Researches On The Ligninolytic Functional Microbial Communities Constructions During Agricultural Waste Composting

Composting has become an important method widely used for treatingagricultural organic waste through the decomposition of biodegradable componentsby bacterial, fungal and actinomycetal microbial communities. The microbialcommunities during composting influence or are influenced by changes of theorganic matter. As the important fraction of the agricultural wastes, lignin islargely resistant against microbial attack because of its complex cross-linkedstructure, protecting the celluloses, which consequently makes the lignindegradation become the limiting step influencing the speed and maturation ofcomposting. As the vital functional enzyme for lignocelluloses degradation,laccases (benzenediol/oxygen oxidoreductases, EC1.10.3.2) or laccase-likemulticopper oxidases (LMCOs) can catalyze the oxidation of various phenolic andsome non-phenolic substrates, concomitantly with the reduction of the molecularoxygen to water. Thus, laccases play an important role in the lignin biodegradationand maturation in the composting process.In this paper, laboratorial composting was set up with agricultural wastes straw,vegetables, soil and bran. Strain C1isolated from the thermophilic-phase sample(Day4) presented the highest laccase activity, using the selection mediumcontaining the substrate guaiacol and antifungal antibiotic. Based on its culturalcharacteristic, morphologic identification,16S rRNA sequence analysis, the strainwas identified as Streptomyces sp. C1. After membrane filtration, precipitatingwith ammonium sulfate, DEAE-52cellulose column chromatography and SephadexG-100column chromatography, the enzyme SCLAC displayed a specific activity of24.42U mg-1protein, with a purification factor of25.70-fold, and with a final yield3.45%. The enzyme SCLAC showed a single protein band on SDS-PAGE gel with amolecular mass of about38kDa. The optimum pH of SCLAC was7.0(2,6-DMP)or8.0(ABTS and guaiacol)，and SCLAC showed alkaline resistance and moderatethermostability (the residual enzyme activity is above60%after incubating for24hat pH10.0or incubating for120min at50℃). DDC and NaN3of1mM wouldinhibit the enzyme activity completely. The enzyme activity was activated by somemetal ions such as Cu2+, Co2+and Fe3+at the concentration of1mM, and therelatively high ion concentration would inhibited the enzyme activity except for Cu2The kinetic constants for SCLAC with ABTS as a substrate are Km=0.43mM(the lowest among the selected substrates) and kcat/Km=1.76×104M-1s-1, whichindicated stronger affinity and highest catalytic efficiency with ABTS. The LMCOcould efficiently decolorize the indigo carmine and diamond black PV withsyringaldehyde as mediator, suggesting a great potential for dye decolorization inthe textile industry. The finding of new laccase-producing Streptomyces sp. C1inthis study will also contribute to the further references for studying the functionalgenes of lignocelluloses degradation microorganisms in the composting process.Substantial efforts have been directed toward characterization of residuecarbon (C) chemistry by solid state cross polarization magic angle spinning13C-NMR (CPMAS13C-NMR) spectroscopy in soil and composting ecosystems;however, the relationship between residue C-chemistry and microbial communitystructure assessed using DNA-based method during lignocellulosic wastecomposting is poorly understood. In this study, residue C-chemistry and microbialcommunities during composting were determined by CPMAS13C-NMR andPCR-denaturing gradient gel electrophoresis (PCR-DGGE), respectively.Redundancy analysis (RDA) was used to assess the relationships betweenenvironmental factors (including physico-chemical parameters and13C-NMR data)and microbial community composition. The results showed that the temporalvariation of bacterial community structure was significantly related to C/N、Aryl-C、pH and O-alkyl-C (P<0.05), among these selected factors, while for fungalcommunity variation, the significantly associated factors were WSC、Di-O-alkyl-C、 N-alkyl-C and pile temperature. Aryl-C was found to havesignificant effect on bacterial community composition, which implies that thelignin degradation was correlated with bacterial communities. These findingswould improve the understanding of the interactions between microbial communitycompositions and carbon availability during composting.Traditional three-domain fungal and bacterial laccases have been extensivelystudied for their significance in various biotechnological applications. Recently,new two-domain laccase-like multicopper oxidases (LMCOs) in Streptomyces spp.have aroused attentions for their novel traits (pH and thermal stability). The aminoacid residues and molecular weight of new Streptomyces two-domain LMCOs wereless than the traditional three-domain LMCOs. Streptomyces are the dominantactinomycetes in the composting ecosystems, could soluble lignin and produce theantibiotics. However, the current knowledge about their ecological role and distribution in natural or artificial ecosystems is insufficient. The aim of this studywas to investigate the diversity and composition of Streptomyces two-domainLMCO genes in agricultural waste composting, which will contribute to theunderstanding of the ecological function of Streptomyces two-domain LMCOs withpotential extracellular activity and ligninolytic capacity. A new specific PCRprimer pair was designed to target the two conserved copper binding r egions (cbr IIand IV) of Streptomyces two-domain LMCO genes. Gene libraries retrieved fromsix composting samples revealed a high diversity and rapid succession ofStreptomyces two-domain LMCO genes during composting. Based on thephylogenetic analysis, the obtained sequences mainly clustered with two-domainlaccases genes of Streptomyces coelicolor, Streptomyces violaceusniger,Streptomyces griseus and Streptomyces ipomoea. The obtained sequence typescluster in8distinct clades, most of which are homologous with Streptomycestwo-domain LMCO genes, but sequences of clades III and VIII do not match withany reference sequence of known streptomycetes. Phenol oxidase activity at pH8.0in the composting process were found to be positively associated with abundance ofStreptomyces two-domain LMCO genes (R2=0.830， P=0.012), however, thesignificant relationships were not found at acid conditions (pH4.5and6.0). Thegene copies of Streptomyces two-domain LMCO were significantly correlated withhemicelluloses degradation ratios (R2=0.651, P=0.05), positively related withcellulose and lignin degradation ratios (R2=0.484and R2=0.466). Theseobservations provide important clues that Streptomyces two-domain LMCOs arepotentially involved in bacterial extracellular phenol oxidase activities andlignocelluloses biodegradation during agricultural waste composting.Traditional researches considered that fungal communities play more importantrole than bacterial communities in the lignocelluloses degradation duringcomposting. Bacterial species could produce lacases and solubilize lignin, andbacterial laccases have advantages over fungal laccases, such as stability at hightemperature and and alkaline pH values, which maybe implies the potentialimportance of bacterial LMCOs microorganisms in the composting systems withalkaine and high temperature condition. Moreover, growing molecular evidencepoints to a wide occurrence of LMCOs-encoding genes among bacterial species,however, the current knowledge of the temporal changes in bacterial LMCO genediversity is insufficient in composting system. Six gene libraries were constructedfrom samples in representative stages (D1, D4, D8, D15, D30, D50) using the bacterial universal specific primers Cu1AF/Cu2R during agricultural wastecomposting. A total of189sequences obtained from sample DNA extracts wereclassified to59OTUs based on10%cutoff. The shannon index reached the highestof3.42on day4based on3%cutoff, indicating a high diversity and rapidsuccession of bacterial LMCO genes during composting. The distribution profile ofbacterial LMCO genes showed-proteobacterial-, γ-proteobacterial-andactinobacterial-associated species were the dominant communities in thecomposting system. They accounted for71.89%of the total sequences. The Pearsoncorrelation analysis indicated that the pile temperature and WSC content weresignificantly positively correlated with bacterial LMCO gene OTU numbers,relative abundance, Chao1and Shannon index, whereas the fulvic acid (HA)content had the most significant effect on the distribution of the bacterial LMCOgenes during composting by redundancy analysis. These findings indicated that theenvironmental changes were the main factors that drives the bacterial LMCOs genecompositions and constructions in the composting process, and it will improve theunderstanding of mutual relationship between environmental factors and bacterialLMCO community compositions.