Background: Large-scale production of effective cellulose hydrolytic enzymes is the key to the bioconversion of agricultural residues to ethanol. The goal of this study was to develop a rice plant as a bioreactor for the large-scale production of cellulose hydrolytic enzymes via genetic transformation, and to simultaneously improve rice straw as an efficient biomass feedstock for conversion of cellulose to glucose. Results: In this study, the cellulose hydrolytic enzyme β-1, 4-endoglucanase (E1) gene, from the thermophilic bacterium Acidothermus cellulolyticus, was overexpressed in rice through Agrobacterium-mediated transformation. The expression of the bacterial E1 gene in rice was driven by the constitutive Mac promoter, a hybrid promoter of Ti plasmid mannopine synthetase promoter and cauliflower mosaic virus 35S promoter enhancer, with the signal peptide of tobacco pathogenesis-related protein for targeting the E1 protein to the apoplastic compartment for storage. A total of 52 transgenic rice plants from six independent lines expressing the bacterial E1 enzyme were obtained that expressed the gene at high levels without severely impairing plant growth and development. However, some transgenic plants exhibited a shorter stature and flowered earlier than the wild type plants. The E1 specific activities in the leaves of the highest expressing transgenic rice lines were about 20-fold higher than those of various transgenic plants obtained in previous studies and the protein amounts accounted for up to 6.1% of the total leaf soluble protein. A zymogram and temperature-dependent activity analyses demonstrated the thermostability of the E1 enzyme and its substrate specificity against cellulose, and a simple heat treatment can be used to purify the protein. In addition, hydrolysis of transgenic rice straw with cultured cow gastric fluid for one hour at 39°C and another hour at 81°C yielded 43% more reducing sugars than wild type rice straw. Conclusion: Taken together, these data suggest that transgenic rice can effectively serve as a bioreactor for the large-scale production of active, thermostable cellulose hydrolytic enzymes. As a feedstock, direct expression of large amount of cellulases in transgenic rice may also facilitate saccharification of cellulose in rice straw and significantly reduce the costs for hydrolytic enzymes.

Acetic acid is a predominant by-product of lignocellulosic biofuel process, which inhibits microbial biocatalysts. Development of bacterial strains that are tolerant to acetic acid is challenging due to poor understanding of the underlying molecular mechanisms. In this study, we generated and characterized two acetic acid-tolerant strains of Zymomonas mobilis using N-methyl-N′-nitro-N-nitrosoguanidine (NTG)-acetate adaptive breeding. Two mutants, ZMA-142 and ZMA-167, were obtained, showing a significant growth rate at a concentration of 244 mM sodium acetate, while the growth of Z. mobilis ATCC 31823 were completely inhibited in presence of 195 mM sodium acetate. Our data showed that acetate-tolerance of ZMA-167 was attributed to a co-transcription of nhaA from ZMO0117, whereas the co-transcription was absent in ATCC 31823 and ZMA-142. Moreover, ZMA-142 and ZMA-167 exhibited a converstion rate (practical ethanol yield to theorical ethanol yield) of 90.16% and 86% at 195 mM acetate-pH 5 stress condition, respectively. We showed that acid adaptation of ZMA-142 and ZMA-167 to 146 mM acetate increased ZMA-142 and ZMA-167 resulted in an increase in ethanol yield by 32.21% and 21.16% under 195 mM acetate-pH 5 stress condition, respectively. The results indicate the acetate-adaptive seed culture of acetate-tolerant strains, ZMA-142 and ZMA-167, could enhance the ethanol production during fermentation.

Soy yogurt has been gaining popularity as a vegan food produced simply by soymilk fermentation with proper microbial manipulation. It is well known that soy containing rich isoflavones is beneficial for ameliorating hyperglycaemic disorders. Soy fermentation can improve the bioavailability of these precious nutrients. Lactiplantibacillus plantarum is one of the most abundant and frequently isolated species in soymilk manufacturing. Soy yogurts produced with efficient GABA (γ-aminobutyric acid)-producing L. plantarum and the deglycosylating activity of L. plantarum were functionally assessed in a STZ-induced hyperglycaemic mouse model. Hyperglycaemic mice were assigned into groups and treated with daily gavage of either dH₂O, soymilk, soy yoghurts produced with high GABA-producing L. plantarum GA30 (LPGA30), low GABA-producing L. plantarum PV30 (LPPV30) or the soy yoghurts fortified with additional 30 mg g⁻¹ GABA counterparts (GA + GABA and PV + GABA groups). Except the dH₂O group, all soy yoghurt groups retained body weight with improved glucose homeostasis, glucose tolerance test results and renal tissue integrity, while the soymilk group shows partial benefits. Plasma GABA concentrations in the daily soy yoghurt-supplemented groups (LPGA30 and LPPV30) plateaued at 5 times higher than the average 0.5 μM in dH₂O and soymilk groups, and their GABA-fortified soy yoghurt counterparts (GA + GABA and PV + GABA) groups were accountable for the restored plasma insulin levels. Gut microbiome analysis revealed dysbiosis in STZ-induced hyperglycemic mice of the dH₂O group with breached out facultative anaerobic Proteobacteria over the normal phyla Firmicutes and Bacteroidetes. Restored gut microbiota with transitionally populated Actinobacteria was demonstrated in the LPGA30 group but not in the LPPV30 group. Soy yoghurts produced with efficient GABA-producing L. plantarum GA30 showed exceptional benefits in modulating gut microbiota with dominant genera of Enterococcus, Lactobacillus and Bifidobacterium, and the presence of some minor beneficial microbial communities including Akkermansia muciniphila, Butyricicoccus pullicaecorum, Corynebacterium spp. and Adlercreutzia spp. Efficient GABA-producing L. plantarum GA30 fermented soymilk to produce soy yoghurts that exhibit profound synergistic protections over rich soy isoflavones to restore pancreatic β-cell functions for insulin production in STZ-induced hyperglycaemic mice. Additionally, the probiotic role of GABA-producing L. plantarum in re-establishing healthy gut microbiota in hyperglycaemic mice implies a possible symbiotic relationship, awaiting further exploration.

Soy isoflavones possess antioxidative, anti-inflammatory, anti-diabetic and phytoestrogenic properties. Soybean residue contains a fair amount of nutrients such as glycosylated isoflavones, minerals and dietary fibers, and is a substantial waste product produced from soymilk and tofu manufacturing. A solid-state fermentation of soybean residue by Rhizopus oligosporus or co-inoculated with Lactiplantibacillus plantarum improves the availability of isoflavones and GABA content which is attributed to ameliorated hyperglycemic symptoms in STZ-induced hyperglycemic mice. The effortless solid-state fermentation with present microbial manipulation supports an anti-hyperglycemia value-added application of soybean residue for functional food development. Background: Due to an awareness of the food crisis and with a rapidly rising prevalence of diabetes, recycling the substantial fibrous soybean residue disposed from soy industries has received consideration. Methods: Lactiplantibacillus plantarum was previously screened for active glutamate decarboxylase, and β-glucosidase activities were adopted for the fermenting of soybean residue using a traditional tempeh solid-state fermenting process with fungal Rhizopus oligosporus. Fermented soybean residue was chemically analyzed and functionally assessed in in vitro and in vivo hyperglycemic conditions. Results: A 48 h longer solid-state fermentation of the soybean residue co-inoculated with R. oligosporus and L. plantarum showed improved contents of isoflavone aglycones and GABA which were attributed to augmented antioxidative capacity, lowered ROS level, improved blood biochemistry, and better blood glucose homeostasis in STZ-induced hyperglycemic mice. Conclusion: The advantages of a food industrial effortless fermentation process, and a health nutritional endorsing anti-hyperglycemic value-added property offer a practical alternative in recycled soybean residue.

The study aimed to investigate the dose tolerance of enzymatically degraded feather meal (EFM) in the diet, and the effect of the two-stage fermented feather meal on the growth performance and amino acid digestibility of broilers. In trial 1, 160 one-day-old broilers were randomly assigned into 0, 10, 15, and 20% EFM groups. In trial 2, 160 one-day-old broilers were randomly assigned into control, 10% EFM, Bacillus subtilis var. natto N21 + B. coagulans L12 fermented EFM (BBEFM), and B. subtilis var. natto N21 + Saccharomyces cerevisiae Y10 fermented EFM (BSEFM) groups. Trial 3 involved 32 twenty-one-day-old male broilers randomly assigned into nitrogen-free diet, highly digestible protein, EFM, and BSEFM groups for a 7-day metabolic trial. During all of the feeding periods, increasing the EFM dosage in the diet linearly and quadratically inhibited weight gain (WG), feed intake, and feed conversion ratio (FCR) (p < 0.05), except the FCR at 22–35 days (p > 0.05). Dietary inclusion of more than 15% resulted in a negative impact on growth performance over days 1–35 (p < 0.05). Therefore, the EFM dose tolerance in the broiler diet is 10%. The WG, FCR, and production efficiency factor of the BSEFM group were better than those of the control group in days 1–35 (p < 0.05). The apparent and standardized ideal amino acid digestibility of BSEFM was higher than EFM in trial 3, except for Met, Cys, and Trp (p < 0.05). In conclusion, the EFM dose tolerance for the broiler diet is 10%. Bacillius subtilis var. natto N21 + S. cerevisiae Y10 fermentation can improve the amino acid digestibility of EFM and enhance broiler growth performance.