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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.

This study applied concentrated xylose (60–250 g/L) medium to produce butanol (acetone butanol ethanol, or ABE). A control batch fermentation of 61 g/L initial glucose using Clostridium beijerinckii P260 resulted in a productivity and yield of 0.33 g/L·h and 0.43 g/g, respectively. Use of 60 g/L xylose in a batch system resulted in productivity and yield of 0.26 g/L·h, and 0.40 g/g, respectively. In these two experiments, the culture fermented 89.3% glucose and 83.6% of xylose, respectively. When ABE recovery was coupled with fermentation for continuous solvent removal, the culture fermented all the added xylose (60 g/L). This system resulted in a productivity and yield of 0.66 g/L·h and 0.44 g/g, respectively. When the sugar concentration was further increased above 100 g/L, only a small fraction of the sugar was fermented in batch cultures without product removal. However, with simultaneous product removal, all the xylose (150 g/L) was fermented provided the culture was fed with nutrients intermittently. In this system, 66.32 g/L ABE was produced from 150 g/L xylose with a productivity of 0.44 g/L·h and yield of 0.44 g/g. Using the integrated culture system allowed sugar consumption to be increased by 300% (150 g/L). The continuous system using xylose as a feed did not sustain and after 36 days (864 h) of fermentation, it produced only 2–3 g/L ABE. Rather, the culture became acidogenic and produced 4–5 g/L acids (acetic and butyric). This study suggested that xylose be fermented in batch reactors coupled with simultaneous product recovery rather than in continuous reactors.

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.

BACKGROUND: Butanol, a superior biofuel to ethanol, can be produced by fermentation of renewable feedstocks such as corn or molasses. Since costs of these feedstocks are high, this biofuel should be produced from economic substrates. Recent studies have identified yellow top (YT) (Physaria fendleri) as an economical feedstock which is currently used for landfill, thus causing environmental pollution. In addition, cutting edge science and technology should be used, such as application of concentrated substrates. This study used concentrated YT ranging from 80 to 150 g L⁻¹. RESULTS: At a YT concentration of 125 g L⁻¹, the culture produced 28.8 g L⁻¹ acetone–butanol–ethanol (ABE) as compared with 18.68 g L⁻¹ using glucose (control) as substrate, which is 154% of the control fermentation. The control fermentation resulted in a productivity of 0.42 g L⁻¹ h⁻¹, while YT resulted in a productivity of 0.70 g L⁻¹ h⁻¹. In YT fermentation, specific ABE productivity was 543% of that obtained in the control run. CONCLUSION: A YT concentration of 125 g L⁻¹ is beneficial for ABE fermentation. At this concentration, ABE production level, productivity and specific productivity were greatly enhanced. Published 2019. This article is a U.S. Government work and is in the public domain in the USA.

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.

The decreasing supply of fossil fuels and increasing environmental concern of food waste disposal have raised interests in food waste conversation to biofuels such as butanol. Apple pomace, a food processing waste rich in carbohydrates, is a good feedstock for butanol production. The goal of this study is to present and evaluate a process to thoroughly convert apple pomace water soluble sugars (WSS) and hydrolyzed sugars from structural carbohydrates to acetone-butanol-ethanol (ABE) by fermentation. WSS was extracted from apple pomace by hot water. The solid residue was pretreated with acid or alkali followed by enzymatic hydrolysis to obtain acid hydrolyzed sugars (ACHS) or alkali hydrolyzed sugars (ALHS). Finally, WSS, ACHS, ALHS, WSS + ACHS, and WSS + ALHS were used as substrates to produce ABE by Clostridium beijerinckii P260, respectively. Acid and alkali pretreated apple pomace showed significantly (p < 0.05) higher glucose yield after cellulase hydrolysis compared with that of unpretreated apple pomace. Addition of pectinase increased hydrolyzed glucose yield by 27.9%, 26.9%, and 33.0% for acid pretreated sample, alkali pretreated sample, and unpretreated sample, respectively. Fermentation results revealed that inhibitors generated during pretreatment could negatively affect the ABE fermentation rate and titers; however, this negative effect could be alleviated by mixing the hydrolyzed sugars with water soluble sugars. A total of 202.8, 42.1, 41.4, 260.1, and 262.2 g of ABE was produced from each kg of dry apple pomace using WSS, ACHS, ALHS, WSS + ACHS, and WSS + ALHS as the substrates, respectively, based on the mass balance. USDA AFRI Foundational Program [2018-67021-27895]; Virginia Agriculture Experiment Station; Hatch Program of the National Institute of Food and Agriculture (NIFA), USDA This work was supported by the USDA AFRI Foundational Program (grant number 2018-67021-27895) and the Virginia Agriculture Experiment Station and the Hatch Program of the National Institute of Food and Agriculture (NIFA), USDA. Public domain – authored by a U.S. government employee

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.

The pulp and paper industry produces about 300–350 million tons of paper mill sludge (PMS) annually and majority is disposed of by landfill. PMS contains up to 75% carbohydrates. In this study, PMS was treated by de-ashing, fiber regeneration and enzymatic hydrolysis. The PMS hydrolysate was used for butyric acid production by Clostridium tyrobutyricum B-67062/RPT 4213. We reported that 8.52 and 8.35 gL−1 butyric acid was produced from 4 L batch fermentation in MRS and RCM4 medium respectively. Moreover, nearly 7 gL−1 butyric acid was produced by using PMS hydrolysates from two different mills when combined with a fraction of MRS, and over 6 gL−1 butyric acid was produced from hydrolysate combined with a fraction of RCM4 medium. This study suggested that beside agricultural lignocellulosic biomass feedstocks, low valued waste materials from the pulp and paper industries could also be used for the sustainable production of butyric acid.

Sweet sorghum syrups represent a renewable raw material that can be available year-round for production of biofuels and biochemicals. Sweet sorghum sugars have been used as sources for butanol production in the past but most often the studies focused on sweet sorghum juice and not on sweet sorghum syrups. Therefore, we investigated the feasibility of using the syrups as feedstock. Initial studies showed that diluted sweet sorghum syrups, to 60 g/L of glucose equivalents, could not be used as a direct replacement of a synthetic growth medium for industrial butanol-producing strain Clostridium beijerinckii. Further studies revealed that supplemental nutrients (most notably, ammonium–nitrogen) were required for successful fermentation. This was found true for two manufacturing sources of sweet sorghum syrups from commercial cultivars and hybrids. Typically, 15 g/L of total acetone, butanol, and ethanol were produced with approximately half of that being butanol. Some minor statistical difference was noted between the production potential of the two sources of syrup. However, aconitic acid, which was present at similar levels in both syrups, was ruled out as a butanol fermentation inhibitor at the fermentation pH > 4.5.

BACKGROUND Butanol is a superior biofuel to ethanol because of its blend properties and higher energy density. However, its recovery by distillation from the fermentation broth is energy intensive. For this reason, butanol recovery by supercritical CO2 extraction from simulated and actual fermentation broths was studied. Recovery of butanol by this technique has numerous advantages including being environmentally favorable, avoiding use of costly chemicals or membranes, and compatible with the microbial culture. RESULTS For the model solution extraction process, feed contained 7.00, 12.00, and 1.00 g L−1 acetone, butanol, ethanol (ABE), respectively, to mimic the actual concentrations obtained in the fermentation broth. In the extract phase, butanol concentration obtained ranged from 460.4 to 573.2 g L−1 with butanol selectivities ranging from 128 to 204.8. ABE recovery was also successfully demonstrated using actual fermentation broth of C. beijerinckii P260. CONCLUSION Supercritical CO2 extraction was successfully used for recovery of all three (ABE) components. Fermentation broth raffinate (feed depleted in ABE after recovery) was also reused for a second fermentation thus allowing process water recycle. Published 2017. This article is a U.S. Government work and is in the public domain in the USA.

Butanol was produced commercially from cornstarch and sugarcane molasses (renewable resources) until 1983, when production of these plants was forced to cease because of unfavorable economics of production caused in part by escalating prices of these feedstocks. During recent years, the focus of research has been on the use of economically available agricultural biomass and residues and cutting-edge science and technology to make butanol production a commercially viable process again. In this study, we produced butanol from sweet sorghum bagasse (SSB) by employing high concentrations of SSB solids and integrated process technology through which simultaneous saccharification, fermentation, and recovery (SSFR) were conducted as one unit operation. The concentrated SSB (16–22% dry wt. basis or 160–220 gL−1) was used to reduce reactor size and potentially reduce fixed and operational costs. Indeed, ABE productivity and yield of 0.21 gL−1h−1 and 0.39 were obtained, respectively, when 160 gL−1 SSB (16%, dry wt.) was used in the SSFR process. In nonintegrated systems, use of >90 gL−1 solid loading is improbable and has not been done until this study.

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.

A novel butanol fermentation process was developed in which sweet sorghum bagasse (SSB) was pretreated using liquid hot water (LHW) pretreatment technique followed by enzymatic hydrolysis and butanol (acetone butanol ethanol (ABE)) fermentation. A pretreatment temperature of 200 °C resulted in the generation of a hydrolyzate that inhibited butanol fermentation. When SSB pretreatment temperature was decreased to 190 °C (0-min holding time), the hydrolyzate was successfully fermented without inhibition and an ABE productivity of 0.51 g L−1 h−1 was achieved which is comparable to the 0.49 g L−1 h−1 observed in the control fermentation where glucose was used as a feedstock. These results are based on the use of 86 g L−1 SSB solid loadings in the pretreatment reactors. We were also able to increase SSB solid loadings from 120 to 200 g L−1 in the pretreatment step (190 °C) followed by hydrolysis and butanol fermentation. As pretreatment solid loadings increased, ABE yield remained in the range of 0.38–0.46. In these studies, a maximum ABE concentration of 16.88 g L−1 was achieved. Using the LHW pretreatment technique, 88.40–96.00 % of polymeric sugars (cellulose + hemicellulose) were released in the SSB hydrolyzate. The LHW pretreatment technique does not require chemical additions and is environmentally friendly, and the hydrolyzate can be used successfully for butanol fermentation.

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 β-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.

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. However, fungal pretreatment can cause carbohydrate loss and it is, therefore, necessary to use the appropriate fungal strain-biomass type combination. In this work, 26 white-rot fungal strains were evaluated under solid state cultivation at 74% moisture level at 28 °C for 30 days for fermentable sugar production from corn stover after enzymatic hydrolysis of each fungal pretreated corn stover using a cocktail of 3 commercial enzyme (cellulase, β-glucosidase, hemicellulase) preparations. The best result was obtained with Cyathus stercoreus NRRL-6573 which gave a sugar yield of 394 ± 13 mg/g of pretreated stover followed by Pycnoporus sanguineus FP-10356-Sp (393 ± 17 mg/g) and Phlebia brevispora NRRL-13108 (383 ± 13 mg/g).

© 2020 by the authors., Worldwide, there are various feedstocks such as straws, corn stover, sugarcane bagasse, sweet sorghum bagasse (SSB), grasses, leaves, whey permeate, household organic waste, and food waste (FW) that can be converted to valuable biofuels such as butanol. For the present studies, an economic analysis was performed to compare butanol production from three feedstocks (SSB; FW; and yellow top presscake, YTP or YT) using a standard process and an advanced integrated process design. The total plant capacity was set at 170,000–171,000 metric tons of total acetone butanol ethanol (ABE) per year (99,300 tons of just butanol per year). Butanol production from SSB typically requires pretreatment, separate hydrolysis, fermentation, and product recovery (SHFR). An advanced process was developed in which the last three steps were combined into a single unit operation for simultaneous saccharification, fermentation, and recovery (SSFR). For the SHFR and SSFR plants, the total capital investments were estimated as $213.72 × 106 and $198.16 × 106, respectively. It was further estimated that the minimum butanol selling price (using SSB as a feedstock) for the two processes were $1.14/kg and $1.05/kg. Therefore, SSFR lowered the production cost markedly compared to that of the base case. Butanol made using FW had an estimated minimum selling price of only $0.42/kg. This low selling price is because the FW to butanol process does not require pretreatment, hydrolysis, and cellulolytic enzymes. For this plant, the total capital investment was projected to be $107.26 × 106. The butanol selling price using YTP as a feedstock was at $0.73/kg and $0.79/kg with total capital investments for SSFR and SHFR of $122.58 × 106 and $132.21 × 106, respectively. In the Results and Discussion section, the availability of different feedstocks in various countries such as Brazil, the European Union, New Zealand, Denmark, and the United States are discussed. Additionally, the use of various microbial strains and product recovery technologies are also discussed.

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.

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.

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.

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.

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.

The production of ethanol from wheat straw (WS) by dilute acid pretreatment, bioabatement of fermentation inhibitors by a fungal strain, and simultaneous saccharification and fermentation (SSF) of the bio-abated WS to ethanol using an ethanologenic recombinant bacterium was studied at a pilot scale without sterilization. WS (124.2 g/L) was pretreated with dilute H 2 SO 4 in two parallel tube reactors at 160 °C. The inhibitors were bio-abated by growing the fungus aerobically. The maximum ethanol produced by SSF of the bio-abated WS by the recombinant Escherichia coli FBR5 at pH 6.0 and 35 °C was 36.0 g/L in 83 h with a productivity of 0.43 g L −1 h −1 . This value corresponds to an ethanol yield of 0.29 g/g of WS which is 86% of the theoretical ethanol yield from WS. This is the first report on the production of ethanol by the recombinant bacterium from a lignocellulosic biomass at a pilot scale.

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 β-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.

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.

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.

This article describes the production of butanol (acetone–butanol–ethanol (also called AB or ABE or solvent)), by fermentation using current technologies from economically available agricultural residues (wheat, and barley straws, corn stover, sweet sorghum bagasse, switchgrass, etc.), and waste feedstocks such as food waste, municipal waste, and domestic organic waste. Production of this biofuel has been revived in the wake of fluctuating and increased gasoline prices. During the last 30–35 years significant progress has been made in this area, including the development of better microbial strains, high productivity reactors, simultaneous recovery of AB from bioreactors, and use of concentrated sugar solutions.

In these studies concentrated sugar solutions of barley straw and corn stover hydrolysates were fermented using Clostridium beijerinckii P260 with simultaneous product recovery and compared with the performance of a control glucose batch fermentation process. The control glucose batch fermentation resulted in the production of 23.25 g L−1 ABE from 55.7 g L−1 glucose solution resulting in an ABE productivity and yield of 0.33 g L−1 h−1 and 0.42, respectively. The control reactor (I) was started with 62.5 g L−1 initial glucose and the culture left 6.8 g L−1 unused sugar due to butanol toxicity resulting in incomplete sugar utilization. Barley straw (BS) hydrolysate sugars (90.3 g L−1) resulted in the production of 47.20 g L−1 ABE with a productivity of 0.60 g L−1 h−1 and a yield of 0.42. Fermentation of corn stover (CS) hydrolysate sugars (93.1 g L−1) produced 50.14 g L−1 ABE with a yield of 0.43 and a productivity of 0.70 g L−1 h−1. These productivities are 182–212% higher than the control run. The culture was able to use 99.4–100% sugars (CS & BS respectively) present in these hydrolysates and improve productivities which were possible due to simultaneous product removal. Use of >100 g L−1 hydrolysate sugars was not considered as it would have been toxic to the culture in the integrated (simultaneous fermentation and recovery) process.

A simultaneous saccharification, fermentation, and recovery (SSFR) process was developed for the production of acetone-butanol-ethanol (AB or ABE), of which butanol is the main product, from corn stover employing Clostridium beijerinckii P260. Of the 86 g L(-1) corn stover provided, over 97% of the sugars were released during hydrolysis and these were fermented completely with an ABE productivity of 0.34 g L(-1)h(-1) and yield of 0.39. This productivity is higher than 0.31 g L(-1)h(-1) when using glucose as a substrate demonstrating that AB could be produced efficiently from lignocellulosic biomass. Acetic acid that was released from the biomass during pretreatment and hydrolysis was also used by the culture to produce AB. An average rate of generation of sugars during corn stover hydrolysis was 0.98 g L(-1)h(-1). In this system AB was recovered using vacuum, and as a result of this (simultaneous product recovery), 100% sugars were used by the culture.

Switchgrass (Panicum virgatum L.) is a perennial C4 grass that is being developed as a bioenergy crop because it has high production yields and suitable agronomic traits. Five switchgrass biomass samples from upland and lowland switchgrass ecotypes harvested at different stages or maturity were used in this study. Switchgrass samples contained 317.0-385.0 g glucans/kg switchgrass dry basis (db) and 579.3-660.2 g total structural carbohydrates/kg switchgrass, db. Carbohydrate contents were greater for the upland ecotype versus lowland ecotype and increased with harvest maturity. Pretreatment of switchgrass with dilute ammonium hydroxide (8% w/w ammonium loading) at 170 degrees C for 20 min was determined to be effective for preparing switchgrass for enzymatic conversion to monosaccharides; glucose recoveries were 66.9-90.5% and xylose recoveries 60.1-84.2% of maximum and decreased with increased maturity at harvest. Subsequently, pretreated switchgrass samples were converted to ethanol by simultaneous saccharification and fermentation using engineered xylose-fermenting Saccharomyces cerevisiae strain YRH400. Ethanol yields were 176.2-202.01/Mg of switchgrass (db) and followed a similar trend as observed for enzymatic sugar yields.

A cost estimation study was performed for a biological butanol production plant with a capacity of 150 × 10 6 kg butanol/year. Wheat straw (WS) was used as a feedstock. In addition to butanol, acetone (78.05 × 10 6 kg/year) and ethanol (28.54 × 10 6 kg/year) would also be produced. The total capital cost for this plant was $193.07 × 10 6 . This exercise was based in part on data generated in our laboratory and in part on data obtained from literature. The design, mass balance, and energy balance simulations were performed using SuperPro Designer (Version 8.5003, 2012). For butanol production wheat straw would be pretreated with dilute (1% v/v) sulfuric acid at 121 °C for 1 h followed by separate hydrolysis (using enzymes), fermentation and recovery. Enzyme cost for wheat straw hydrolysis was adapted from literature ($0.16/kg butanol). Utilities which included steam/high pressure steam, cooling/chilling water, and electricity represented the major cost of the operation (49.18%) followed by raw materials (26.81%). Based on batch fermentation of wheat straw hydrolysate and distillative recovery of acetone butanol ethanol (ABE), butanol production cost was estimated to be $1.30/kg for a grass-rooted/green-field plant. Application of a membrane recovery process could reduce this price to $1.00/kg for a plant annexed to an existing distillery.

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