Isolation Of Lignocellulolytic Microorganisms To Develop A Composite Inoculum For Green Waste Composting

The yield of green waste is increased as the deveploment of landscaping in urban China,how to realize efficient disposal of green waste has become the main problem of municipal afforesting.Composting is an economic and practical method for green waste disposal,however,the recalcitrance of green waste,caused by its high lignocellulose content,is a technical challenge obstracting the composting process.Composite inoculum is a superior solution for the technical challenge,however,there is a lack of lignocellulolytic microorganisms isolated from compost of green waste,composite inoculum specially used for green waste composting is rarely reported.Furthermore,current inocula are designed for adding at the beginning of composting,functions of inocula may be weakened because microorganisms of inocula cannot adapt the entire composting.In order to isolate lignocellulolytic microorganisms,develop the composite inoculum specially used for green waste composting,and find out the optimal time of inoculation and the relative mechanisms,finally promote lignocellulose degradation.The current study isolated lignocellulolytic microorganisms from different phases of green waste composting,using classical cultivation method,which was identified using 16S rDNA and ITS sequence analyses.Statistic methods were used to optimize the conditions of fermentation and enzymes production,and the combination of microorganisms,which were to develop the composite inocula used for different phases.Finally,the inocula were added to green waste composting to investigate their influences.Details are as follows:(1)Isolation of lignocellulolytic microorganisms:Two lignocellulolytic microorganisms were isolated from compost of thermophilic phase,marked as B1 and B2,three were isolated from compost of mature phase,marked as B3,B4,and A.After verification using sequence analyses of 16S rDNA and ITS,and morphological observation,the strains of B1-B4 were identified as Bacillus sp.,A was identified as Aspergillus sp.The test of antagonism and inhibition zone showed that there were no antagonism between B1 and B2,and among B3,B4,and A.After verification using dyeing of congo red,aniline blue,and guaiacol,B1-B3 are capable of producing cellulase,lignin peroxidase or manganese peroxidase,B4 can only produce cellulase,A can only produce lignin peroxidase or manganese peroxidase.(2)Cultivation optimization of microorganisms:An orthogonal experiment was used to optimize the fermentation conditions of microorganisms,the results showed that fermentation period of 20(B1-B4)and 48 hours(A),inoculum size of 4%(B1,A)and 3%(B2-B4),rotate speed of 200 rpm,temperature of 37℃(B1-B4)/28℃(A),initial pH of 7,corn steep powder of 20 g/L(B1-B4)and 17 g/L(A),glucose of 7 g/L,molass of 12 g/L,and soybean cake powder of 17 g/L constituted the optimal cultivation conditions of the microorganisms.After the optimization,biomass and activities of lignocellulolytic enzymes were significantly increased,in which biomass was increased by 38.60-128.37%,cellulase,lignin peroxidase,and manganese peroxidase were increased by 84.50-187.31%,86.47-221.90%,and 50.48-277.78%.(3)Development of composite inocula:An orthogonal experiment was used for combination optimization of B1 and B2,which was to develop the composite inoculum used for thermophilic phase.The results showed that the ratio of 6:3(B1:B2)and addtive amount of 15%achieved relatively stronger lignocellulose degradation abilities.Quadratic regressive orthogonal model was used to optimize the combination of B3,B4,and A to develop the composite inoculum used for mature phase,a regression equation was obtained as y=11.99+2.2621+1.33z2+3.71z3+0.13z1z3+0.16z2z3-0.36z12-0.15z22-0.67z32,with p=0.0108,R2=0.85,using stepwise regression.The optimal additive amounts of the three microorganisms turned out to be B3:2.97×104 cfu/g,B4:6.69×106 cfu/g,and A:1.83×105 cfu/g,by the extremum method,which is the formula of microbial inoculum specially used in mature phase.After the combination,degradation abilities of the microorganisms were signficantly increased because of the synergistic effects.Correlation analysis showed that the strain of A and the synergistic effects between A and B4 were the main factors promoting lignin degradation,the strains of B3,B4,A,and their synergistic effects enhanced cellulose degradation.(4)Verification of the composite inocula:1 m3 sized heaps were used for composting,the composite inocula obtained were respectively added at thermophilic and mature phases.Because the microbial and enzymatic activities were inhibited under thermophilic conditions,and the effects of inoculum were covered by indigenous microorganisms,both the composite inocula used for thermophilic and mature phase cannot significantly influence the composting at themophilic phase,but increased the activities of lignocellulolytic enzymes,lignin and cellulose degradation at mature phase,the composite inoculum used for mature phase can also promote humification.Compared to the treatment without inoculum,the composite inocula promoted the activities of cellulase,lignin peroxidase,manganese peroxidase,and laccase by 27.67-112.68%,37.29-126.93%,10.70-70.80%,and 33.37-80.26%,increased the lignin and cellulose degradation rates by 1.70-10.37%and 4.60-13.14%,and enhanced the content of humic substances by 7.80%.These results indicated that temperature is the critical factor influencing the effects of inoculum.Therefore,it is recommoned to add the inoculum to mature phase to increase the adaptation of microorganisms and insure their deserved effects.A total of 5 lignocellulolytic microorganisms were isolated from green waste composting,relative germplasm resources are supplemented.A composite inoculum specially used for green waste composting is developed,which alleviates the problem that composite inoculum is lacked in green waste composting.The idea that adding inoculum in mature phase ensures its effects is presented,which alleviates the problem that current inocula are added at the beginning of composting,functions of inocula are easy to be weakened,it is proved that temperature is the critical factor influencing the effects of inoculum.Application of the composite inoculum alleviates the problem of low efficiency of green waste composting.