Variation in microbial population during composting of agro-industrial waste

Two compost piles were prepared, using two ventilation systems: forced ventilation and ventilation through mechanical turning. The material to compost was a mixture of orange waste, olive pomace, and grass clippings (2:1:1 v/v). During the composting period (375 days), samples were periodically taken from both piles, and the enumeration of fungi, actinomycetes, and heterotrophic bacteria was carried out. All studied microorganisms were incubated at 25 and 55 °C after inoculation in appropriate growth media. Fungi were dominant in the early stages of both composting processes; heterotrophic bacteria proliferated mainly during the thermophilic stage, and actinomycetes were more abundant in the final stage of the composting process. Our results showed that the physical and chemical parameters: temperature, pH, moisture, and aeration influenced the variation of the microbial population along the composting process. This study demonstrated that composting of these types of wastes, despite the prolonged mesophilic stage, provided an expected microbial variation.     

一株分离自绿化废弃物堆肥枯草芽孢杆菌的常压室温等离子诱变及其发酵条件

【背景】为了提高堆肥降解有机废弃物的效率,高效堆肥菌剂成为了研究热点,其中以真菌应用的研究为多,但真菌也有对氧气和底物敏感等缺点,细菌对堆肥的作用开始被研究。本实验室以羧甲基纤维素钠(CMC-Na)为底物,从绿化废弃物堆肥中筛选得到枯草芽孢杆菌(Bacillussubtilis,B.subtilis) BL03,它具有较好的纤维素分解能力,能提高绿化废弃物堆肥中纤维素降解和腐殖质合成的速度。【目的】进一步提高B.subtilisBL03的纤维素酶生产能力。【方法】利用常压室温等离子(Atmospheric and room temperature plasma,ARTP)诱变BL03菌,通过CMC-刚果红固体培养基观察水解透明圈,以及液体发酵后检测酶活力的方法进行3轮筛选;通过连续多代培养观察突变株的遗传稳定性;通过梯度温度、p H培养研究突变株发酵的最适生长温度、培养基初始pH;利用正交设计方法研究适合突变株发酵培养的工业级原料配方。【结果】筛选到2株正突变株,酶活力分别提高了69%和72%;连续10代培养稳定,验证了突变株的遗传稳定性;其中酶活力最高的突变株BLA3890最适培养温度为37°C、培养基初始pH为5.0-6.5,研究得到较经济的发酵培养基配方。【结论】ARTP诱变B. subtilis BL03后得到的突变株BLA1973和BLA3890在绿化废弃物堆肥或其他纤维素降解行业具有进一步研究和应用的价值。     

Low pH as an inhibiting factor in the transition from mesophilic to thermophilic phase in composting

During composting of household waste, the acidity of the material affects the process during the initial phase of rising temperature. In this study, the effects of temperature (36-46 degrees C) and pH (4.6-9.2) on the respiration rate during the early phase of composting were investigated in two different composts. A respiration method where small compost samples were incubated at constant temperature was used. The respiration rate was strongly reduced at 46 degrees C and pH below 6, compared to composts with a higher pH or lower temperature. The combination of high temperature and low pH is a possible adverse factor in large-scale composting of food waste.     

静态好氧高温牛粪堆肥中nirK型反硝化细菌群落动态变化

【背景】堆肥过程中,不同反硝化微生物相互作用产生大量气态氮,不仅导致氮素流失使得堆肥肥效降低,而且造成环境污染。但是目前关于堆肥中反硝化细菌群落结构变化,尤其是群落结构与堆肥理化因子间相关性方面的报道较为欠缺。【目的】对堆肥中反硝化细菌进行研究,旨在揭示反硝化细菌群落动态变化,为深入理解堆肥氮循环机理提供科学数据。【方法】设计一种静态好氧高温堆肥技术处理牛粪和水稻秸秆,利用高通量测序技术研究堆肥中nirK型反硝化细菌群落组成的动态变化,并分析优势反硝化细菌菌属与理化指标之间的相关性。【结果】堆肥全程共17d,各项堆肥理化指标以及生物学指标表明堆肥已经基本腐熟。高通量测序结果表明,在堆肥的不同阶段nirK型反硝化细菌群落结构差异显著。门水平上,堆肥中反硝化细菌属于变形菌门(Proteobacteria)和一未分类门;目水平上,优势类群主要属于根瘤菌目(Rhizobiales)、红杆菌目(Rhodobacterales)和伯克氏菌目(Burkholderiales),其中根瘤菌目(Rhizobiales)的种类最多,而伯克氏菌目(Burkholderiales)的相对丰度最高。Spearman相关性分析表明未分类门的反硝化细菌和未分类科根瘤菌目的反硝化细菌与全碳、碳氮比、含水率以及pH呈显著负相关(P0.05),与凯氏氮和硝态氮呈显著正相关(P0.05);其他优势菌属与全碳、碳氮比、含水率以及pH呈显著正相关(P0.05),与凯氏氮和硝态氮呈显著负相关(P0.05);未分类科伯克氏菌目的反硝化细菌、未分类纲变形菌门的反硝化细菌、产碱杆菌科的Pusillimonas属和副球菌属(Paracoccus)与铵态氮显著相关(P0.05)。【结论】静态好氧高温堆肥技术可以缩短堆肥周期。在堆肥的不同阶段nirK型反硝化细菌群落结构差异显著,并且该菌群落结构的变化受到堆肥理化因子的显著影响。本研究有助于揭示堆肥中氮素转化规律,并为改进堆肥工艺提供理论依据。     

Microbial characterization during composting of municipal solid waste

This study investigates the prevailing physico-chemical conditions and microbial community; mesophilic bacteria, yeasts and filamentous fungi, bacterial spores, Salmonella and Shigella as well as faecal indicator bacteria: total coliforms, faecal coliforms and faecal Streptococci, present in a compost of municipal solid waste. Investigations were conducted in a semi-industrial pilot plant using a moderate aeration during the composting process. Our results showed that: (i) auto-sterilization induced by relatively high temperatures (60–55°C) caused a significant change in bacterial communities. For instance, Escherichia coli and faecal Streptococci populations decreased, respectively, from 2×10⁷ to 3.1×10³ and 10⁷ to 1.5×10³ cells/g waste dry weight (WDW); yeasts and filamentous fungi decreased from 4.5×10⁶ to 2.6×10³ cells/g WDW and mesophilic bacteria were reduced from 5.8×10⁹ to 1.8×10⁷ bacteria/g WDW. On the other hand, the number of bacterial spores increased at the beginning of the composting process, but after the third week their number decreased notably; (ii) Salmonella disappeared completely from compost by the 25th day as soon as the temperature reached 60°C; and (iii) the bacterial population increased gradually during the cooling phase. While Staphylococci seemed to be the dominant bacteria during the mesophilic phase and at the beginning of the thermophilic phase, bacilli predominated during the remainder of the composting cycle. The appearance of gram-negative rods (opportunistic pathogens) during the cooling phase may represent a serious risk for the sanitary quality of the finished product intended for agronomic reuse. Compost sonication for about 3 min induced the inactivation of delicate bacteria, in particular gram-negatives. By contrast, gram-positive bacteria, especially micrococcus, spores of bacilli, and fungal propagules survived, and reached high concentrations in the compost.     

The study of the aerobic bacterial microbiota and the biotoxicity in various samples of olive mill wastewaters (alpechin) during their composting process

Five different piles were prepared by mixing olive mill wastewater (alpechin) and alpechin sludge with two bulking agents (cotton waste and maize straw) and two organic wastes with high content of nitrogen (sewage sludge and poultry manure), which were composted by the Rutgers static pile composting system in a pilot plant. The aim of this work was to study the evolution of total nitrogen and different forms of organic matter and evaluate the variation in the aerobic bacterial microbiota present and biotoxicity during the composting process.In piles prepared with alpechin, the use of the maize straw as a bulking agent reduced the nitrogen losses whereas the use of sewage sludge, instead of poultry manure, with cotton waste originated the highest degradation of organic matter. In piles prepared with alpechin sludge a similar evolution of the composting process was observed. There were not great variations during composting in the aerobic bacterial microbiota present in the mixtures. However, the pile prepared with alpechin sludge and maize straw was only one to present bacteria capable of growing in alpechin, and the toxicity study showed that this was only present in the starting mixtures.     

鸡粪好氧堆肥过程中细菌群落结构和功能分析

为了明确鸡粪好氧堆肥过程中细菌群落结构和功能的变化,采用高通量测序技术测定了好氧堆肥前、中、后3个时期样品的16S rRNA基因序列,并进行了生物信息学分析。结果表明, 3个堆肥阶段中仅有10%左右的分类操作单元(operational taxonomic units, OTUs)具有阶段特异性;不同发酵阶段细菌α多样性指数ACE、Chao1和Simpson均呈现先升高后降低的趋势,但各阶段间差异不显著(P<0.05)。3个发酵阶段优势菌门类相同,但丰度存在差异。线性判别分析[line discriminant analysis (LDA) effect size, LEfSe]法对细菌生物标志分析表明,从门到属水平共有49个物种,堆肥前期样品组中显著富集的物种最多,后期最少。原核分类群功能注释(functional annotation of prokaryotic taxa,FAPROTAX)对细菌功能多样性分析表明,堆肥前期细菌功能多样性最高,而随着发酵进行细菌功能富集程度增加、多样性降低。该研究为畜禽粪污好氧堆肥过程调控提供理论支撑和技术指导。     

Successions of bacterial community in composting cow dung wastes with or without hyperthermophilic pre-treatment

Comparative analyses of bacterial community successions in the composting materials were done for a conventional windrow post-treatment (WPOT) process with the hyperthermophilic pre-treatment (HTPRT) and simple windrow composting (SWC; without the HTPRT). Multidimensional scaling profiles based on data of terminal restriction fragment length polymorphisms of the bacterial population in the samples of every 7 days composting material and analyses of the 16S rRNA gene-based clone library of the 7 and 21 days composting materials suggested that bacterial communities of the composting materials differed much between these two processes until the 35 days of composting, whereas that they were closely related to each other at the final composting stage (42 days of composting). Detailed phylogenetic analysis clarified that all WPOT clone libraries contained many clones of the lineages of aerobic bacteria (for example, bacilli). However, the most abundant clones retrieved from all SWC materials were affiliated with a clone cluster closely related to identified and classified members of the phylum Firmicutes that have strictly anaerobic metabolism pathways. From these results, we conclude that the HTPRT process contributed to easily establish an aerobic ecosystem from the early stage to the final stage of WPOT composting with plowing the materials only once a week.     

The influence of yeast on thermophilic composting of food waste

The microbial changes during thermophilic composting of food waste were investigated at 50 °C using a laboratory-scale composter. The results showed that an early increase in the growth of yeast was followed by the growth of thermophilic bacteria, and that subsequently, the yeast declined rapidly. The growth interactions of selected strains of yeast and bacteria were studied in culture media containing organic acids. The results demonstrated that the acidity of the media was reduced during the growth of the yeast. Therefore, it is concluded that in the early stages of composting, the growth of thermophilic bacteria is inhibited by organic acids, and that the growth of yeast enables subsequent bacterial growth by removing organic acids from food waste.     

Long-term survival of pathogenic and sanitation indicator bacteria in experimental biowaste composts

For economic, agricultural, and environmental reasons, composting is frequently used for organic waste recycling. One approach to limiting the potential risk from bacterial food-borne illnesses is to ensure that soil amendments and organic fertilizers are disinfected. However, more knowledge concerning the microbiological safety of composted substrates other than sludge and manure is necessary. Experimental in-vessel biowaste composts were used to study the survival of seeded Listeria monocytogenes, Salmonella enterica subsp. enterica serotype Enteritidis, and Escherichia coli. Four organic waste mixtures, containing various proportions of paper and cardboard, fruits and vegetables, and green waste, were composted in laboratory reactors with forced aeration. The physicochemical and microbiological parameters were monitored for 12 weeks during composting. The survival of bacteria over a 3-month period at 25 degrees C was assessed with samples collected after different experimental composting times. Strain survival was also monitored in mature sterilized composts. Nonsterile composts did not support pathogen growth, but survival of seeded pathogens was observed. Salmonella serovar Enteritidis survived in all composts, and longer survival (3 months) was observed in mature composts (8 and 12 weeks of composting). Mature biowaste composts may support long-term survival of Salmonella serovar Enteritidis during storage at room temperature. E. coli and L. monocytogenes survival was observed only in 4-week-old composts and never in older composts. Proper composting may prevent long-term survival of E. coli and L. monocytogenes. These results suggest that like composted sewage sludge or manure, domestic waste composts may support pathogen survival. Survival was not related to the physicochemical characteristics of the composts.     

Succession of microbial communities during hot composting as detected by PCR-single-strand-conformation polymorphism-based genetic profiles of small-subunit rRNA genes

A cultivation-independent technique for genetic profiling of PCR-amplified small-subunit rRNA genes (SSU rDNA) was chosen to characterize the diversity and succession of microbial communities during composting of an organic agricultural substrate. PCR amplifications were performed with DNA directly extracted from compost samples and with primers targeting either (i) the V4-V5 region of eubacterial 16S rRNA genes, (ii) the V3 region in the 16S rRNA genes of actinomycetes, or (iii) the V8-V9 region of fungal 18S rRNA genes. Homologous PCR products were converted to single-stranded DNA molecules by exonuclease digestion and were subsequently electrophoretically separated by their single-strand-conformation polymorphism (SSCP). Genetic profiles obtained by this technique showed a succession and increasing diversity of microbial populations with all primers. A total of 19 single products were isolated from the profiles by PCR reamplification and cloning. DNA sequencing of these molecular isolates showed similarities in the range of 92.3 to 100% to known gram-positive bacteria with a low or high G+C DNA content and to the SSU rDNA of gamma-Proteobacteria. The amplified 18S rRNA gene sequences were related to the respective gene regions of Candida krusei and Candida tropicalis. Specific molecular isolates could be attributed to different composting stages. The diversity of cultivated bacteria isolated from samples taken at the end of the composting process was low. A total of 290 isolates were related to only 6 different species. Two or three of these species were also detectable in the SSCP community profiles. Our study indicates that community SSCP profiles can be highly useful for the monitoring of bacterial diversity and community successions in a biotechnologically relevant process.     

Metaproteomics reveals major microbial players and their biodegradation functions in a large-scale aerobic composting plant

Composting is an appropriate management alternative for municipal solid waste; however, our knowledge about the microbial regulation of this process is still scare. We employed metaproteomics to elucidate the main biodegradation pathways in municipal solid waste composting system across the main phases in a large-scale composting plant. The investigation of microbial succession revealed that Bacillales, Actinobacteria and Saccharomyces increased significantly with respect to abundance in composting process. The key microbiologic population for cellulose degradation in different composting stages was different. Fungi were found to be the main producers of cellulase in earlier phase. However, the cellulolytic fungal communities were gradually replaced by a purely bacterial one in active phase, which did not support the concept that the thermophilic fungi are active through the thermophilic phase. The effective decomposition of cellulose required the synergy between bacteria and fungi in the curing phase.     

Strategies to reduce short-chain organic acids and synchronously establish high-rate composting in acidic household waste

The aim of this study was to document whether addition of lime or increased amount of bulking agent would ensure, efficiently, a predictable composting process in acidic SSOW applicable in full scale plants. The results show that both lime addition and increasing the amount of bulking agent relative to waste support the development of high-rate respiration in composting. Both strategies are considered efficient in establishing desired microbial composting processes of acid household waste. Reduction in the content of different organic acids and loss on ignition were higher when more bulking agent was used compared with adding 5% lime to the acidic SSOW. Respiration was completely repressed in samples with 10% lime, where pH remained high. In addition fat and protein seem to degrade faster with increasing amount of bulking agent.     

DGGE and T-RFLP Analysis of Bacterial Succession during Mushroom Compost Production and Sequence-aided T-RFLP Profile of Mature Compost

The amount of button mushroom (Agaricus bisporus) harvested from compost is largely affected by the microbial processes taking place during composting and the microbes inhabiting the mature compost. In this study, the microbial changes during the stages of this specific composting process were monitored, and the dominant bacteria of the mature compost were identified to reveal the microbiological background of the favorable properties of the heat-treated phase II mushroom compost. 16S ribosomal deoxyribonucleic acid (rDNA)-based denaturing gradient gel electrophoresis (DGGE) and terminal restriction fragment length polymorphism (T-RFLP) molecular fingerprinting methods were used to track the succession of microbial communities in summer and winter composting cycles. DNA from individual DGGE bands were reamplified and subjected to sequence analysis. Principal component analysis of fingerprints of the composting processes showed intensive changes in bacterial community during the 22-day procedure. Peak temperature samples grouped together and were dominated by Thermus thermophilus. Mature compost patterns were almost identical by both methods (DGGE, T-RFLP). To get an in-depth analysis of the mature compost bacterial community, the sequence data from cultivation of the bacteria and cloning of environmental 16S rDNA were uniquely coupled with the output of the environmental T-RFLP fingerprints (sequence-aided T-RFLP). This method revealed the dominance of a supposedly cellulose-degrading consortium composed of phylotypes related to Pseudoxanthomonas, Thermobifida, and Thermomonospora.     

Bacterial community profiles on feathers during composting as determined by terminal restriction fragment length polymorphism analysis of 16S rDNA genes

Composting is one of the more economical and environmentally safe methods of recycling feather waste generated by the poultry industry, since 90% of the feather weight consists of crude keratin protein, and feathers contain 15% N. However, the keratin in waste feathers is resistant to biodegradation and may require the addition of bacterial inocula to enhance the degradation process during composting. Two keratin-degrading bacteria isolated from plumage of wild songbirds and identified as Bacillus licheneformis (OWU 1411T) and Streptomyces sp. (OWU 1441) were inoculated into poultry feather composts (1.13×10⁸ cfu g^–1 feathers) and co-composted with poultry litter and straw in 200-l compost vessels. Composting temperatures, as well as CO₂ and NH₃ evolution, were measured in these vessels to determine the effects of inoculation on the rate and extent of poultry feather decomposition during composting. Terminal restriction fragment length polymorphisms of 16S rRNA genes were used to follow changes in microbial community structure during composting. The results indicated that extensive carbon conversion occurred in both treatments (55.5 and 56.1%). The addition of the bacterial inocula did not enhance the rate of waste feather composting. The microbial community structure over time was very similar in inoculated and uninoculated waste feather composts.     

Vermicompost derived from mushroom residues improves soil C/P cycling,bacterial community, and fungal abundance

The utilization of agricultural waste organic materials through composting technology has gained significant traction in agricultural production as an effective means of crop nutrient management. However, the differences in the impact of organic amendments prepared by traditional composting and vermicomposting on soil properties still deserve further research. Based on field experiments conducted in greenhouse, compared to chemical fertilizer treatments as control, we utilized traditional compost (OF) and vermicompost (VcF) derived from agricultural organic waste edible mushroom bran and cow manure (2:8). Variations in soil physiochemical properties, activities of soil enzymes related C and P cycling, abundances and diversities of bacterial 16S rRNA and fungal ITS gene at total DNA level were analyzed. Both compost treatments enhanced soil organic carbon, soil total phosphorus, and soil available P content significantly and also increased the activities of soil α-glucosidase, β-glucosidase, acid phosphomonoesterase, and alkaline phosphomonoesterase significantly. The above results suggested that soil C and P transformations were stimulated effectively by both organic amendments. OF and VcF increased the fungal ITS absolute abundances significantly while diversity indices of soil bacterial community increased significantly under both treatments. Correlation analysis indicated that bacterial community composition was strongly correlated with several soil property indexes while fungal community composition was only significantly correlated with soil total phosphorous content. In conclusion, similar to traditional compost, vermicompost significantly improved soil nutrient cycling (especially C and P aspects). In terms of soil microbes, bacteria and fungi showed different responding mechanism to vermicompost: bacteria adjust microbial structure, while fungi tend to proliferated. In consideration of the advantages of vermicompost in technology and economic cost, it could be applied in the subsequent agricultural production more frequently.     

Diversity of Two-Domain Laccase-Like Multicopper Oxidase Genes in Streptomyces spp.: Identification of Genes Potentially Involved in Extracellular Activities and Lignocellulose Degradation during Composting of Agricultural Waste

Traditional three-domain fungal and bacterial laccases have been extensively studied for their significance in various biotechnological applications. Growing molecular evidence points to a wide occurrence of more recently recognized two-domain laccase-like multicopper oxidase (LMCO) genes in Streptomyces spp. However, the current knowledge about their ecological role and distribution in natural or artificial ecosystems is insufficient. The aim of this study was to investigate the diversity and composition of Streptomyces two-domain LMCO genes in agricultural waste composting, which will contribute to the understanding of the ecological function of Streptomyces two-domain LMCOs with potential extracellular activity and ligninolytic capacity. A new specific PCR primer pair was designed to target the two conserved copper binding regions of Streptomyces two-domain LMCO genes. The obtained sequences mainly clustered with Streptomyces coelicolor, Streptomyces violaceusniger, and Streptomyces griseus. Gene libraries retrieved from six composting samples revealed high diversity and a rapid succession of Streptomyces two-domain LMCO genes during composting. The obtained sequence types cluster in 8 distinct clades, most of which are homologous with Streptomyces two-domain LMCO genes, but the sequences of clades III and VIII do not match with any reference sequence of known streptomycetes. Both lignocellulose degradation rates and phenol oxidase activity at pH 8.0 in the composting process were found to be positively associated with the abundance of Streptomyces two-domain LMCO genes. These observations provide important clues that Streptomyces two-domain LMCOs are potentially involved in bacterial extracellular phenol oxidase activities and lignocellulose breakdown during agricultural waste composting.     

Isolation and characterization of alginate-degrading bacteria for disposal of seaweed wastes

Aims:  Isolation of novel alginate degrading bacteria for the disposal of seaweed waste in composting process.
Methods and Results:  Decomposition of alginate polymers was checked by the 3,5-dinitrosalicylic acid (DNS) method for reducing sugar, and absorbance at 235 nm for unsaturated sugar. A bacterium A7 was isolated from wakame compost and confirmed to belong to the genus Gracilibacillus by partial 16S rDNA analysis. The optimum condition for the growth of A7 in a medium containing 5 g l⁻¹ of sodium alginate is as follows: pH, 8·5–9·5; NaCl, 0·5 mol l⁻¹; temperature, 30°C and polypeptone as nutrient content, 2–5 g l⁻¹. In a laboratory-scale composting experiment, the alginate content in wakame compost decreased to 14·3% after 72 h of composting from an initial value of 36%, indicating the effectiveness of alginate decomposition of A7 in wakame composting.
Conclusions:  The bacterium A7 was found to be alginate lyase-producing in genus Gracilibacillus and effective in degrading alginate to oligosaccharides in wakame during composting process.
Significance and Impact of the Study:  Development of new methods for the disposal of marine wastes and production of functional products.     

Laboratory composting of extruded poly(lactic acid) sheets

Composting of extruded poly(lactic acid) (PLA) in combination with pre-composted yard waste in a laboratory composting system was studied. Yard waste and PLA mixtures containing 0%, 10%, or 30% PLA (dry weight basis) were placed in composting vessels for four weeks. Exhaust gases were analyzed for carbon dioxide concentration twice per week. After the first week, significantly greater (P < 0.05) amounts of carbon dioxide were generated in vessels with 10% or 30% PLA than in control (0% PLA) vessels. Data indicated that microbial degradation of PLA occurred. There was no significant difference (P > 0.05) in carbon dioxide emission between 10% and 30% PLA mixtures. Compost pH dropped (from 6.0 to 4.0) after 4 weeks of composting for 30% PLA, but remained unchanged (6.3) for 0% or 10% PLA. Most likely, in the case of 30% PLA, substantial chemical hydrolysis and lactic acid generation lowered the compost pH. The lowered pH likely suppressed microbial activity, thus explaining the lack of difference in carbon dioxide emissions between 10% and 30% PLA mixtures. Gel permeation chromatography showed a notable decrease in PLA molecular weight as a result of composting. It was demonstrated that PLA can be efficiently composted when added in small amounts (<30% by weight) to pre-composted yard waste.