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In the U.S., 30–40% of all food produced ends up as waste. By taking advantage of biological processes this waste nutrients can be converted into value-added 2,3-butanediol (2,3-BDO). However, food waste contains live bacteria and fungi which must first be inactivated, a process typically done with autoclaving under high pressure steam. If contaminants are not removed they can out-compete added industrial microbes for nutrients, resulting in lower yields of bio-products. Unfortunately, sterilization methods such as autoclaving greatly increase production time, and increase energy consumption. This creates a demand for novel microorganisms that can out-compete food waste microbiota by natural means without the need for sterilization of food waste. For this study, several Bacillus licheniformis isolates from Yellowstone National Park were investigated for their potential use in the 2,3-BDO industry. B. licheniformis species are a strain of industrial importance as of late, due to the wide metabolic functions and sustainable growth conditions above 45 °C. Whole genomic sequencing data and biochemical classification of B. licheniformis YNP1-TSU, B. licheniformis YNP2-TSU, and B. licheniformis YNP3-TSU were assessed and revealed a wide assortment of anti-microbial peptides and antibodies as well as catabolic genes for assimilation of glucose, fructose, galactose, xylose, arabinose, mannose, starch and sucrose, and linoleic acid. All three B. licheniformis strains had an optimal growth temperature of 50 °C, and were identified as high 2,3-BDO producers. Each strain YNP1-TSU, YNP2-TSU, and YNP3-TSU yielded 0.44 g/g, 0.45 g/g, and 0.43 g/g from mixed sugars found in non-sterilized food waste, respectively.

2,3-Butanediol (2,3-BD) is an important platform chemical that can be produced biologically. It is currently fermented below 40 °C using mesophilic strains at acid to neutral pH conditions, but the...

Conversion of food waste into 2,3-butanediol (2,3-BDO) via microbial fermentation provides a promising way to reduce waste disposal to landfills and produce sustainable chemicals. However, sterilization of food waste, an energy- and capital-costly process, is generally required before fermentation to avoid any contamination, which reduces the energy net output and economic feasibility of food waste fermentation. In this study, we investigated the non-sterile fermentation of food waste to produce 2,3-BDO using a newly isolated thermophilic and alkaliphilic B. licheniformis YNP5-TSU. Three unitary food waste samples (i.e., pepper, pineapple, cabbage wastes) and one miscellaneous food waste mixture were respectively inoculated with B. licheniformis YNP5-TSU under non-sterile conditions. At 50 °C and an initial pH of 9.0, B. licheniformis YNP5-TSU was able to consume all sugars in food waste and produce 5.2, 5.9, 5.9 and 4.3 g/L of 2,3-BDO within 24 h from pepper, pineapple, cabbage and miscellaneous wastes, respectively, corresponding to a yield of 0.40, 0.38, 0.41 and 0.41 g 2,3-BDO/g sugar. These 2,3-BDO concentrations and yields from the non-sterile fermentations were comparable to those from the traditional sterile fermentations, which produced 4.0–6.8 g/L of 2,3-BDO with yields of 0.31–0.48 g 2,3-BDO/g sugar. Moreover, B. licheniformis was able to ferment various food wastes (pepper, pineapple and miscellaneous wastes) without any external nutrient addition and produce similar 2,3-BDO quantities. The non-sterile fermentation of food waste using novel thermophilic and alkaliphilic B. licheniformis YNP5-TSU provides a robust and energy-efficient approach to convert food waste to high-value chemicals.

To better evaluate potential uses for grape pomace (GP) waste, a comprehensive chemical composition analysis of GP in Virginia was conducted. Eight commercial white and red pomace samples (cv. Viognier, Vidal Blanc, Niagara, Petit Manseng, Petit Verdot, Merlot, Cabernet Franc, and Chambourcin) obtained from different wineries in Virginia, USA were used. For extractives, GPs contained 2.89%&ndash, 4.66% titratable acids, 4.32%&ndash, 6.60% ash, 4.62%&ndash, 12.5% lipids with linoleic acid being the predominant (59.0%&ndash, 70.9%) fatty acid, 10.4&ndash, 64.8 g total phenolic content (gallic acid equivalents)/kg GP, 2.09&ndash, 53.3 g glucose/kg GP, 3.79&ndash, 52.9 g fructose/kg GP, and trace sucrose. As for non-extractives, GPs contained 25.2%&ndash, 44.5% lignin, 8.04%&ndash, 12.7% glucan, 4.42%&ndash, 7.05% xylan, and trace amounts of galactan, arabinan, and mannan (less than 3% in total). Potential usages of these components were further examined to provide information on better valorization of GP. Considering the valuable extractives (e.g., polyphenols and oil) and non-extractives (e.g., lignin), designing a biorefinery process aiming at fully recover and/or utilize these components is of future significance.

Background Bacillus cereus is a bacterial species which grows efficiently on a wide range of carbon sources and accumulates biopolymer poly-hydroxybutyrate (PHB) up to 80% cell dry weight. PHB is an aliphatic polymer produced and stored intracellularly as a reservoir of carbon and energy, its mobilization is a key biological process for sporulation in Bacillus spp. Previously, B. cereus tsu1 was isolated and cultured on rapeseed cake substrate (RCS), with maximum of PHB accumulation reached within 12 h, and depleted after 48 h. Fore-spore and spore structure were observed after 24 h culture. Results Quantitative proteomic analysis of B. cereus tsu1 identified 2952 quantifiable proteins, and 244 significantly changed proteins (SCPs) in the 24 h:12 h pair of samples, and 325 SCPs in the 48 h:12 h pair of samples. Based on gene ontology classification analysis, biological processes enriched only in the 24 h:12 h SCPs include purine nucleotide metabolism, protein folding, metal ion homeostasis, response to stress, carboxylic acid catabolism, and cellular amino acid catabolism. The 48 h:12 h SCPs were enriched into processes including carbohydrate metabolism, protein metabolism, oxidative phosphorylation, and formation of translation ternary structure. A key enzyme for PHB metabolism, poly(R)-hydroxyalkanoic acid synthase (PhaC, KGT44865) accumulated significantly higher in 12 h-culture. Sporulation related proteins SigF and SpoEII were significantly higher in 24 h-samples. Enzymes for nitrate respiration and fermentation accumulated to the highest abundance level in 48 h-culture. Conclusions Changes in proteome of B. cereus tsu1 during PHB intracellular mobilization were characterized in this study. The key enzyme PhaC for PHB synthesis increased significantly after 12 h-culture which supports the highest PHB accumulation at this time point. The protein abundance level of SpoIIE and SigF also increased, correlating with sporulation in 24 h-culture. Enzymes for nitrate respiration and fermentation were significantly induced in 48 h-culture which indicates the depletion of oxygen at this stage and carbon flow towards fermentative growth. Results from this study provide insights into proteome profile changes during PHB accumulation and reuse, which can be applied to achieve a higher PHB yield and to improve bacterial growth performance and stress resistance.

Optimization of cellulase activity is vital for synthesizing the end-products of second generation biofuel production. The slightest change in fermentation parameters can reduce the secretion of necessary enzymes to degrade cellulosic biomass. Determining the ecological effects of certain key media components is essential to understand how bacterial species will respond in a fluid environment. For our experiment a cellulosic media was designed to enhance the industrially important thermophile, Bacillus licheniformis YNP5-TSU. After several attempts to simplify the carboxymethylcellulose (CMC) media composition, impaired biofilm maturation and cellulase activity was noticed. This negative artifact occurred only when magnesium sulphate was removed from media. To analyze the shift in gene expression caused by magnesium stress, biofilm associated proteins were extracted from both control (4.0 mM MgSO4) and magnesium depleted (0.0 mM MgSO4) media at 24 hr and 48 hr incubation periods. These proteins were quantified through isobaric labeling and raw data generated from nanoLC-MS/MS identified over 2,000 proteins from the Bacillus licheniformis YNP5-TSU proteome (NCBI accession number MEDD00000000). After statistical normalization and false discovery rate were calculated, a total of 161 proteins from magnesium depleted media and 238 proteins from control media were deemed statistically relevant. A closer look through STRING interconnected webs, data mining, and NCBI annotations revealed several up/down regulated proteins that had linkage to biofilm formation and cellulase secretion. In this study we are able to provide significant evidence that; (1) biofilm maturation and cellulase production are highly correlated and (2), their optimization is dependent on the expression of several key proteins.

Gram’s Iodine Assay is currently used to detect endoglucanase, a cellulase enzyme which cleaves β-1,4-glycosidic bonds in carboxymethylcellulose (CMC). When a zone of degradation was observed around non-cellulolytic Escherichia coli JM109 with the use of Gram’s Iodine, the assay was further examined. Additional cellulolytic bacteria were chosen to display these inconsistencies. Bacillus thuringiensis subsp. Kurstaki (Btk) and Cellulomonas persica, known to possess endoglucanase, were incubated at 30 °C for 48 h on several different media; 1.5 % agar only, Davis minimal media, CMC agar, non-CMC agar, and Phytagel CMC. After the incubation period all plates were flooded with Gram’s Iodine for 3–5 min. A clear ring of degradation was identified around colonies in plates containing no cellulose and plates containing agar only. When agar was removed as the solidifying agent and substituted with Phytagel, zones of clearing around cellulolytic organisms were altered. These findings indicate that agar alone can cause additional artifacts and should be replaced by other solidifying agents as well as supplementation of the Congo Red Assay. Taking these precautions can overcome inconsistency problems for future cellulolytic screenings.