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Multispecies solid-state fermentation is a traditional processing technique for the traditional Chinese food, such as cereal vinegar, Baijiu, etc. Generally, few abundant and many rare microbes were involved in such processes, and the necessity and roles of the latter are less studied. Here the co-occurrence patterns of abundant and rare bacterial community and abiotic factors influencing their community assembly were investigated in acetic acid fermentation following starter inoculation, using Zhenjiang aromatic vinegar as a model system. Abundant taxa that contribute to the function of accumulating acid exhibited a ubiquitous distribution while the distribution of rare taxa along the fermentation process unraveled. The species composition of the rare taxa significantly altered, but abundant taxa were maintained after inoculation. Moreover, the diversity of rare taxa changed more significantly than that of abundant taxa. Both abundant and rare sub-communities, which were contributed more with species turnover than species richness, were demonstrated to be driven by pH, acetic acid, ammonium nitrogen, and ethanol. Stochastic processes regulated the assembly of both sub-communities, but more prominent on rare sub-communities. Co-occurrence network was more governed by rare sub-communities, and the co-variations between microbial communities were predominantly positive, implying that rare taxa played more important role in the fermentation stability and network robustness. Furthermore, seven network connectors were identified, and three of them belonged to rare taxa. These microbes of different modules were enriched at particular phases of fermentation. These results demonstrate the ecological significance of rare bacteria and provide new insights into understanding the abiotic factors influence microbial structure in traditional fermented foods.

The dietary supplementation of prebiotics is considered a promising strategy for the modulation of gut microbiota. Due to the wide variety of animal models and tremendous inter-individual variability from human investigations, the prebiotic effect of fibers is often difficult to compare between studies. Here, the effects of 11 dietary fibers on human fecal microbiota were studied using an in vitro human fecal fermentation model under well-controlled conditions. All fibers showed positive regulatory effects on short chain fatty acids (SCFAs) and several beneficial bacteria, including Parabacteroides distasonis and Bifidobacterium spp. Cultures supplemented with xylo-oligosaccharide and konjac flour showed the highest SCFAs. According to regulatory effects, fibers were divided into three groups, with 13 indicator OTUs (operational taxonomic units) identified. Fecal microbiota regulated by isomalto-oligosaccharide and chitosan-oligosaccharide were similar to fructo-oligosaccharide and inulin outputs. As a supplement to in vivo studies, our results comprehensively summarized the similarities and distinctiveness of fibers in regulating fecal microbiota structures. KEY POINTS: • Fibers were divided into three groups based on the regulatory effects in microbiota. • Thirteen indicator OTUs were identified using pairwise comparisons. • Fiber similarities and distinctive traits in regulating microbiota effect were identified.

Phosphatidylserine is widely used in food, health, chemical and pharmaceutical industries. The phospholipase D-mediated green synthesis of phosphatidylserine has attracted substantial attention in recent years. In this study, the phospholipase D was heterologously expressed in Bacillus subtilis, Pichia pastoris, and Corynebacterium glutamicum, respectively. The highest activity of phospholipase D was observed in C. glutamicum, which was 0.25 U/mL higher than these in B. subtilis (0.14 U/mL) and P. pastoris (0.22 U/mL). System engineering of three potential factors, including (1) signal peptides, (2) ribosome binding site, and (3) promoters, was attempted to improve the expression level of phospholipase D in C. glutamicum. The maximum phospholipase D activity reached 1.9 U/mL, which was 7.6-fold higher than that of the initial level. The enzyme displayed favorable transphosphatidylation activity and it could efficiently catalyze the substrates l-serine and soybean lecithin for synthesis of phosphatidylserine after optimizing the conversion reactions in detail. Under the optimum conditions (trichloromethane/enzyme solution 4:2, 8 mg/mL soybean lecithin, 40 mg/mL l-serine, and 15 mM CaCl2, with shaking under 40 °C for 10 h), the reaction process showed 48.6% of conversion rate and 1.94 g/L of accumulated phosphatidylserine concentration. The results highlight the use of heterologous expression, system engineering, and process optimization strategies to adapt a promising phospholipase D for efficient phosphatidylserine production in synthetic application.

An α-keratinase producing strain was isolated with wool as the sole carbon and nitrogen source and identified as Bacillus pumilus K9. The major amino acids liberated from the keratin degradation of wool by B. pumilus K9 were glutamic acid and leucine. The α-keratinase was purified to electrophoretic homogeneity with a molecular weight of 32000. The purified enzyme exhibits an optimum activity at 60 °C and pH=9.0. It was stable at pH values between 8 and 11. Bacillas pumilus keratinase displays a high activity towards casein, keratin, wool and feather, which indicates its wide application range. The keratinase was completely inhibited by phenylmethyl-sulfonyl fluoride(PMSF) and β-mercaptoethanol, and moderately inhibited by ethylemediamine-tetraacetic acid(EDTA), suggesting it is a metallo-cysteine keratinase. This enzyme could remain stable that could even be promoted in the presence of surfactants, including sodium dodecyl sulfate(SDS), Tween and Triton. And Tween 40 and Triton X-100 could substantially enhance the activity of the enzyme by 54% and 35%, respectively. It may indicate the prominent feature of the keratinase to tolerate surfactants. The enzymatic properties distinguish this keratinase from others in the literature. Furthermore, this enzyme is extremely stable in the presence of a commercially available detergent with 1% concentration. Detergents ARIEL, Bluemoon and WhiteCat can enhance the activity of the keratinase by 43.56%, 15.22%, and 22.48%, respectively.

Comparative studies of immobilized and free cells of Gibberella intermedia CA3-1 in bioconversion of 3-cyanopyridine to nicotinic acid were performed. Entrapping method was chosen based on the advantages in enzymatic activity recovery, mechanical strength and preparation procedure. Four entrapment matrices were investigated and sodium alginate was screened to be the most suitable material. Maximal nitrilase activity of alginate immobilized cells was obtained under conditions of 2 % alginate, 0.6 % CaCl2, 0.4 g cell/g alginate, 1.8 mm bead size. The immobilized cells showed excellent substrate tolerance even when the 3-cyanopyridine concentration was 700 mM. The half-lives of immobilized cells at 30, 40 and 50 °C were 315, 117.5 and 10.9 h, respectively, correspondingly 1.4, 1.6 and 1.7-fold compared with that of the free cells. Efficient reusability of immobilized cells up to 28 batches was achieved and 205.7 g/(g dcw) nicotinic acid was obtained with 80.55 % enzyme activity preserved.

Nitrilase is one of the most important members in the nitrilase superfamily and it is widely used for bioproduction of commodity chemicals and pharmaceutical intermediates as well as bioremediation of nitrile-contaminated wastes. However, its application was hindered by several limitations. Searching for new nitrilases and improving their application performances are the driving force for researchers. Genetic data resources in various databases are quite rich in post-genomic era. Besides, more than 99 % of microbes in the environment are unculturable. Metagenomic technology and genome mining are thus becoming burgeoning areas and provide unprecedented opportunities for searching more useful novel nitrilases due to the abundance of already existing but unexplored gene resources, namely uncharacterized genome information in the database and unculturable microbes in the natural environment. These techniques seem to be innovative and highly efficient. This study reviews the current status and future directions of metagenomics and genome mining in nitrilase exploration. Moreover, it discussed their utilization in coping with the challenges for nitrilase application. In the next several years, with the rapid development of nitrile biocatalysis, these two techniques would be bound to attract increasing attentions and even become a dominant trend for finding more novel nitrilases. Also, this review would provide guidance for exploitation of other commercially important enzymes.

Antrodia camphorata is a well-known Chinese medicinal mushroom that protects against diverse health-related conditions. Submerged fermentation of A. camphorata is an alternative choice for the effective production of bioactive metabolites, but the effects of nutrition and environment on mycelial morphology are largely unknown. In this study, we show that A. camphorata American Type Culture Collection 200183 can form arthrospores in the end of liquid fermentation. Different morphologies of A. camphorata in submerged culture were analyzed using scanning electron microscopy. The optimal carbon and nitrogen sources for sporulation were soluble starch and yeast extract. We found that a carbon-to-nitrogen ratio (C/N) of 40:1, MgSO4 (0.5 g/l), KH2PO4 (3.0 g/l), an initial pH 5.0, and an inoculum size of 1.5×10(5) spores/ml led to maximum production of arthroconidia. Our results will be useful in the regulation and optimization of A. camphorata cultures for efficient production of arthroconidia in submerged culture, which can be used as inocula in subsequent fermentation processes.

Microbial transformation of glycinonitrile into glycine by nitrile hydrolase is of considerable interest to green chemistry. A novel fungus with high nitrile hydrolase was newly isolated from soil samples and identified as Fusarium oxysporum H3 through 18S ribosomal DNA, 28S ribosomal DNA, and the internal transcribed spacer sequence analysis, together with morphology characteristics. After primary optimization of culture conditions including pH, temperature, carbon/nitrogen sources, inducers, and metal ions, the enzyme activity was greatly increased from 326 to 4,313 U/L. The preferred carbon/nitrogen sources, inducer, and metal ions were glucose and yeast extract, caprolactam, and Cu(2+), Mn(2+), and Fe(2+), respectively. The maximum enzyme formation was obtained when F. oxysporum H3 was cultivated at 30 °C for 54 h with the initial pH of 7.2. There is scanty report about the optimization of nitrile hydrolase production from nitrile-converting fungus.

Surfactant-stable keratinases with good properties are promising candidates for extensive applications in detergent industries. A novel fungal keratinase-producing strain, Gibberella intermedia CA3-1, is described in this study. The keratinase production medium was optimized and composed of 10 g L−1 of wool powder, 5 g L−1 of tryptone, 10 g L−1 of maltodextrin and 0.5 g L−1 of NaCl. Keratinase activity was increased up to 109 U mL−1 from 15 U mL−1 by culture optimization. The optimal reaction pH and temperature of the enzyme were 9.0 and 60°C, respectively. The keratinase activity could be improved by sodium dodecyl sulphate (SDS), and it remained stable in the presence of several surfactants and commercial detergents. G. intermedia keratinase was proved to completely remove blood stains from cotton cloth when combined with detergents. These findings indicate that this fungal keratinase is a promising catalyst for the application in detergent industry. To our knowledge, this is the first report on keratinase production by Gibberella genus.

Pseudomonas putida CGMCC3830 harboring nitrilase was used in isonicotinic acid production from 4-cyanopyridine. This nitrilase showed optimum activities towards 4-cyanopyridine at pH 7.5 and 45°C. The half-life of P. putida nitrilase was 93.3 h, 33.9 h, and 9.5 h at 30°C, 38°C, and 45°C, respectively. 4-Cyanopyridine (100 mM) was fully converted into isonicotinic acid within 20 min. The bench-scale production of isonicotinic acid was carried out using 3 mg of resting cells per mL in a 1 L system at 30°C and finally, 123 g L−1 of isonicotinic acid were obtained within 200 min without any by-products. The conversion reaction suffered from the product inhibition effect after the tenth feeding. The volumetric productivity was 36.9 g L−1 h−1. P. putida shows significant potential in nitrile hydrolysis for isonicotinic acid production. This paper is the first report on isonicotinic acid biosynthesis using Pseudomonas putida and it represents the highest isonicotinic acid production reported so far.