Purification and characterization of solvent stable,alkaline protease from Bacillus firmus CAS 7 by microbial conversion of marine wastes and molecular mechanism underlying solvent stability

A marine bacterium Bacillus firmus CAS 7 produced protease in the medium supplemented with 3:1 shrimp and crab shell powder at 55 °C and was purified with the specific activity of 473.4 U/mg. The purified protease was highly stable up to 70 °C, pH 11.0 and 30% NaCl. The protease purified was quite stable in the presence of anionic and non-ionic surfactants and organic solvents. The molecular dynamics simulation confirmed that the competition between organic solvent and water for the enzyme surface was comparatively higher in water–miscible organic solvent which is responsible for organic solvent stability. The purified protease from B. firmus CAS 7 could be greatly useful to develop industrial processes performed under harsh conditions or with denaturants and organic solvents. The protease production by microbial conversion of marine wastes suggested its potential utilization to generate high value-added products.     

<Emphasis Type="Italic">Polyporus tenuiculus</Emphasis>: a new naturally occurring mushroom that can be industrially cultivated on agricultural waste

Polyporus tenuiculus is a naturally occurring species from Central and South America that is consumed by different ethnic groups in the region. To determine the optimal conditions for fruiting body production, two strains were assayed on wheat straw and sawdust with or without supplements. Sixty days of incubation at 25°C were needed to produce a solid block. The highest yield was obtained with strain ICFC 383/00 grown on supplemented willow sawdust. In a second experiment the strain ICFC 383/00 and different supplements were used to improve the biological efficiency (BE) and to determine the quality traits and its biodegradation capacity. The highest yields were obtained on sawdust with 25% of supplements reaching 82.7% of BE. Supplements raised the number of flushes, generally from four to five, contributing to increased yields. The type of substrate had a significant effect on fruiting body diameters of P. tenuiculus, and the largest mushrooms were harvested on supplemented substrate with the highest BE coinciding with the highest dry matter loss in substrates. P. tenuiculus showed a capacity to degrade sawdust, causing a decrease of 67.2–74.5% in cellulose, 80.4–85.7% in hemicellulose, and 60.6–66.2% in lignin content at the end of the cultivation cycle. The decrease in hemicellulose was relatively greater than that of cellulose and lignin on supplemented substrates. This is the first report of the cultivation of P. tenuiculus on lignocellulosic waste, and it is a promising species both for commercial production and for its potential use in the degradation of other biowastes.     

Bioremediation of Agricultural Wastes through Vermicomposting

The post-harvest residues of some local crops, e.g., wheat (Triticum aestivum), millets (Penniseum typhoides and Sorghum vulgare), and a pulse (Vigna radiata) were subjected to recycle through vermicomposting. The crop residues were amended with animal dung, and three types of vermibeds were prepared: (i) millet straw (S. vulgare + P. typhoides in equal quantity) + sheep manure (1: 2 ratio) (MS); (ii) pulse bran (V. radiata) + wheat straw (T. aestivum) + cow dung (1: 1: 2 ratio) (PWC); and (iii) mixed crop residues (mixing of all types crop residues, used in this study) + cow dung in 1:1 ratio (MCR + CD). The fourth treatment was cattle shed manure (CSM). Vermicomposting resulted in a significant increase in total N (97.3% to 155%), available P (67.5% to 123.5%), exchangeable K (38.3% to 112.9%), and exchangeable Ca (23.3% to 53.2%), and decrease in organic C content (20.4% to 29.0%) in the different vermibeds. The earthworm showed the higher biomass gain, growth rate (mg wt. worm^?1 day^?1) and cocoon numbers in the CSM vermibed. The quality of crop residues was directly related to the rate of organic matter mineralization during the vermicomposting. This study suggests that agriculture wastes could be converted into some value-added products, e.g., vermicompost and worm biomass through vermicomposting technology. The higher concentrations of plant nutrients in end products indicate a potential for using agriculture wastes in sustainable crop production.     

Construction and evaluation of self‐cloning bottom‐fermenting yeast with high SSU1 expression

Aim: To construct a self‐cloning brewer’s yeast that can minimize the unfavourable flavours caused by oxidation and certain kinds of sulfur compounds. Methods and Results: DNA fragments of a high‐expression promoter from the TDH3 gene originating from Saccharomyces cerevisiae were integrated into the promoter regions of the S. cerevisiae‐type and Saccharomyces bayanus‐type SSU1 genes of bottom‐fermenting brewer’s yeast. PCR and sequencing confirmed the TDH3 promoter was correctly introduced into the SSU1 regions of the constructed yeasts, and no foreign DNA sequences were found. Using the constructed yeasts, the concentration of sulfite in fermenting wort was higher when compared with the parent strain. In addition, the concentrations of hydrogen sulfide, 3‐methyl‐2‐buten‐1‐thiol (MBT) and 2‐mercapto‐3‐methyl‐1‐butanol (2M3MB) were lower when compared with the parent strain. Conclusion: We successfully constructed a self‐cloning brewer’s yeast with high SSU1 expression that enhanced the sulfite‐excreting ability and diminished the production ability of hydrogen sulfide, MBT and 2M3MB. Significance and Impact of the Study: The self‐cloning brewer’s yeast with high SSU1 expression would contribute to the production of superior quality beer with a high concentration of sulfite and low concentrations of hydrogen sulfide, MBT and 2M3MB.     

Laccase and Manganese Peroxidase Activities of Phellinus Robustus and Ganoderma Adspersum Grown on Food Industry Wastes in Submerged Fermentation

Phellinus robustus produced both laccase (700–4,000 U l⁻¹) and manganese peroxidase (MnP) (1,000–11,300 U l⁻¹) in fermentation of nine food wastes, whereas Ganoderma adspersum produced only laccase (600–34,000 U l⁻¹). Glucose provided high laccase and MnP activity of P. robustus but repressed enzyme production by G. adspersum. Ammonium sulphate and ammonium tartrate increased the P. robustus laccase yield (3-fold), whereas the accumulation of MnP was not enhanced by additional nitrogen.     

虻粪二次堆肥生化特性的动态变化趋势及其微生物演替规律

[目的] 通过培养试验动态观察了虻粪在后续二次堆肥过程中生化特性的动态变化趋势,研究了堆体微生物生态的动态演替规律,讨论了虻粪二次堆肥的稳定化机理。[方法] 采取盛有虻粪的圆桶埋入大条垛发酵堆体的动态培养试验,在为期49 d的测试期内间隔7 d动态采样,采用16S rRNA高通量测序开展虻粪堆体样品的微生物多样性与群落结构分析以及代谢功能预测。[结果] 虻粪堆体经历2周快速升温至68.7℃后,逐步降温至第49天的37.7℃,总有机碳、溶解性有机质（DOM）、pH分别达到67.3%、18.4 g/kg、8.0。在堆体熟腐过程中,Shannon指数、Simpson指数和Chao1指数等微生物多样性水平均随堆肥进程而快速增加,而后缓慢下降至结束时的7.45、0.98和985。16S rRNA测序发现,Bacteroidetes（拟杆菌门）、Firmicutes（厚壁菌门）、Proteobacteria（变形菌门）与Actinobacteria（放线菌门）等是堆体微生物群落的主要菌群。与此同时,Corynebacteriaceae（棒状杆菌科）从初始的35.7%下降至结束时的6.5%,Flavobacteriaceae（黄杆菌科）则从3.22%逐步增长至12.6%,Porphyromonadaceae（单胞菌科）快速增加至11.5%之后缓慢下降至与初始水平相当的5.1%。PICRUSt预测发现,在整个虻粪堆肥过程中,氨基糖和核苷酸糖代谢、碳固定通路、甲烷代谢等功能平均相对丰度保持较强水平,且均在堆肥中期（第15–35天）出现峰值,后续均呈现下降态势。脂肪代谢、不饱和脂肪酸生物合成代谢、多环芳烃降解以及硫代谢等平均相对丰度较弱的代谢功能呈现先降后升的特征。[结论] 虻粪二次堆肥过程中,物料含水量与DOM快速下降、有机质降解逐步完成以及物料微生物结构与功能趋于稳定等,堆制腐熟后的虻粪生化性质稳定,有利于虻粪有机肥农田再利用。     

Effects of Composts from Agroindustrial Wastes on Microbial Activity of a Typic Xerofluvent Soil Under Mediterranean Conditions,SE Turkey

Supplementing the nutrient requirement of crops through organic manures as compost derived from agroindustrial wastes plays a key role in sustaining soil fertility, and crop productivity and reducing use of chemical fertilizers. Therefore, this work was conducted for investigating the effects of addition of oily cumin compost (CC) and oily oregano compost (OC) (these composts were derived from oily cumin and oily oregano wastes of aromatic plant factory) at rates of 40 t ha^?1 to identify those potential organic amendments that might improve the quality of an Entisol. Additionally, those effects on the biochemical properties of a Typic xerofluvent soil were compared to chemical fertilization (CF) and also control (CT) during a cotton vegetation period under a Mediterranean climatic condition. Soil biological status was evaluated by measuring the soil microbial biomass carbon (MBC), basal soil respiration (BSR), N-mineralization (N_min), soil metabolic quotient (qCO₂) and soil enzymatic activities (dehydrogenase-DHG, urease-UA, protease-PRO, and alkaline phosphate-ALKPA) in soil samples that were collected on the 19th, 78th and 190th days followed by compost application to the experimental soils. The MBC, BSR and qCO₂, as well as soil enzyme activities, increased significantly in the compost-treated soils compared with the CF-treated soil and nontreated soils (CT) with respect mean values. The level of microbial activity of soil applied chemical fertilizer was almost the same to those of control soil. As a result of cumin compost (CC) application 137-1810% increase of the level of microbial activity with respect to the CT and CF, followed by OC, 47-314% occurred at the end of the experiment. Because of this there were no toxic effects caused by composts observed. The application of these composts to the soil resulted in the most increase in DHG activity significantly. The application of CC with a C/N ratio of 23 resulted a more favorable soil biological properties than the application of OC (C/N ratio = 32) during cotton vegetation period (190 days). Results from this study suggest that composted aromatic plant wastes can be used to enhance the soil microbial activity, thereby promoting plant growth.     

Thermophilic co-digestion of organic fraction of municipal solid wastes with FOG wastes from a sewage treatment plant: reactor performance and microbial community monitoring

Working at thermophilic conditions instead of mesophilic, and also the addition of a co-substrate, are both the ways to intend to improve the anaerobic digestion of the source-collected organic fraction of municipal solid wastes (SC-OFMSW). Addition of sewage treatment plant fat, oil and grease wastes (STP-FOGW), that are nowadays sent to landfill, would represent an opportunity to recover a wasted methane potential and, moreover, improve the whole process. In this study, after a first period feeding only SC-OFMSW, a co-digestion step was performed maintaining thermophilic conditions. During the co-digestion period enhancements in biogas production (52%) and methane yield (36%) were achieved. In addition, monitoring of microbial structure by using PCR-DGGE and cloning techniques showed that bacterial community profiles clustered in two distinct groups, before and after the extended contact with STP-FOGW, being more affected by the STP-FOGW addition than the archaeal one.     

Enhancement options for the utilisation of nitrogen rich animal by-products in anaerobic digestion

This study focuses on the enhancement of an Austrian anaerobic digestion plant at a slaughterhouse site which exclusively uses animal by-products as substrate. High ammonia concentrations from protein degradation cause severe inhibitions of anaerobic microorganisms. For improving the current situation the COD:TKN ratio is widened by (a) ammonia stripping directly out of the process and (b) addition of a C source to the substrate. Different OLR and HRT were tested in continuous experiments to simulate new operating conditions. The results show that the addition of carbon cannot improve fermentation capacity. The reduction of ammonia boosts the degradation: After reduction of TKN from 7.5 to 4.0 g kg⁻¹ the initially high VFA concentration decreased and the COD degradation was improved by 55.5%. Hence, the implementation of the new N reduction process facilitates either the increase of the OLR by 61% or the reduction of the HRT by 25%.