, 40.5 g/L of glucose (initial glucose 56.7 g/L) was used to produce 14.2 g/L of ABE with a fermentation productivity and a yield of 0.22 g/L/h and 0.35 g/g, respectively. In a similar fermentation 81 g/L of food waste (containing equivalent glucose of 60.1 g/L) was used as substrate, and the culture produced 18.9 g/L ABE with a high ABE productivity of 0.46 g/L/h and a yield of 0.38 g/g. Fermentation of food waste at higher concentrations (129, 181 and 228 g/L) did not remarkably increase ABE production but resulted in high residual glucose due to the culture butanol inhibition. An integrated vacuum stripping system was designed and applied to recover butanol from the fermentation broth simultaneously to relieve the culture butanol inhibition, thereby allowing the fermentation of food waste at high concentrations. ABE fermentation integrated with vacuum stripping successfully recovered the ABE from the fermentation broth and controlled the ABE concentrations below 10 g/L during fermentation when 129 g/L food waste was used. The ABE productivity with vacuum fermentation was 0.49 g/L/h, which was 109 % higher than the control fermentation (glucose based). More importantly, ABE vacuum recovery and fermentation allowed near-complete utilization of the sugars (~98 %) in the broth. CONCLUSIONS: In these studies it was demonstrated that food waste is a superior feedstock for producing butanol using Clostridium beijerinckii. Compared to costly glucose, ABE fermentation of food waste has several advantages including lower feedstock cost, higher productivity, and less residual sugars.

Irradiation of Yarrowia lipolytica NRRL YB-567 creating novel strains with enhanced ammonia and oil production on protein and carbohydrate substrates.

Increased interest in sustainable production of renewable diesel and other valuable bioproducts is redoubling efforts to improve economic feasibility of microbial-based oil production. Yarrowia lipolytica is capable of employing a wide variety of substrates to produce oil and valuable co-products. We irradiated Y. lipolytica NRRL YB-567 with UV-C to enhance ammonia (for fertilizer) and lipid (for biodiesel) production on low-cost protein and carbohydrate substrates. The resulting strains were screened for ammonia and oil production using color intensity of indicators on plate assays. Seven mutant strains were selected (based on ammonia assay) and further evaluated for growth rate, ammonia and oil production, soluble protein content, and morphology when grown on liver infusion medium (without sugars), and for growth on various substrates. Strains were identified among these mutants that had a faster doubling time, produced higher maximum ammonia levels (enzyme assay) and more oil (Sudan Black assay), and had higher maximum soluble protein levels (Bradford assay) than wild type. When grown on plates with substrates of interest, all mutant strains showed similar results aerobically to wild-type strain. The mutant strain with the highest oil production and the fastest doubling time was evaluated on coffee waste medium. On this medium, the strain produced 0.12 g/L ammonia and 0.20 g/L 2-phenylethanol, a valuable fragrance/flavoring, in addition to acylglycerols (oil) containing predominantly C16 and C18 residues. These mutant strains will be investigated further for potential application in commercial biodiesel production.

Enhancement of xylose utilization from corn stover by a recombinant Escherichia coli strain for ethanol production.

Effects of substrate-selective inoculum prepared by growing on glucose, xylose, arabinose, GXA (glucose, xylose, arabinose, 1:1:1) and corn stover hydrolyzate (dilute acid pretreated and enzymatically hydrolyzed, CSH) on ethanol production from CSH by a mixed sugar utilizing recombinant Escherichia coli (strain FBR5) were investigated. The initial ethanol productivity was faster for the seed grown on xylose followed by GXA, CSH, glucose and arabinose. Arabinose grown seed took the longest time to complete the fermentation. Delayed saccharifying enzyme addition in simultaneous saccharification and fermentation of dilute acid pretreated CS by the recombinant E. coli strain FBR5 allowed the fermentation to finish in a shorter time than adding the enzyme simultaneously with xylose grown inoculum. Use of substrate selective inoculum and fermenting pentose sugars first under glucose limited condition helped to alleviate the catabolite repression of the recombinant bacterium on ethanol production from lignocellulosic hydrolyzate.