Enzyme Production-Based Approach for Determining the Functions of Microorganisms within a Community

The functions of specific microorganisms in a microbial community were investigated during the composting process. Cerasibacillus quisquiliarum strain BLx^T and Bacillus thermoamylovorans strain BTa were isolated and characterized in our previous studies based on their dominance in the composting system. Strain BLx^T degrades gelatin, while strain BTa degrades starch. We hypothesized that these strains play roles in gelatinase and amylase production, respectively. The relationship between changes in the abundance ratios of each strain and those of each enzyme activity during the composting process was examined to address this hypothesis. The increase in gelatinase activity in the compost followed a dramatic increase in the abundance ratio of strain BLx^T. Zymograph analysis demonstrated that the pattern of active gelatinase bands from strain BLx^T was similar to that from the compost. Gelatinases from both BLx^T and compost were partially purified and compared. Homologous N-terminal amino acid sequences were found in one of the gelatinases from strain BLx^T and that of compost. These results indicate strain BLx^T produces gelatinases during the composting process. Meanwhile, the increase in the abundance ratio of strain BTa was not concurrent with that of amylase activity in the compost. Moreover, the amylase activity pattern of strain BTa on the zymogram was different from that of the compost sample. These results imply that strain BTa may not produce amylases during the composting process. To our knowledge, this is the first report demonstrating that the function of a specific microorganism is directly linked to a function in the community, as determined by culture-independent and enzyme-level approaches.     

Control of type IV collagenase activity by components of the urokinase-plasmin system: a regulatory mechanism with cell-bound reactants.

The urokinase-type plasminogen activator (uPA) and the matrix-degrading metalloproteinases MMP-2 and MMP-9 (type IV collagenases/gelatinases) have been implicated in a variety of invasive processes, including tumor invasion, metastasis and angiogenesis. MMP-2 and MMP-9 are secreted in the form of inactive zymogens that are activated extracellularly, a fundamental process for the control of their activity. The physiological mechanism(s) of gelatinase activation are still poorly understood; their comprehension may provide tools to control cell invasion. The data reported in this paper show multiple roles of the uPA-plasmin system in the control of gelatinase activity: (i) both gelatinases are associated with the cell surface; binding of uPA and plasmin(ogen) to the cell surface results in gelatinase activation without the action of other metallo- or acid proteinases; (ii) inhibition of uPA or plasminogen binding to the cell surface blocks gelatinase activation; (iii) in soluble phase plasmin degrades both gelatinases; and (iv) gelatinase activation and degradation occur in a dose- and time-dependent manner in the presence of physiological plasminogen and uPA concentrations. Thus, the uPA-plasmin system may represent a physiological mechanism for the control of gelatinase activity.     

高通量测序技术分析猪粪堆肥过程中微生物群落结构变化

为了解猪粪堆肥过程中微生物群落结构组成及多样性的变化,采集猪粪堆肥过程的三个代表性样品—新鲜猪粪、高温堆肥、腐熟堆肥,利用Illumina Miseq高通量测序技术对16S rRNA V4～V5可变区序列进行测序,分别获得37 009、42 470、36 713条有效序列及328、280、160个操作分类单元(OTU)。Alpha多样性分析表明,在堆肥过程中微生物群落丰富度呈现降低趋势,而多样性呈现先上升后下降趋势。随着堆肥的进行,在门水平上,厚壁菌门、拟杆菌门和软壁菌门相对丰度降低,而变形菌门和放线菌门相对丰度升高;在属水平上,Turicibacter、Terrisporobacter、Parabacteroides、Clostridium sensu stricto、Corynebacterium等来自动物肠道的微生物相对丰度明显下降,Thermopolyspora、Thermomonospora、Thermobifida、Halocella等耐热耐盐微生物成为最主要优势菌。堆肥过程不同菌群优势度的变化是微生物与堆肥中各理化因子相互作用的结果。     

Comparison of characterization and microbial communities in rice straw- and wheat straw-based compost for <Emphasis Type="Italic">Agaricus bisporus</Emphasis> production

Rice straw (RS) is an important raw material for the preparation of Agaricus bisporus compost in China. In this study, the characterization of composting process from RS and wheat straw (WS) was compared for mushroom production. The results showed that the temperature in RS compost increased rapidly compared with WS compost, and the carbon (C)/nitrogen (N) ratio decreased quickly. The microbial changes during the Phase I and Phase II composting process were monitored using denaturing gradient gel electrophoresis (DGGE) and phospholipid fatty acid (PLFA) analysis. Bacteria were the dominant species during the process of composting and the bacterial community structure dramatically changed during heap composting according to the DGGE results. The bacterial community diversity of RS compost was abundant compared with WS compost at stages 4–5, but no distinct difference was observed after the controlled tunnel Phase II process. The total amount of PLFAs of RS compost, as an indicator of microbial biomass, was higher than that of WS. Clustering by DGGE and principal component analysis of the PLFA compositions revealed that there were differences in both the microbial population and community structure between RS- and WS-based composts. Our data indicated that composting of RS resulted in improved degradation and assimilation of breakdown products by A. bisporus, and suggested that the RS compost was effective for sustaining A. bisporus mushroom growth as well as conventional WS compost.     

Response of compost maturity and microbial community composition to pentachlorophenol (PCP)-contaminated soil during composting

Two composting piles were prepared by adding to a mixture of rice straw, vegetables and bran: (i) raw soil free from pentachlorophenol (PCP) contamination (pile A) and (ii) PCP-contaminated soil (pile B). It was shown by the results that compost maturity characterized by water soluble carbon (WSC), TOC/TN ratio, germination index (GI) and dehydrogenase activity (DA) was significantly affected by PCP exposure, which resulted in an inferior degree of maturity for pile B. DGGE analysis revealed an inhibited effect of PCP on compost microbial abundance. The bacteria community shifts were mainly consistent with composting factors such as temperature, pH, moisture content and substrates. By contrast, the fungal communities were more sensitive to PCP contamination due to the significant correlation between fungal community shifts and PCP removal. Therefore, the different microbial community compositions for properly evaluating the degree of maturity and PCP contamination were suggested.     

Changes in the microbial community structure during thermophilic composting of manure as detected by the quinone profile method

Quinone profiles and physico-chemical properties were measured to characterize the microbial community structure during a 14-day thermophilic composting of cattle manure mixed with rice straw as a bulking agent. The change in total quinone content (TQ) and the divergence of quinone (DQ) indicated that the microbial biomass reached a peak followed by a decrease, whereas the microbial community diversity increased continuously during the composting process. The high mole fraction of ubiquinones in the raw materials, and at the beginning of the composting period suggested that fungi and/or Proteobacteria were present. The predominance of MK-7 from days 3 to 7 suggested that Bacillus spp. were the main microbial species. An increase in partially saturated and long-chain menaquinones during the latter composting period indicated that the proliferation of various species of Actinobacteria was occurring. The microbial community structure, as expressed by TQ and DQ, corresponded well to physico-chemical properties such as the C/N ratio, pH, O₂ consumption and compost mass reduction.     

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.     

Airborne and indigenous microbiomes co-drive the rebound of antibiotic resistome during compost storage

Composting is widely used to reduce the abundance of antibiotic resistance genes (ARGs) in solid waste. While ARG dynamics have been extensively investigated during composting, the fate and abundance of residual ARGs during the storage remain unexplored. Here, we tested experimentally how ARG and mobile genetic element (MGE) abundances change during compost storage using metagenomics, quantitative PCR and direct culturing. We found that 43.8% of ARGs and 39.9% of MGEs quickly recovered already during the first week of storage. This rebound effect was mainly driven by the regrowth of indigenous, antibiotic-resistant bacteria that survived the composting. Bacterial transmission from the surrounding air had a much smaller effect, being most evident as MGE rebound during the later stages of storage. While hyperthermophilic composting was more efficient at reducing the relative abundance of ARGs and MGEs, relatively greater ARG rebound was observed during the storage of hyperthermophilic compost, exceeding the initial levels of untreated sewage sludge. Our study reveals that residual ARGs and MGEs left in the treated compost can quickly rebound during the storage via airborne introduction and regrowth of surviving bacteria, highlighting the need to develop better storage strategies to prevent the rebound of ARGs and MGEs after composting.     

Effects of Elevated Tetracycline Concentrations on Aerobic Composting of Human Feces: Composting Behavior and Microbial Community Succession

The effects of antibiotics on aerobic composting are investigated by dosing of tetracycline (TC) in fresh human feces with sawdust as biomass carrier. Variability in process parameters such as temperature, pH, water-soluble carbon, germination index (GI) and dehydrogenase activity (DHA) are evaluated at TC dosages of 0, 100, 250 and 500 mg/kg in a 21-day composting. Moreover, microbial community succession is examined by high-throughput 16S rRNA gene sequencing. Findings indicate significant impacts to the process parameters with the increase of TC concentration such as inhibition of temperature increases during aerobic composting, lowering of pH, increasing of water-soluble carbon residue, a decrease of GI, and hindering of DHA. Furthermore, elevated TC concentrations significantly alter the microbial community succession and reduce the community diversity and abundance. Therefore, interference in microbial community structures and a hindrance to biological activity are believed to be the main adverse effects of TC on the composting process and maturity of the composting products.     

Diversity and Abundance of Ammonia-Oxidizing Bacteria and Ammonia-Oxidizing Archaea During Cattle Manure Composting

Ammonia-oxidizing bacteria (AOB) and ammonia-oxidizing archaea (AOA) play important roles in nitrification in various environments. They may also be key communities for ammonia oxidation in composting systems, although few studies have discussed their presence. We investigated the relative diversity and abundance of AOB and AOA using cloning procedures, denaturing gradient gel electrophoresis analysis, and real-time PCR during several stages in the process of cattle manure composting. Our results revealed that the AOB community structure changed during the process. At the high-temperature stage (>60°C), a member of the Nitrosomonas europaea/eutropha cluster dominated while the uncultured Nitrosomonas spp. cluster appeared after the temperature decreased. Additionally, our analysis indicated that AOA sequences, which were classified into a soil/sediment cluster, were present after the temperature decreased during the composting process. At these stages, the number of the archaeal amoA gene copies (3.2 or 3.9?×?10⁷ copies per gram freeze-dried compost) was significantly higher than that of bacterial amoA gene copies (2.2–7.2?×?10⁶ copies per gram freeze-dried compost). Our results suggest that both AOB and AOA are actively involved in nitrification of composting systems.     

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。     

Community structure and population dynamics of ammonia oxidizers in composting processes of ammonia-rich livestock waste

This study investigated the relationship between the population dynamics of ammonia-oxidizing bacteria (AOB) and archaea (AOA), and changes in the concentrations of nitrogenous compounds during ammonia-rich livestock waste-composting processes. The data showed that ammonia in beef and dairy cow livestock waste-composting piles was slowly oxidized to nitrite and nitrate after approximately 21–35 days under thermophilic or moderately thermophilic and mesophilic conditions. Real-time quantitative PCR (qPCR) assays showed a relative abundance of betaproteobacterial AOB during ammonia oxidation but did not detect AOA in any composting stage. Furthermore, real-time qPCR and terminal-restriction fragment length polymorphism (T-RFLP) analyses for the AOB in two composting processes (beef and dairy cow livestock waste) out of the three studied found that thermophilic or moderately thermophilic uncultured betaproteobacterial AOB from the “compost AOB cluster” contributed to ammonia oxidation during hot composting stages. Non-metric multidimensional scaling analyses of the data from T-RFLP showed that only a few analogous species predominated during composting of beef, dairy cow and pig livestock wastes, and thus, the AOB community structures in the three composting piles operating under different conditions were similar. AOB-targeted clone library analyses revealed that uncultured members of the “compost AOB cluster”, which could be clearly distinguished from the authentic species of the genus Nitrosomonas, were the major constituents of the AOB populations. These results suggested that a limited and unique species of AOB played a role in ammonia oxidation during the composting of ammonia-rich livestock waste.     

Dynamics of pruning waste and spent horse litter co-composting as determined by chemical parameters

Co-composting of pruning waste and horse manure was monitored by different parameters. A windrow composting pile, having the dimensions 2.5m (height) x 30m (length) was established. The maturation of pruning waste and horse manure compost was accompanied by a decline in NH(4)(+)-N concentration, water soluble C and an increase in NO(3)(-)-N content. Organic matter (OM) content during composting followed a first-order kinetic equation. This result was in agreement with the microbiological activity measured by the CO(2) respiration during the process. The correlation at a high level of probability found between the OM loss and CO(2) evolution showed that both parameters could be used to indicate the degree of OM degradation that is the maturity and stability phases of the compost studied. Humification parameters data from the organic matter fractionation did not show a clear tendency during the composting time, suggesting that these parameters are not suitable for evaluating the dynamics of the process.     

彩绒革盖菌在猪粪堆肥中应用的初步研究

在以猪粪为原料的静态条垛堆肥的堆体试验中添加了彩绒革盖菌,研究其对堆肥发酵的影响。研究表明彩绒革盖菌在堆肥二次发酵时期有利于堆体温度的提升和保温,说明了在进入堆肥后期彩绒革盖菌对其中剩余的木质素等成分有很好的分解能力,有利于堆肥的腐熟和养分的释放,初步表明彩绒革盖菌是一株理想的堆肥发酵菌株。     

Identification of structural elements important for matrix metalloproteinase type V collagenolytic activity as revealed by chimeric enzymes. Role of fibronectin-like domain and active site of gelatinase B

Digestion of type V collagen by the gelatinases is an important step in tumor cell metastasis because this collagen maintains the integrity of the extracellular matrix that must be breached during this pathological process. However, the structural elements that provide the gelatinases with this unique proteolytic activity among matrix metalloproteinases had not been thoroughly defined. To identify these elements, we examined the substrate specificity of chimeric enzymes containing domains of gelatinase B and fibroblast collagenase. We have found that the addition of the fibronectin-like domain of gelatinase B to fibroblast collagenase is sufficient to endow the enzyme with the ability to cleave type V collagen. In addition, the substitution of the catalytic zinc-binding active site region of fibroblast collagenase with that of gelatinase B increased the catalytic efficiency of the enzyme 3- to 4-fold. This observation led to the identification of amino acid residues, Leu(397), Ala(406), Asp(410), and Pro(415), in this region of gelatinase B that are important for its efficient catalysis as determined by substituting these amino acids with the corresponding residues from fibroblast collagenase. Leu(397) and Ala(406) are important for the general proteolytic activity of the enzyme, whereas Asp(410) and Pro(415) specifically enhance its ability to cleave type V collagen and gelatin, respectively. These data provide fundamental information about the structural elements that distinguish the gelatinases from other matrix metalloproteinases in terms of substrate specificity and catalytic efficiency.     

Humic substances change during the co-composting process of municipal solid wastes and sewage sludge

The change of the degree of stability of compost during the composting process was a kind of guideline for our study. This stability was estimated by monitoring the chemical fractionation (extraction of humic and fulvic acids, and humin) during two cycles of composting. Change of humin (H), humic-like acid carbon (CHA) and fulvic-like acid carbon (CFA) fractions during the composting process of municipal solid wastes were investigated using two windrows W1 (100% of municipal solid wastes) and W2 (60% of municipal solid wastes and 40% of dried sewage sludge). Humin and fulvic acid fractions in the two windrows decreased since the start of composting process and tend to stabilize. At the end of composting process, humic acid fraction is more important in the windrow without sludge (W1) than the one with sludge (W2). The humification indexes used in this study showed that the humic-like acid carbon fraction production takes place largely during the phase of temperature increase (thermophilic phase), and it appeared very active in the windrow W2. At the end of composting process, the E₄/E₆ ratio value indicated that the compost of W1 is more mature than the compost of W2. The humification ratio (HR) allowed a correct estimation of compost organic matter stabilization level.     

Changes in Bacterial and Fungal Communities across Compost Recipes,Preparation Methods,and Composting Times

Compost production is a critical component of organic waste handling, and compost applications to soil are increasingly important to crop production. However, we know surprisingly little about the microbial communities involved in the composting process and the factors shaping compost microbial dynamics. Here, we used high-throughput sequencing approaches to assess the diversity and composition of both bacterial and fungal communities in compost produced at a commercial-scale. Bacterial and fungal communities responded to both compost recipe and composting method. Specifically, bacterial communities in manure and hay recipes contained greater relative abundances of Firmicutes than hardwood recipes with hay recipes containing relatively more Actinobacteria and Gemmatimonadetes. In contrast, hardwood recipes contained a large relative abundance of Acidobacteria and Chloroflexi. Fungal communities of compost from a mixture of dairy manure and silage-based bedding were distinguished by a greater relative abundance of Pezizomycetes and Microascales. Hay recipes uniquely contained abundant Epicoccum, Thermomyces, Eurotium, Arthrobotrys, and Myriococcum. Hardwood recipes contained relatively abundant Sordariomycetes. Holding recipe constant, there were significantly different bacterial and fungal communities when the composting process was managed by windrow, aerated static pile, or vermicompost. Temporal dynamics of the composting process followed known patterns of degradative succession in herbivore manure. The initial community was dominated by Phycomycetes, followed by Ascomycota and finally Basidiomycota. Zygomycota were associated more with manure-silage and hay than hardwood composts. Most commercial composters focus on the thermophilic phase as an economic means to insure sanitation of compost from pathogens. However, the community succeeding the thermophilic phase begs further investigation to determine how the microbial dynamics observed here can be best managed to generate compost with the desired properties.     

Modulation of gelatinase activity correlates with the dedifferentiation profile of regenerating salamander limbs.

Remodeling of extracellular matrix (ECM) is one of the key events in many developmental processes. In the present study, a temporal profile of gelatinase activities in regenerating salamander limbs was examined zymographically. In addition, the effect of retinoic acid (RA) on these enzyme activities was examined to relate the pattern-duplicating effect of RA in limb regenerates with gelatinase activities. During regeneration, various types of gelatinase activities were detected, and these activities were at their maximum levels at the dedifferentiation stage. Upon treatment with chelating agents EDTA and 1,10-phenanthroline, the enzyme activities were inhibited indicating that those enzymes are likely matrix metalloproteinases (MMPs). Considering the molecular sizes and the decrease of molecular sizes by treatment with p-aminophenylmercuric acetate, an artificial activator of proMMP, some of the gelatinases expressed during limb regeneration are presumed to be MMP-2 and MMP-9. In RA-treated regenerates, overall gelatinase activities increased, especially the MMP-2-like gelatinase activity which increased markedly. These results suggest that MMP-2-like and MMP-9-like gelatinases play a role in ECM remodeling during regeneration, and that gelatinases are involved in the excessive dedifferentiation after RA treatment.     

Cytokines Regulate Gelatinase A and B (Matrix Metalloproteinase 2 and 9) Activity in Cultured Rat Astrocytes

Abstract: Under a tightly regulated expression mechanism, matrix metalloproteinases degrade extracellular matrix proteins and are though to play a role in injury repair and tumor metastasis in peripheral tissues. Little is known about the function of matrix metalloproteinases or agents that regulate their production in adult brain; however, it has been shown that the activity of a calcium-dependent metalloproteinase is elevated in Alzheimer's hippocampus. The goals of this study were to determine whether cultured rat astrocytes produce matrix metalloproteinases and to identify agents that regulate protease activity. Enriched astrocyte cultures were prepared from brains of 1-day-old rat pups, and experiments were performed 13 days later. Gelatinase activity in astrocyte conditioned medium was determined using zymography with gelatin copolymerized with acrylamide in the gel. Under basal conditions after a 24-h incubation, rat astrocytes produce gelatinases of 58 and 66 kDa. On stimulation of astrocytes with lipopolysaccharide, interleukin-1α or -β, or tumor necrosis factor-α for 24 h, a dose-dependent increase in the activity of the 58- and 66-kDa gelatinases and the induction of a 94-kDa gelatinase occurred. All three astrocyte-derived proteases showed maximal activity in the presence of millimolar levels of Ca²⁺, their activity was inhibited in the presence of 1,10-phenanthroline, and their proenzymes were cleaved and activated after incubation with p-aminophenylmercuric acetate. Using immunoblotting, immunopositive bands at the respective molecular sizes indicated that the 58-kDa gelatinase was gelatinase A (matrix metalloproteinase 2) and the 94-kDa activity was gelatinase B (matrix metalloproteinase 9). Induction of the 94-kDa gelatinase by lipopolysaccharide was not influenced when interleukin-1 receptor antagonist was included during the 24-h incubation period; however, the antagonist completely blocked interleukin-1β-induced 94-kDa activity and diminished the activity of the 58- and 66-kDa gelatinases. Dexamethasone inhibited both lipopolysaccharide and interleukin-1β stimulation of the 94-kDa gelatinase. These results indicate that cytokines regulate matrix metalloproteinase expression in cultured rat astrocytes. Because astrocytes become “activated” (are hypertrophic and express increased levels of glial fibrillary acidic protein) in the presence of several inflammatory cytokines, it is possible that these astrocyte-derived enzymes contribute to the activation process and may participate in tissue remodeling after brain injury.