Researches On The Relationships Between Functional Microbial Communities With Sample Properities In Compost Based On Molecular Biological Techniques

Bacteria and fungi species play significant roles in the decomposition andmineralization of agricultural organic wastes during composting. Their communitycompositions are likely influenced by several physico-chemical parameters incomposting systems. So far, the physico-chemical parameters have not beenevaluated simultaneously with the bacterial and fungal composition changes toseparate out their relative importance. It is of interest to conduct such research todetermine to what extent of differences in bacterial and fungal communities areinfluenced by these parameters, respectively. The goal of this study was to identifyand prioritize some of the physico-chemical parameters that contributed to bacterialand fungal community compositions during agricultural waste composting. Ricestraw, vegetables, soil and bran were homogenized at a ratio of11:3:8:2to simulateagrcultural wastes composting. Bacterial and fungal community compositions weredetermined by polymerase chain reaction-denaturing gradient gel electrophoresis（PCR-DGGE）. DGGE banding profiles both for bacterial and fungal community weredigitized after average background subtraction for the entire gel.The relativeintensity of a specific band was transformed according to the sum of intensities of allbands in a pattern. Redundancy analysis （RDA） was conducted to determine therelationships between microbial community structure and physico-chemicalparameters by Canoco （version4.5）. The results showed that approximate73.2%and75.5%of the variation in the bacterial and fungal species data were explained by allparameters. The temporal variation of bacterial community composition wassignificantly related to WSC （19.5%, P=0.002）, ammonium （14.1%, P=0.004）andnitrate （8.5%, P=0.002）, while the most variation in the distribution of fungalcommunity composition was statistically explained by pile temperature （17.0%,P=0.002）, WSC （11.8%, P=0.002）, and moisture content （8.5%, P=0.01）. Thoseparameters were the most likely ones to influence, or be influenced by the bacterialand fungal species. NH₃and its unincorporated form NH4+are the most important nitrogenouscompounds available in the compost materials. An alkaline pH during thecomposting process may lead to substantial loss of nitrogen as gaseous NH3. Theoxidation of ammonia is the first and rate-limiting step of nitrification, anddetermines the transformation balance between oxidized and reduced forms ofnitrogen. Both ammonia oxidizing bacteria （AOB） and archaea （AOA） employ thesame functional amoA gene, encoding a subunit of the ammonia monooxygenaseenzyme responsible for the first step of the nitrification process. The roles of AOAand AOB, and to what extent these ammonia oxidizers might affect nitrogentransformation balance during the composting process are not well understood yet.The aim of this study was to compare the relative contribution of AOA and AOB tonitrification during agricultural waste composting. Potential ammonia oxidation（PAO） rate was also determined. The amoA gene abundance and composition weredetermined using quantitative PCR （qPCR） and DGGE, respectively. Relationshipsbetween PAO rates and the amoA gene abundance were determined to assess thecontribution of AOA and AOB to nitrification.The results showed that the PAO ratevaried between29.3and89.9ng NO2-min-1g-1DW compost sample, with samplescollected on day2to day6displaying the highest activities. The PAO rate decreasedsignificantly during the thermophilic stage and increased further during thematuration stage and reached66.1ng NO2-min-1g-1DW compost sample by the endof the process. The archaeal amoA gene was abundant throughout the compostingprocess. Phylogenetic analyses of the archaeal amoA gene fragments for day4showed that all AOA sequences fell within the soil/sediment lineage. While thebacterial amoA gene abundance decreased to undetectable level during thethermophilic and cooling stages. The high temperature and low oxygenconcentration during the thermophilic and cooling stages might have provided anunfavourable condition for the AOB populations. Phylogenetic analyses of thebacterial amoA gene fragments for day50showed that most AOB sequences showinghigher sequence similarity to Nitrosomonas europaea/communis. DGGE showedmore diverse archaeal amoA gene composition when the PAO rate reached peak values. A significant positive relationship was observed between the PAO rate andthe archaeal amoA gene abundance （R2=0.554; P<0.001）, indicating that archaeadominated ammonia oxidation during the thermophilic and cooling stages. Bacteriawere also related to ammonia oxidation activity （R2=0.503; P=0.03） especiallyduring the mesophilic and maturation stages.As an important fraction of the total organic matter in the compost materials,the lignocellulose are resistant to biodegradation by microbial communities, addingadds difficulties to wastes disposal. Several species of basidiomycetes designated aswhite-rot fungi can efficiently decompose lignocellulose into carbon dioxide andwater in a variety of lignocellulosic materials. The inoculation with thoselignocellulolytic microorganisms has been widely used as a strategy that couldpotentially speed up the composting process and ultimately improve the quality ofcompost product. Phanerochaete chrysosporium （P. chrysosporium） has been widelyassumed one of the most active ligninolytic organisms among white-rot fungidescribed to date. Much attention has currently been drawn to the effects of P.chrysosporium inoculation on the sample maturity index, the enzyme activities andthe reduction of heavy metal toxicity. However, little information is available on theeffect of inoculation with P. chrysosporium on the indigenous microbialcommunities during agricultural waste composting. This research was conducted todistinguish between the separate effects of the P. chrysosporium inoculation andsample property heterogeneity induced by different inoculation regimes on theindigenous bacterial communities during agricultural waste composting. Fourseparate experiments, each in triplicate, were conducted in this experiment. Run Awas uninoculated with P. chrysosporium as control. Run B and Run C wereinoculated with1%of P. chrysosporium mycelium （fresh weight） during the firstfermentation phase （day2） and the second fermentation phase （day20）, respectively.Run D was inoculated with the same amount of P. chrysosporium mycelium bothduring the first and the second fermentation phases. The bacterial communityabundance and structure were determined by the quantitative PCR and denaturinggradient gel electrophoresis analysis, respectively. Results showed that different inoculation regimes changed pile temperature, ammonium, nitrate, WSC, pH andC/N. The ratios of lignocellulose degradation for all runs reached final values ofabout50%,45%and40%for cellulose, hemicellulose and lignin, respectively.Statistically significant higher bacterial16S rDNA gene abundance was obtained intreatments with P. chrysosporium inoculation treatments, indicating a significantstimulatory effect of P. chrysosporium inoculation on the bacterial communityabundance. The bacterial community abundance significantly coincided with piletemperature, ammonium and nitrate （P<0.006）. Variance partition analysis showedthat the P. chrysosporium inoculation directly explained20.5%（P=0.048） of thevariation in the bacterial communities, whereas the sample property changes inducedby different inoculation regimes indirectly explained up to35.1%（P=0.002）. Thebacterial community structure was significantly related to pile temperature, WSCand C/N ratio when P. chrysosporium were inoculated. The C/N ratio solelyexplained7.9%（P=0.03） of the variation in community structure, whereas piletemperature, WSC explained7.7%（P=0.026） and7.5%（P=0.034）, respectively. P.chrysosporium inoculation affected the indigenous bacterial communities mostprobably indirectly through increasing pile temperature, enhancing the substrateutilizability and changing other physico-chemical factors.The fungal community structure and diversity were obtained by sequencingtechnology and the quantitative PCR （qPCR） method, respectively. By sequencing,956effective sequences in total were yielded for all20samples, which consisted of13unique OTUs at99%sequence identity. The dominant phylum across all pooledsamples was Ascomycota, specifically from the species Acremonium chrysogenum,Galactomyces geotrichum and Scytalidium thermophilum. Species of Coprinopsiscinerea and Mortierella angusta that affiliated with Basidiomycota and Fungiincertae sedis, respectively, were also extensively represented. Cladosporiumcladosporioides, Cryptococcus podzolicus, Debaryomyces nepalensis,Cystofilobasidium macerans, Eurotium amstelodami, Nigrospora oryzae, andPlectosphaerella cucumerina were present in samples collected during the earlystage （from day1to day7）, but at low abundance. The white-rot fungi inoculants P. chrysosporium were present in relatively higher abundance in samples collectedduring the second fermentation phase （17.8-26.7%of total fungal communities）. P.chrysosporium only accounted for4.4%of the total fungal population on day7forRun D. Principal component analysis based on relative OUT abundance indicatesthat the indigenous fungal communities might apparently not be changed by P.chrysosporium inoculants during the thermophilic stage （day4to day12, exceeding50°C）. Indigenous fungal community structure significantly changed afterinoculation during the second fermentation phase （day13to day50）. Samplesinoculated with P. chrysosporium during the second fermentation phase （i.e., Runs Cand D） contained higher proportion of A. chrysogenum and G. geotrichum affiliatedwith Ascomycota compared with the control （i.e., Run A） on Day50. Thosenon-inoculated samples （i.e., Runs A and B） that dominated by C. cinerea and S.thermophilum were well separated from those P. chrysosporium inoculated ones. Thetotal and indigenous fungal community abundances in samples with P.chrysosporium inoculation were statistically lower compared to the control （Run A）during the first7days. The regressions between fungal community abundance andsample properties showed that there were significant negative relationships betweenindigenous fungal18S rDNA gene abundance and C/N ratio （R2=0.4483, P<0.01）and WSC （R2=0.691, P<0.001）, as well as a significant positive relationship betweenindigenous fungal18S rDNA gene abundance and moisture content （R2=0.2058,P=0.045）. The results of redundancy analysis showed that the most variation indistribution of indigenous fungal community structure was statistically explained byC/N ratio （F=34.546, P=0.002）, moisture content （F=3.471, P=0.032）, and nitrateconcentration （F=5.219, P=0.012）. Those factors as well as the interactions amongthem were the most likely ones to influence, or be influenced by the indigenousfungal communities in samples with different P. chrysosporium inoculation regimes.