Community dynamics of methanotrophic bacteria during composting of organic matter

In this study, we describe the effects of composting on the diversity, abundance and activity of the methanotrophic community present in the compost. Composting was allowed to proceed for 10 weeks in an in-vessel reactor. Self-heating capacity (Rottegrad) indicated that compost maturity was reached after 4 weeks. After 6 weeks, a second thermophilic phase was induced by manually increasing temperature to investigate whether or not the methanotrophs shifted back to the thermophilic population. Polymerase chain reaction (PCR)-denaturing gradient gel electrophoresis (DGGE) with primers specific for type I methanotrophs of 16S rDNA and 16S rRNA were used to characterize the composition of the microbial community. Cluster and diversity analyses of RNA DGGE patterns were more sensitive than those of DNA DGGE patterns, and revealed that mesophilic and thermophilic methanotrophic communities could be differentiated. Moreover, it was seen that the diversity of the community was low during the thermophilic phase and increased during the final maturation phase. Real-time PCR analysis was also performed on the DNA and RNA extracts and showed no changes in the abundance of type I methanotrophs during the composting process (10(9) DNA copies/g compost). However, RNA-related activity did change, with the lowest activity (10(7) cDNA copies/g compost) observed during the thermophilic phase, subsequently increasing to its maximum value (10(9) cDNA copies/g compost), and finally decreasing during the maturation phase. This study confirmed the population dynamics, as seen for general groups such as bacteria and fungi during composting, for a very specific and sensitive group of bacteria, it is the type I methanotrophs.     

Dynamics of the co‐composting of barley waste with liquid poultry manure

Co‐composting of barley waste with liquid poultry manure was assessed by monitoring several chemical and biological parameters related to compost stability/maturity throughout a 103 day period. A compost of solid poultry manure was used as the control, because a compost could not be obtained from the liquid manure. The materials composted reached thermophilic stage temperatures within 4 days, lasting for more than 10 days; thereafter the temperature decreased rapidly to around ambient levels. The initial pH value was around 9 for both mixtures and, despite an initial decrease, high values were maintained over the entire co‐composting process. Electrical conductivity increased with composting time, while the opposite behaviour was observed for total carbon content. Organic matter loss in the co‐composting of barley waste with liquid poultry manure was 35%. In general, the final products showed physicochemical characteristics considered normal for such materials, but pH and ammonia content values were high. The results of a bioassay test performed with seeds of three plant species (ryegrass, wheat and barley) indicated that the co‐compost could be considered mature from a biological point of view, since phytotoxicity was absent for ryegrass and barley. Comparison of these data with those obtained for solid poultry manure indicated that barley waste notably improved the composting efficiency as well as the quality of the final product. © 2003 Society of Chemical Industry     

Survival of coliform bacteria in static compost piles of dairy waste solids intended for freestall bedding

Dairy waste solids separated from a slurry by a centrifugal separator were composted in 12 static piles. Seven of the compost piles were naturally aerated, and five were aerated by a fan that forced air through the piles of solids. The natural aeration process aged the manure solids in an unconfined pile. The fan in the forced aeration process forced air into a perforated plenum beneath the compost piles. Dairy waste solids in each compost pile were heated into the thermophilic temperature range and generally composted well. At most sampling points, coliform bacteria declined to low or undetectable numbers early in the composting period. However, as the composting process proceeded, bacterial numbers increased to approximately those present in raw dairy waste solids. Findings of this study suggest that composting offers little benefit toward net reduction in coliform bacterial numbers in dairy waste solids.     

Denaturing gradient gel electrophoresis analyses of microbial community from field-scale composter

The diversity of microbial community during the decomposition of waste in a field-scale composter (Hazaka system) was investigated by denaturing gradient gel electrophoresis (DGGE). The composter operates at a high temperature through a self-heating system, creating a thermophilic (60-76 degrees C) stage during the initial phase and a mesophilic (45 degrees C) stage towards the later phase of the composting period. The pH of the system (pH 7.75-8.10) did not vary significantly during the process while moisture content was reduced from 48.8% to 25.1%. DGGE and 16S rDNA analyses showed that the following genera were found throughout the process: Propionibacterium sp., Methylobacterium sp., Pseudomonas sp., and Bradyrhizobium sp. Different Bacillus spp. thrive at the thermophilic or the mesophilic stage while Clostridium sp. was only found at the initial phase of the process. Staphylococcus sp. and Caulobacter sp. or Brevundimonas sp. existed during the later phase of the composting period.     

Enterococcus faecium AS8及其胞外多糖对发酵乳流变学特性的影响

通过红外光谱、气相色谱-质谱联用和流变仪的检测,探究产自屎肠球菌（Enterococcus faecium）AS8的胞外多糖（exopolysaccharide,EPS）（AS8-EPS）的结构组成和流变性能。采用3 种发酵乳作为样品,分别为Streptococcus thermophilus＋Lactobacillus bulgaricus、EPS＋S. thermophilus＋L. bulgaricus和E. faecium AS8。结果表明,在贮藏期间,不同发酵乳样品具有不同的流变学性质。同时,补充添加EPS和原位EPS对发酵乳流变性能具有不同的影响。通过Sephadex G-100和Sephadex G-50的分离纯化,获得2 种多糖,分别为AS8-1-EPS和AS8-2-EPS。AS8-1-EPS主要单糖组成为甘露糖、葡萄糖和半乳糖（分别占59.1%、26.8%、7.9%）,还有含量很少的其他单糖;AS8-2-EPS主要单糖组成为甘露糖、葡萄糖、半乳糖和鼠李糖（分别占65.4%、21.3%、8.9%、4.4%）。红外光谱检测结果表明AS8-1-EPS和AS8-2-EPS均为杂多糖。     

Microbial communities in the garbage composting with rice hull as an amendment revealed by culture-dependent and -independent approaches

The diversity and succession of microbial communities during the garbage composting with rice hull as an amendment were studied by denaturing gradient gel electrophoresis (DGGE) and clone library analysis of PCR-amplified 16S ribosomal DNA (rDNA) with universal primers. Based on temperature changes, the composting process could be divided into thermophilic, cooling-down, and maturing stages. The DGGE profiles and clone library analysis revealed that the microbial community drastically changed during the composting process from the thermophilic to the maturing stages. The dominant bacterial group changed from the phylum Firmicutes in the thermophilic stage to the phylum Bacteroidetes in the maturing stage. This change in microbial communities may be significant for the composting process. The diversity of cultivated bacteria isolated from samples taken at various stages of the composting process was low. A total of 87 isolates were classified as belonging to only four different groups. These groups were also detected in the DGGE profiles and by the clone library analysis. Our study indicated that a combination of culture-dependent and -independent approaches could be very useful for monitoring both bacterial diversity and the succession of communities during the composting process. This study would be beneficial for assessing the ecological consequences of disposal of organic waste.     

Response of anaerobic granular sludge to a shock load of zinc oxide nanoparticles during biological wastewater treatment

The increasing use of zinc oxide nanoparticles (ZnO NPs) in consumer and industrial products highlights a need to understand their potential environmental impacts. In this study, the response of anaerobic granular sludge (AGS) to a shock load of ZnO NPs during anaerobic biological wastewater treatment was reported. It was observed that the extracellular polymeric substances (EPS) of AGS and the methane production were not significantly influenced at ZnO NPs of 10 and 50 mg per gram of total suspended solids (mg/g-TSS), but they were decreased when the dosage of ZnO NPs was greater than 100 mg/g-TSS. The visualization of EPS structure with multiple fluorescence labeling and confocal laser scanning microscope revealed that ZnO NPs mainly caused the decrease of proteins by 69.6%. The Fourier transform infrared spectroscopy analysis further indicated that the C-O-C group of polysaccharides and carboxyl group of proteins in EPS were also changed in the presence of ZnO NPs. The decline of EPS induced by ZnO NPs resulted in their deteriorating protective role on the inner microorganisms of AGS, which was in correspondence with the observed lower general physiological activity of AGS and the death of microorganisms. Further investigation showed that the negative influence of ZnO NPs on methane production was due to their severe inhibition on the methanization step.     

利用酿酒废弃物生产有机肥及肥效研究

该文以泸州老窖股份有限公司产生的污泥和酒糟为原料,研究高温发酵生产有机肥技术及对高粱的肥效。结果表明,污泥、酒糟、腐熟剂按不同比例混合堆肥,历经升温期,高温期和降温期,水分含量持续降低,氮、磷、钾和腐殖酸含量逐渐增加,堆肥物料由黄褐色变为褐色、黑褐色或黑色。其中,酒糟与污泥按2∶1(C∶N≈25∶1,水分质量分数为65%,pH 6.5)混合腐熟最快,肥质最佳,符合NY525—2012有机肥质量标准。50%有机肥与50%的化肥配施(以氮计),高粱产量、籽粒含氮量和植株磷钾吸收量分别比单施等养分的化肥增加12.55%、36.70%、22.22%和29.20%;土壤有效氮,磷酸酶、脲酶和脱氢酶活性也显著提高。因此,污泥和酒糟适量混合,添加菌剂,经高温发酵生产的有机肥符合国家标准,与化肥适量配施显著增加高粱产量,改善植物营养,提高土壤酶活。     

Reduction of nitrous oxide emission from pig manure composting by addition of nitrite-oxidizing bacteria

Nitrous oxide (N2O) is emitted from pig manure composting, and the emission correlates with nitrite (NO2-) accumulation in the composting material. In the present study, we added nitrite-oxidizing bacteria (NOB)to inhibit NO2- accumulation and evaluated its effect on N2O emission in a laboratory-scale composting experiment. Mature pig manure compost (MPMC) containing NOB at 10(6) MPN g(-1) WM or cultured MPMC (cul-MPMC) NOB at 10(11) MPN g(-1) WM was added after the thermophilic phase of composting. The addition of these materials prevented NO2- accumulation, promoting oxidation to nitrate (NO3-), whereas the accumulation of NO2- occurred in the material to which NOB was not added as the result of the delayed growth of indigenous NOB compared with that of ammonia-oxidizing bacteria (AOB). The pattern of NO2- in the material agreed with that of N2O emission; therefore, N2O emission ceased rapidly when NOB was added. Emission rates of N2O were 88.5 (no addition), 17.5 (MPMC addition), and 20.2 (cul-MPMC addition) g N-N2O kg(-1) TNinitial, respectively. Improving composition of nitrifying communities for complete nitrification promotion would be useful to establish a composting method with low N2O emission.     

造纸污泥好氧堆肥处理技术研究

造纸污泥是一种生物固体废弃物，通过调节水分与C／N比，在强制通风与定期翻堆情况下，由于微生物作用，有机质发生降解，C／N比在不断下降，经过2个月左右高温堆肥，可以转化为高效的有机肥料。从堆肥过程的物理、化学及生物学指标变化可以看出，添加富含纤维素降解菌的发酵料，可以加速造纸污泥的腐熟，堆肥的阳离子交换量(CEC)与盆栽玉米增产率之间具有较好的相关性，CEC值可以作为造纸污泥堆肥腐熟程度的控制指标，CEC值≥80mmol／100g时，认为造纸污泥堆肥已经腐熟。     

Lactobacillus casei HS4胞外多糖对发酵乳组织结构和流变特性的影响

研究干酪乳杆菌（Lactobacillus casei）HS4所产乳酸菌胞外多糖（exopolysaccharide,EPS）及其对发酵乳微观结构和流变性的影响。通过Sephadex G-50柱纯化得到两种类型的EPS,分别命名为HS4-1-EPS和HS4-2-EPS。HS4-1-EPS主要由葡萄糖组成,峰面积为0.940。HS4-2-EPS主要由葡萄糖和甘露糖组成,峰面积比为0.3830.364。红外光谱结果显示,HS4-1-EPS和HS4-2-EPS均为杂多糖。分别采用干酪乳杆菌HS4、嗜热链球菌-保加利亚乳杆菌（11）复合菌株以及复合菌株添加纯化EPS制成的不同发酵乳作为样品,通过测定流变特性及微观结构观察,研究补充纯化EPS和原位EPS对发酵乳流变特性及微观结构的影响。结果显示,其在4 ℃贮存期间显示出不同的流变特性及微观结构。基于扫描电镜下样品的微观结构,可以推知,EPS的类型和空间阻挡效应与发酵乳的流变性质相关。     

Effect of temperature on reaction rate and microbial community in composting of cattle manure with rice straw

The effects of temperature on microbial communities and mass reduction in composting were studied by comparing the mesophilic process with the thermophilic process of cattle manure-rice straw composting for 21 d. The respiratory quinone content (indicator of microbial biomass) continuously increased in the mesophilic process, but in the thermophilic process, it was much higher after 3 d and then decreased to the same level as that in the mesophilic process after 21 d. The diversity of quinones increased continuously in both the mesophilic and thermophilic processes with a higher value in the thermophilic process. The mesophilic microbial community was characterized by the predominance of ubiquinones and menaquinone (MK-8), which correspond to Proteobacteria and fungi. The thermophilic microbial community was characterized by the dominance of MK-7 in the initial period, and increases in the amounts of menaquinones with long and partially-saturated side chains in the later period, corresponding to Firmicutes and Actinobacteria, respectively. DNA fingerprinting by denaturing gradient gel electrophoresis (DGGE) of 16S ribosomal RNA genes also confirmed that diversity of microbial communities increased but differently in the two processes. Our results suggest that mesophilic composting is more effective for mass reduction in cattle manure composting than thermophilic composting because of the higher decomposition activity of the microbial community characterized by the predominance of Proteobacteria and fungi.     

Removal of Pseudomonas putida biofilm and associated extracellular polymeric substances from stainless steel by alkali cleaning

Alkali (NaOH)-based compounds are commonly used in the food industry to clean food contact surfaces. However, little information is available on the ability of alkali and alkali-based cleaning compounds to remove extracellular polymeric substances (EPS) produced by biofilm bacteria. The objectives of this study were to determine the temperature and NaOH concentration necessary to remove biofilm EPS from stainless steel under turbulent flow conditions (clean-in-place simulation) and to determine the ability of a commercial alkaline cleaner to remove biofilm EPS from stainless steel when applied under static conditions without heat. Biofilms were produced by growing Pseudomonas putida on stainless steel for 72 h at 25 degrees C in a 1:10 dilution of Trypticase soy broth. The biofilms were treated using NaOH at concentrations of 1.28 to 6.0% and temperatures ranging from 66 to 70 degrees C. Other biofilms were treated with commercial alkaline cleaner at 25 or 4 degrees C for 1 to 30 min. Removal of EPS was determined by direct microscopic observation of samples stained with fluorescent-labeled peanut agglutinin lectin. Treatment with 1.2% NaOH at 66 degrees C for 3 min was insufficient to remove biofilm EPS. A minimum of 2.5% NaOH at 66 degrees C and 2.0% NaOH at 68 degrees C for 3 min were both effective for EPS removal. Commercial alkaline cleaner removed over 99% of biofilm EPS within 1 min at 4 and 25 degrees C under static conditions. Selection of appropriated cleaning agent formulation and use at recommended concentrations and temperatures is critical for removal of biofilm EPS from stainless steel.     

乳酸菌胞外多糖的研究进展

乳酸菌胞外多糖是一种天然的、具有诸多生理功能的生物高分子聚合物。乳酸菌可以产生不同结构的胞外多糖,根据多糖的组成不同可以将其分为同型多糖和异型多糖。本文对乳酸菌胞外多糖的种类、结构、生物合成、生理功能、分离纯化方法以及应用前景进行了综述。     

Temperature and nutrient effects on Campylobacter jejuni attachment on multispecies biofilms on stainless steel

Campylobacterjejuni is a thermophilic microaerophilic pathogen that is commonly found in the intestinal tract of chickens. In this study, attachment of C. jejuni 1221gfp in biofilms on stainless steel was assessed at various temperatures and with reduced nutrients. Bacteria collected from a saline rinse of processed broiler chicken carcasses were used to form initial biofilms. The whole carcass rinse (WCR) biofilms were formed by incubation of the bacteria for 16 h at 13, 20, 37, and 42 degrees C on stainless steel coupons in tryptic soy broth (TSB). The resulting biofilms were stained with Hoechst 33258 stain and visualized by epifluorescence microscopy. WCR biofilms formed at 13 degrees C yielded the highest surface area coverage (47.6%), and the lowest coverage (2.1%) was attained at 42 degrees C. C. jejuni transformed to produce green fluorescent protein (gfp) was allowed to attach to the preexisting biofilms (from WCR incubated for 16 h) at each of the four temperatures, and attached cells were enumerated by visualization with an epifluorescence microscope. Attachment of C. jejuni 1221gfp did not significantly differ (P > 0.05) among the four temperatures. C. jejuni 1221gfp was cultured only from coupons with biofilms formed at 13 and 20 degrees C. For nutrient limitation experiments, WCR biofilms were allowed to grow in 10- and 50-fold diluted TSB at 20 and 37 degrees C for 48 h. The WCR biofilm surface area coverage (approximately 2%) was greater at 37 degrees C than at 20 degrees C for both TSB concentrations. C. jejuni 1221gfp was incubated with the WCR biofilm for 48 h at 20 and 37 degrees C, and attached cells were enumerated. Attachment was significantly higher (P < 0.05) only for the treatments with 1:10 TSB at 20 degrees C and 1:50 TSB at 37 degrees C. Under reduced-nutrient conditions, C. jejuni 1221gfp was cultured only from biofilms formed at 20 degrees C. Under the conditions tested, the attachment of C. jejuni 1221gfp on stainless steel and biofilms was affected by a combination of temperature and nutrient availability, but C. jejuni culturability was affected solely by temperature.     

Composting cattle dung wastes by using a hyperthermophilic pre-treatment process: characterization by physicochemical and molecular biological analysis

To solve malodorous odor problems by ammonia emission in composting of cattle dung wastes, we developed an alternative composting method consisting of a hyperthrmophilic pre-treatment reactor (HTPRT) (first step) combined with a general windrow post-treatment system (WPOT) (second step). In this study, physicochemical and microbiological differences in compost materials during the HTPRT-WPOT process and a simple windrow composing process (SWC) were investigated. The HTPRT-WPOT process removed excess ammonia in the compost materials by physical ammonia stripping, and controlled the malodorous ammonia emission. The organic matter evolution index showed that the HTPRT-WPOT process also contributed to accelerate formation of humic acids in composting. Quantitative real-time PCR analyses using Bacterial-, Archaeal- and fungal-protozoan-specific primer sets showed that small subunit ribosomal RNA (SSU rRNA) gene copy numbers differed much between composting materials of these two processes. Particularly, the SSU rRNA gene copy of eukaryotic microbes (fungi-protozoa) in the HTPRT-WPOT process was much higher than in the SWC process. From these results, we conclude that the HTPRT-WPOT process has great advantages for the control of malodorous odor problems caused by ammonia emission, and for high rate of composting evaluated by the humification rate and microbial characterization of the composting materials.     

Cellulose as an extracellular matrix component present in Enterobacter sakazakii biofilms

Cellulose was identified and characterized as an extracellular matrix component present in the biofilm of an Enterobacter sakazakii clinical isolate grown in nutrient-deficient (M9) medium. Using a bacterial artificial cloning approach in Escherichia coli and subsequent screening of transformants for fluorescence on calcofluor plates, nine genes organized in two operons were identified as putatively responsible for the biosynthesis of cellulose. In addition to the genes already described for cellulose production, two more genes were identified, putatively transcribed together with the genes from the first operon. Putative cellulose in E. sakazakii ES5 biofilm grown on glass coverslips was visualized by calcofluor staining and confocal fluorescence laser scanning microscopy. For the first time, the presence of cellulose in biofilms produced by E. sakazakii was confirmed by methylation analysis.     

Fate of Escherichia coli O157:H7 during composting of bovine manure in a laboratory-scale bioreactor

Inactivation profiles of Escherichia coli O157:H7 in inoculated bovine manure-based compost ingredients were determined by composting these ingredients in a bioreactor under controlled conditions. A 15-liter bioreactor was constructed to determine the fate of E. coli O157:H7 and changes in pH, moisture content, temperature, and aerobic mesophilic and thermophilic bacterial counts during composting. Fresh cow manure, wheat straw, cottonseed meal, and ammonium sulfate were combined to obtain a moisture content of ca. 60% and a carbon/nitrogen ratio of 29:1. The compost ingredients were held in the bioreactor at a constant external temperature of 21 or 50 degrees C. Self-heating of the ingredients due to microbial activity occurred during composting, with stratified temperatures occurring within the bioreactor. At an external temperature of 21 degrees C, self-heating occurred for 0 to 3 days, depending on the location within the bioreactor. E. coli O157:H7 populations increased by 1 to 2 log10 CFU/g during the initial 24 h of composting and decreased by ca. 3.5 log10 CFU/g near the bottom of the bioreactor and by ca. 2 log10 CFU/g near the middle and at the top during 36 days of composting. At an external temperature of 50 degrees C. E. coli O157:H7 was inactivated rapidly (by ca. 4.9 log10 CFU/g at the top of the bioreactor, by 4.0 log10 CFU/g near the middle, and by 5.9 log10 CFU/g near the bottom) within 24 h of composting. When inoculated at an initial level of ca. 10(7) CFU/g. E. coli O157:H7 survived for 7 days but not for 14 days at all three sampling locations, as indicated by either direct plating or enrichment culture. At the top of the bioreactor a relatively constant moisture content of 60% was maintained, whereas the moisture content near the bottom decreased steadily to 37 to 45% over 14 days of composting. The pH of the composting mixture decreased to ca. 6 within 1 to 3 days and subsequently increased to 8 to 9. Results obtained in this study indicate that large populations (10(4) to 10(7) CFU/g) of E coli O157:H7 survived for 36 days during composting in a bioreactor at an external temperature of 21 degrees C but were inactivated to undetectable levels after 7 to 14 days when the external temperature of the bioreactor was 50 degrees C. Hence, manure contaminated with large populations (e.g., 10(7) CFU/g) of E. coli O157:H7 should be composted for more than 1 week, and preferably for 2 weeks, when held at a minimum temperature of 50 degrees C.     

In situ characterization and analysis of Salmonella biofilm formation under meat processing environments using a combined microscopic and spectroscopic approach

Salmonella biofilm on food-contact surfaces present on food processing facilities may serve as a source of cross-contamination. In our work, biofilm formation by multi-strains of meat-borne Salmonella incubated at 20 °C, as well as the composition and distribution of extracellular polymeric substances (EPS), were investigated in situ by combining confocal laser scanning microscopy (CLSM), scanning electron microscope (SEM), attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) and Raman spectroscopy. A standard laboratory culture medium (tryptic soy broth, TSB) was used and compared with an actual meat substrate (meat thawing-loss broth, MTLB). The results indicated that Salmonella grown in both media were able to form biofilms on stainless steel surfaces via building a three-dimensional structure with multilayers of cells. Although the number of biofilm cells grown in MTLB was less than that in TSB, the cell numbers in MTLB was adequate to form a steady and mature biofilm. Salmonella grown in MTLB showed “cloud-shaped” morphology in the mature biofilm, whereas when grown in TSB appeared “reticular-shaped”. The ATR-FTIR and Raman analysis revealed a completely different chemical composition between biofilms and the corresponding planktonic cells, and some important differences in biofilms grown in MTLB and in TSB. Importantly, our findings suggested that the progress towards a mature Salmonella biofilm on stainless steel surfaces may be associated with the production of the EPS matrix, mainly consisting of polysaccharides and proteins, which may serve as useful markers of biofilm formation. Our work indicated that a combination of these non-destructive techniques provided new insights into the formation of Salmonella biofilm matrix.