An Improved Sequence-aided T-RFLP Analysis of Bacterial Succession During Oyster Mushroom Substrate Preparation

While oyster mushroom (Pleurotus spp.) is one of the most popular cultivated edible mushrooms, there is scanty information about the microbial community taking part in mushroom substrate production. In this study, an improved sequence-aided terminal restriction fragment length polymorphism (T-RFLP) was used to identify and (semi-)quantify the dominant bacteria of oyster mushroom substrate preparation. The main features of the improved T-RFLP data analysis were the alignment of chromatograms with variable clustering thresholds, the visualization of data matrix with principal component analysis ordination superimposed with cluster analysis, and the search for stage-specific peaks (bacterial taxa) with similarity percentage (analysis of similarity) analysis, followed by identification with clone libraries. By applying this method, the dominance of the following bacterial genera was revealed during oyster mushroom substrate preparation: Pseudomonas and Sphingomonas at startup, Bacillus, Geobacillus, Ureibacillus, Pseudoxanthomonas, and Thermobispora at the end of partial composting, and finally several genera of Actinobacteria, Thermus, Bacillus, Geobacillus, Thermobacillus, and Ureibacillus in the mature substrate. As the proportion of uncultured bacteria increased during the process, it is worth establishing strain collections from partial composting and from mature substrate for searching new species.     

Biomass and Biological Activity during the Production of Compost Used as a Substrate in Mushroom Cultivation

The production of a suitable substrate for the cultivation of the common white button mushroom, Agaricus bisporus, is referred to as composting. High microbiological activity causes temperatures of the composting material to rise as high as 80°C. At stacking, an optimal oxygen consumption rate of 140 μmol of O₂ h⁻¹ g (dry weight)⁻¹ was found in the compost at 50°C, whereas the oxygen consumption rate of the end product was lower at all temperatures tested. No significant differences were observed between biomass content and mineralization rate of ¹⁴C-labeled glutamate of the two composts. Biomass content was shown to be a major function of both temperature and the sampling site position in the stack. On the basis of the results reported here, a minimal composting time of 3.3 days for the phase I process was calculated. Further suggestions are made to reduce the time necessary for the production of a substrate for A. bisporus considerably.     

Odorous Sulfur Compounds Emitted during Production of Compost Used as a Substrate in Mushroom Cultivation

Large-scale composting facilities are known to cause environmental problems, mainly through pungent air emitted by composting material. In air samples taken above stacks set up to prepare compost used as a substrate in mushroom cultivation, several volatile compounds were identified by means of the coupled techniques of gas chromatography and mass spectrography. Among the compounds identified, sulfur-containing compounds [H₂S, COS, CH₃SH, CS₂, (CH₃)₂S, (CH₃)₂S₂, and (CH₃)₂S₃] are the most conspicuous in causing a nuisance. Quantification of these compounds was performed by concentrating a relatively small air sample on Tenax GC. The sampling method appeared to be very useful under field conditions. During the composting process, the concentration of the volatile sulfur compounds in emitted air ranged from 1 to 35 μmol/m³. The highest concentrations were obtained at the end of the outdoor process. Total sulfur emission amounted to 8.3 mg of sulfur per kg (fresh weight) of compost. The end product still contained 2.58 g of sulfur per kg (fresh weight) of compost. Suggestions about the origin of the volatile sulfur compounds are made.     

Succession of Bacterial Communities during Early Plant Development: Transition from Seed to Root and Effect of Compost Amendment

Compost amendments to soils and potting mixes are routinely applied to improve soil fertility and plant growth and health. These amendments, which contain high levels of organic matter and microbial cells, can influence microbial communities associated with plants grown in such soils. The purpose of this study was to follow the bacterial community compositions of seed and subsequent root surfaces in the presence and absence of compost in the potting mix. The bacterial community compositions of potting mixes, seed, and root surfaces sampled at three stages of plant growth were analyzed via general and newly developed Bacteroidetes-specific, PCR-denaturing gradient gel electrophoresis methodologies. These analyses revealed that seed surfaces were colonized primarily by populations detected in the initial potting mixes, many of which were not detected in subsequent root analyses. The most persistent bacterial populations detected in this study belonged to the genus Chryseobacterium (Bacteroidetes) and the family Oxalobacteraceae (Betaproteobacteria). The patterns of colonization by populations within these taxa differed significantly and may reflect differences in the physiology of these organisms. Overall, analyses of bacterial community composition revealed a surprising prevalence and diversity of Bacteroidetes in all treatments.     

Comparison of T-RFLP and DGGE techniques to assess denitrifier community composition in soil

Terminal restriction fragment length polymorphism (T-RFLP) and denaturing gradient gel electrophoresis (DGGE) and subsequent statistical analysis were compared with assess denitrifier community composition in agricultural soil based on the nosZ gene, encoding the nitrous oxide reductase. Analysis of binary or relative abundance-based metric and semi-metric distance matrices provided similar results for DGGE, but not for T-RFLP. Moreover, DGGE had a higher resolution than T-RFLP and binary data was better for discriminating between samples.     

土壤细菌DNA提取及多样性分析的T-RFLP方法

获得高质量的土壤总DNA是土壤细菌生态学的关键步骤之一.实验通过综合应用两个试剂盒(Soilmaster kit和DNA IQTM系统)的优点进行土壤样品总DNA的提取,结果证明该方法是一种快速、有效、灵敏、稳定的土壤DNA提取方法.另外尝试将16S rDNA序列和T-RFLP(Terminal restriction fragment 1ength polymorphism)技术引入土壤细菌DNA群落多样性的研究中,证明T-RFLP是一种有力的土壤细菌多样性分析工具.     

Growth Characteristics of the Thermophilic Fungus Scytalidium thermophilum in Relation to Production of Mushroom Compost

Scytalidium thermophilum is an important thermophilic fungus in the production of mushroom compost. I investigated the characteristics of this organism and present a simple model with which fungal growth in compost can be described. The model is used to predict better circumstances for rapid indoor production of mushroom compost. I conclude that inoculation of the starting material with prepared compost either before or after the pasteurization phase has only a minor effect on the shortening of the composting process. This is because the initial growth rate of the fungus is much higher than its growth rate later. A lower temperature (53.5°C instead of the usual 56 to 58°C) during the pasteurization phase may be most profitable for rapid compost production; such a temperature may reduce the time that is needed for the last phase of the production process by at least 1 day.     

Succession of microbial communities during a biostimulation process as evaluated by DGGE and clone library analyses

AIMS: The objective of this study was to investigate the changes in the indigenous bacterial community structure for assessing the impact of biostimulation on spilled oil. METHODS AND RESULTS: Changes in the bacterial community structure were monitored by denaturing gradient gel electrophoresis (DGGE) and clone library methods based on 16S rRNA gene (rDNA) sequences. The results of DGGE, coupled with the use of the Shannon index and principal component analysis (PCA) and clone library analyses, were consistent. In the treated (fertilized) area, one operational taxonomic unit (OTU) became dominant during the fertilization period, and it was most closely related to Pseudomonas putida. CONCLUSIONS: The bacterial community structure in the treated area was markedly different from that in the control (non-fertilized) area during the fertilization period, but in the two areas it became similar at 14 weeks after the end of fertilization. SIGNIFICANCE AND IMPACT OF THE STUDY: The results suggest that the bacterial community structure was disrupted by the biostimulation treatment, but that it recovered immediately after the end of fertilization.     

Microbiological and Biochemical Investigation of Succession in Lignin-Containing Compost Piles

The investigation of microbiological succession and changes in the enzymatic activity, temperature, pH, and phytotoxicity of lignin during its composting showed that the addition of a starter culture (a specially developed association of microorganisms) affects degradational succession in the compost pile. The process of composting can be monitored either microbiologically or biochemically, by measuring the activity of some enzymes. The compost is ready for use when the activity of oxidoreductases (particularly polyphenol oxidases) falls and the activity of invertase stabilizes at a certain level.     

Comparative studies to assess bacterial communities on the clover phylloplane using MLST, DGGE and T-RFLP

Denaturing gradient gel electrophoresis (DGGE) and terminal restriction fragment length polymorphism (T-RFLP) were used to characterise the changes that occurred in Bacillus cereus group strains present in the phylloplane of clover Trifolium hybridum over 4 months. These strains had previously been analysed by multiple locus sequence typing (MLST). DGGE displayed many equally intense bands which indicated many equally abundant ribotypes. The bacterial community composition was variable and the leaves sampled as little as a week apart were found to have some temporal variability, indicating that diverse phylloplane bacterial communities follow sequential patterns from time to time. The B. cereus group community clearly clustered into early, mid and late branches, possibly due to multiple successional sequences occurring during growing seasons. The functionally and phylogenetically diverse microbial communities appeared to exhibit predictable successional patterns over shorter time scales. DGGE analysis with the molecular marker rpoB gave better resolution than 16S rRNA amplicons. There were no strong similarities between the dendrograms produced by DGGE, MLST and T-RFLP and the clustering produced by the automated T-RFLP method was variable even between the three restriction enzymes used. The DGGE–MLST method emerged as a superior method to T-RFLP–MLST for rapid typing of bacterial communities.     

Succession of Bacterial Populations during Plant Residue Decomposition in Rice Field Soil

The incorporation of rice residues into paddy fields strongly enhances methane production and emissions. Although the decomposition processes of plant residues in rice field soil has been documented, the structure and dynamics of the microbial communities involved are poorly understood. The purpose of the present study was to determine the dynamics of short-chain fatty acids and the structure of bacterial communities during residue decomposition in a rice field soil. The soil was anaerobically incubated with the incorporation of rice root or straw residues for 90 days at three temperatures (15, 30, and 45°C). The dynamics of fatty acid intermediates showed an initial cumulative phase followed by a rapid consumption phase and a low-concentration quasi-steady state. Correspondingly, the bacterial populations displayed distinct successions during residue decomposition. Temperature showed a strong effect on the dynamics of bacterial populations. Members of Clostridium (clusters I and III) were most dominant in the incubations, particularly in the early successions. Bacteroidetes and Chlorobi were abundant in the later successions at 15 and 30°C, while Acidobacteria were selected at 45°C. We suggest that the early successional groups are responsible for the decomposition of the easily degradable fraction of residues, while the late successional groups become more important in decomposing the less-degradable or resistant fraction of plant residues. The bacterial succession probably is related to resource availability during residue decomposition. The fast-growing organisms are favored at the beginning, while the slow-growing bacteria are better adapted in the later stages, when substrate availability is limiting.Rice residues, including root and straw residues, serve as the major carbon source in paddy fields. It has been estimated that the amounts of organic matter supplied annually to paddy fields range from 1,700 to 3,470 kg ha⁻¹, and more than 65% of them were derived from plant residues (19, 23). The incorporation of rice residues into paddy fields helps sustain soil organic matter, improve physical and chemical properties, and increase nutrient availability (15, 39). However, it also strongly enhances methane production and emissions (6, 45, 46).Numerous studies have been carried out on residue decomposition and CH₄ production in rice field soils (11-13, 22). The rate of decomposition usually is separated into a fast phase and a slowdown phase (24). According to the dynamics of the intermediates H₂ and fatty acids and the activities of polysaccharolytic enzymes, Glissmann and Conrad (13) proposed five stages for residue decomposition, including the production and consumption of reducing sugars, the production and consumption of H₂ and fatty acids, and the production of CH₄. The rate of decomposition was affected by the composition of residues (11, 18). Root residues generally are decomposed slower than straw residues (24). The fermentation pathway also could differ depending on the residue material, resulting in different fatty acid intermediates (13).A complex microbial assemblage consisting of hydrolytic, fermenting, homoacetogenic, syntrophic, and methanogenic microorganisms are involved in the anaerobic decomposition of organic residues (5, 41, 48). Plant residues are composed of complex components. With the decomposition process, the proportion of labile component decreases while the resistant components relatively accumulate. Changes in the activity and structure of the microbial community thus are anticipated during the processes of residue decomposition. However, little has been known about the dynamics of microbial populations during residue decomposition in anoxic rice soil. Using culture-independent methods, Weber et al. (47) showed that the structure of the bacterial community shifted between early and late stages. Microscopic observation revealed the differential colonizations of bacterial populations on different parts of straw residue, indicating the effects of residue quality and niche condition (18). The microbial community appears to form a spatially well organized architecture, with the fermenting bacteria colonizing the residue particles, while the syntrophic bacteria and methanogens mainly inhabit the adjacent soil during the decomposition process (12).Air temperature exhibits a large seasonal variation in southeastern Asia, where rice is widely cultivated. The lowest and highest records in our research site, for instance, were −5 and 40°C, respectively, in 2006, when we collected the soil samples. It has been demonstrated that temperature has a strong effect on residue decomposition and CH₄ production (8, 22, 33). However, it is uncertain whether the effect also is reflected in the structure and function of degrading microbial communities in the soil. Therefore, the purpose of our project was to determine the effect of temperature on microbial communities during the processes of plant residue decomposition in a Chinese rice field soil. The dynamics of methanogenesis and methanogenic archaea have been reported previously (33). Here, we show the results on fatty acid intermediates and the responsible bacterial communities.     

双孢蘑菇培养料发酵过程中细菌群落动态变化

双孢蘑菇产量的最主要影响因素是培养料的堆肥发酵过程,而微生物是其中的主要推动力。为了揭示工厂化生产双孢蘑菇堆肥发酵过程中的微生物群落结构变化及演替规律,深入了解细菌在堆肥发酵过程中作用,文章以工厂化生产双孢蘑菇堆肥为研究对象,采用16S rRNA基因高通量测序技术分析堆肥前、一次发酵、二次发酵过程中细菌群落特征。共产生有效操作分类单元(OTUs) 854个,涵盖了19门259属的细菌。堆肥前优势类群为Proteobacteria,一次发酵优势类群为Firmicutes、Deinococcus-Thermus等,二次发酵时绿弯菌门Chloroflexi为主要类群。研究结果表明:微生物种类和数量随堆肥过程不断升高,并在二次发酵后降低,且微生物群落结构呈现连续变化的规律。高通量测序还发现了很多未分类细菌,说明双孢蘑菇培养料堆肥样品中还蕴含着大量未知的微生物种类。     

Inoculation of Scytalidium thermophilum in Button Mushroom Compost and Its Effect on Yield

Scytalidium thermophilum isolates in culture, as well as the endogenous strain(s) in mushroom compost, were inactivated at 70°C. This temperature was used to pasteurize composts for experiments. Of nine thermophilic fungal species, only S. thermophilum and Myriococcum thermophilum grew well on pasteurized compost in test tubes. The effect of both species on the crop yield of Agaricus bisporus mushrooms was studied. In solid-state fermentation rooms called tunnels, compost was pasteurized and inoculated. After incubation, the inoculated organisms were reisolated and counted, showing their successful colonization. The yield of mushrooms on inoculated composts was almost twice that on the pasteurized control. This result demonstrates the effectiveness of S. thermophilum in compost preparation. Inoculation is not necessary for traditional compost preparation. Naturally occurring strains of S. thermophilum, present in ingredients, readily colonize compost during preparation. Inoculation may be vital if compost is pretreated at a high temperature in tunnels. This finding is of relevance for the environmentally controlled production of high-yielding compost.     

Evolution of Volatile Sulfur Compounds during Laboratory-Scale Incubations and Indoor Preparation of Compost Used as a Substrate in Mushroom Cultivation

Volatile sulfur compounds are known to be produced during the preparation of compost used as a substrate in mushroom cultivation. Because they cause odor problems, attempts have been made to reduce the production of these compounds. The influences of temperature and various additions on the production of volatile sulfur compounds from composting material were tested on laboratory-scale preparations. The production of H₂S, COS, CH₃SH, and (CH₃)₂S was proven to be a biological process with an optimal temperature that coincides with the optimal temperature for biological activity. The formation of CS₂ and (CH₃)₂S₂ was shown to be a nonbiological process. The emission of volatile sulfur compounds during the indoor preparation of mushroom compost appeared to be remarkably reduced (about 90%) as compared with the emission during the conventional outdoor process. Introduction of this indoor composting process would result in a significant reduction in environmental pollution.     

Analysis of the K+ Current Profile of Mature Rat Oligodendrocytes in situ

Previous studies have reported that mature oligodendrocytes (OLGs) in vitro display various voltage-dependent K+ currents while in situ OLGs show only voltage-independent K+ currents. Given this discrepancy and the lack of information on myelinating OLG ion channel expression in situ, we characterized mature OLG currents in myelinating corpus callosum slices from 17 to 36-day old rats. OLGs were recorded in cell-attached and whole-cell patch-clamp configurations, displayed morphology typical of mature OLGs, and stained positive for myelin basic protein. OLGs displayed large voltage-independent currents that decayed during the voltage pulse and small voltage-activated outward currents. The latter were blocked by TEA, activated between -40 and -50 mV, and decayed slowly. The former were composed of large voltage-independent, time-dependent Ba2+ (1 mM)-sensitive currents, and voltage-dependent Cs+ (5 mM) and Ba2+ (100 mM)-sensitive currents that reversed near the K+ equilibrium potential and inactivated at hyperpolarized potentials, identifying them as inwardly rectifying K+ currents. Inwardly rectifying single-channel K+currents could be recorded in the cell-attached configuration. The estimated single-channel slope conductance was 30 pS. The steady-state open probability was voltage-dependent and declined from 0.9 to 0.5 between -80 and -150 mV. Overall, mature OLGs in situ possess time- and also voltage-dependent K+ currents, which may facilitate clearance of K+ released during axonal firing.     

Estimation of nitrifier abundances in a partial nitrification reactor treating ammonium-rich saline wastewater using DGGE, T-RFLP and mathematical modeling

The bacterial community in a partial nitrification reactor was analyzed on the basis of 16S rRNA gene by cloning–sequencing method, and the percentages of ammonia-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB) in the activated sludge were quantified by three independent methods, namely, denaturing gradient gel electrophoresis (DGGE), terminal restriction fragment length polymorphism (T-RFLP) and Double Monod modeling. The clone library results suggested that there were only a dominant AOB and a dominant NOB species in the reactor, belonging to Nitrosomonas genus and Nitrospira genus, respectively. The percentages of NOB in total bacterial community increased from almost 0% to 30% when dissolved oxygen (DO) levels were changed from 0.15 mg/L to 0.5 mg/L, coinciding with the accumulation and conversion of nitrite, while the percentages of AOB changed little in the two phases. The results confirmed the importance of low DO level for inhibiting NOB to achieve partial nitrification. Furthermore, the percentages of AOB and NOB in the total bacteria community were estimated based on the results of batch experiments using Double Monod model, and the results were comparable with those determined according to profiles of DGGE and T-RFLP.     

Colony-Forming Analysis of Bacterial Community Succession in Deglaciated Soils Indicates Pioneer Stress-Tolerant Opportunists

We investigated the response of bacterial communities inhabiting two deglaciated soils (10 and 100 years post-deglaciation) to two stimuli: (i) physical disruption (mixing), and (ii) disruption plus nutrient addition. PCR/DGGE analysis of 16S rRNA genes extracted from soil during a 168-h incubation period following the stimuli revealed that more bacterial phylotypes were stimulated in the 10-y soil than in the 100-y soil. In addition to 10-y and 100-y soils, two additional soils (46 and 70 y) were further differentiated using colony-forming curve (CFC) analysis during a 168-h incubation period, which revealed that younger soils contained a higher proportion of rapidly colonizing bacteria than successively older soils. Eco-collections of CFC isolates that represented colonies that formed fast (during the first 24 h) and slow (final 36 h) were harvested from 10-y and 100-y soils and differentiated according to response to three stress parameters: (i) tolerance to nutrient limitation, (ii) tolerance to temperature change, and (iii) resistance to antibiotics. The tested parameters distinguished fast from slow bacteria regardless of the age of the soil from which they were isolated. Specifically, eco-collections of fast bacteria exhibited greater nutrient- and temperature-stress tolerance as well as more frequent antibiotic resistance than slow bacteria. Further DGGE analysis showed that several eco-collection phylotype bands matched (electrophoretically) those of soil phylotypes enriched by mixing and nutrient stimulus. Overall, the results of this study indicated that the succession of colony-forming bacteria was differentiated by bacterial opportunism and temporal response to stimuli. Furthermore, although stress tolerance strategies are associated with opportunistic bacteria regardless of successional age, it appears that the proportion of opportunistic bacteria distinguishes early vs late succession forefield bacterial populations.     

Development of Bacterial Contamination during Production of Yeast Extracts

Baker’s yeast suspensions having bacterial populations of 10⁶ and 10⁸ CFU/ml were subjected to autolysis processes designed to obtain yeast extracts (YE). The bacterial contaminants added to the yeast cell suspensions were produced with spent broths obtained from a commercial yeast production plant and contained 59% cocci (Leuconostoc, Aerococcus, Lactococcus) as well as 41% bacilli (Bacillus). Autolyses were conducted at four different pH levels (4.0, 5.5, 7.0, and 8.5) and with two autolysis-promoting agents (ethyl acetate and chitosan). Processing parameters were more important than the initial bacterial population in the development of contaminating bacteria during manufacture of YE. Drops in the viable bacterial population after a 24-h autolysis were observed when pH was adjusted to 4.0 or when ethyl acetate was added. A significant interaction was found between the effects of pH and autolysis promoters on the bacterial population in YE, indicating that the activity of ethyl acetate, as opposed to that of chitosan, was not influenced by pH.     

Effect of biocontrol agent Pseudomonas fluorescens 2P24 on soil fungal community in cucumber rhizosphere using T-RFLP and DGGE

Fungi and fungal community play important roles in the soil ecosystem, and the diversity of fungal community could act as natural antagonists of various plant pathogens. Biological control is a promising method to protect plants as chemical pesticides may cause environment pollution. Pseudomonas fluorescens 2P24 had strong inhibitory on Rastonia solanacearum, Fusarium oxysporum and Rhizoctonia solani, etc., and was isolated from the wheat rhizosphere take-all decline soils in Shandong province, China. However, its potential effect on soil fungal community was still unknown. In this study, the gfp-labeled P. fluorescens 2P24 was inoculated into cucumber rhizosphere, and the survival of 2P24 was monitored weekly. The amount decreased from 10(8) to 10(5) CFU/g dry soils. The effect of 2P24 on soil fungal community in cucumber rhizosphere was investigated using T-RFLP and DGGE. In T-RFLP analysis, principle component analysis showed that the soil fungal community was greatly influenced at first, digested with restriction enzyme Hinf I and Taq I. However, there was little difference as digested by different enzymes. DGGE results demonstrated that the soil fungal community was greatly shocked at the beginning, but it recovered slowly with the decline of P. fluorescens 2P24. Four weeks later, there was little difference between the treatment and control. Generally speaking, the effect of P. fluorescens 2P24 on soil fungal community in cucumber rhizosphere was just transient.