Development Of A Novel Microbial Based Lignocellulolytic Enzymatic System For Simultaneous Composting Of Rice Straw With Cow Manure In Two-step Fermentation Process

The lignocelluloses are complex biopolymers and the degradation of such complex biopolymers is one of the great limitations during the composting process.Many advanced strategies are now being used to increase the efficiency of composting process,which also include the pretreatment of lignocellulose before co-composting with livestock manure.Pretreatment of complex lignocelluloses is a crucial step in the conversion of lignocellulosic biomass to simple sugars.Compared to various pretreatment strategies chemical pretreatment and microbial pretreatment are most practiced.Among diverse microbial pretreatment agents,fungal pretreatment is most dominant,because it reduces the recalcitrance of lignocellulosic biomass by using a biochemical oxidation process of lignin degradation,which means it is potentially an environmentally friendly and energy efficient pretreatment technology prior to co-composting.That’s why a study was conducted,which comprised two fermentation processes,including rice straw（RS）pretreatment in which the effects of fungal or chemical pretreatment on RS lignocellulose degradation was explored,while the other fermentation focused on the impacts of pretreated RS on lignocellulose degradation and humic substances formation during 35 days of co-composting with cow manure.In detail,the 35 fungal isolate were purified from degrading wood logs and bamboo shoots.The 35 fungal isolates were assessed for three important lignocellulolytic extracellular enzymes production,which included CMCase（for cellulose degradation）,xylanase（for hemicellulose degradation）,and laccase（for lignin degradation）.All enzymes screening were performed in potato dextrose agar plates and potato dextrose broth supplemented with the specialized substrate,such as carboxymethylcellulose,birchwood xylan,or guaiacol for CMCase,xylanase,and laccase activity analysis,respectively.The results of screening showed among 35 fungi,only five fungi were capable to produce a high concentration of CMCase,xylanase,and laccase enzymes,which were further identified using the ITS1/ITS4 techniques.The ITS identification revealed that five lignocellulolytic fungi were Aspergillus nidulans（AN）,Aporospora terricola（AT）,Trametes hirsuta（TH）,Arthrinium saccharicola（AS）,and Phanerochaete chrysosporium（PC）.These fungal isolates significantly differed in their lignocellulolytic enzyme production（P<0.05）,such as PC and TH showed significantly（P<0.05）higher CMCase,xylanase,and laccase activities than AT,AS,and AN.The five fungal species were tested for RS pretreatment and results showed that,all fungal species significantly（P<0.05）increased the degradation rate of RS compared to the control,and different fungal species also showed significantly different degradation abilities.Such as,the degradation rate for cellulose was 30.1%in TH,which was 1.9-,1.7-and 1.5-fold higher,than in AT,AS,and AN.After a single fungal specie pretreatment study,a fungal consortium study was also conducted.For that,three fungal consortiums were set up to avoid antagonism between chosen fungal species within the consortium:FT1（AT+AN+AS+PC）,FT2（TH+AN+PC）,and FT3（AN+AS+PC）.The pretreatment of RS with fungal consortium showed that the combination of multiple fungal species（consortium）enhanced the degradation of RS biomass,relative to single species application.The degradation rate of total weight,cellulose,hemicellulose,and lignin in FT2 treatment was 2.5-,0.9-,1.6-and 1.4-fold higher,respectively,than in FT1 treatment,1.3-,1.4-,2.0-and 1.2-fold higher,respectively,than in FT3 treatment,5.0-,2.6-,7.2-and 9.9-fold higher,respectively,than in control.Similar to fungal pretreatment,a parallel chemical pretreatment experiment of RS was also conducted,in which a comparative analysis of alkali（Na OH）,dilute acid（H₂SO₄）,and Fenton reagent was done.The chemical pretreatment results revealed that,overall,Fenton pretreatment showed significantly（P<0.05）high cellulose,hemicellulose,and lignin degradation in RS as compared to acid,alkali,and CK pretreatments,thus making it a suitable approach to utilize with co-composting experiments further.After pretreatment,the next step was to compare the composting efficiency of RS pretreated with FT2 fungal consortium（FPT）relative to that in non-pretreated RS（CK）and chemically pretreated（Fenton reagent）RS（CPT）.The FPT had a significant impact on lignocellulose degradation（84%）of RS by producing higher lignocellulolytic enzymes than chemical pretreatments（79%）or the control（61%）.The FPT composting pile showed a significantly high composting temperature in the late mesophilic stage,which enhanced the degradation of lignocellulose.The fluorescence excitation-emission spectroscopy revealed that significantly more humic acid-like compounds were formed in FPT as compared to CPT and CK.The mature 1%compost was introduced into the soil to grow rice seedlings,which also showed FPT mature compost（CMP3）significantly increased plant height and total biomass,which indicate fungal pretreatment impact positively on RS co-composting.The current study provided a demonstration of the promising full-scale pretreatment technology prior to co-composting for comprehensive management of the RS and cow manure.The findings of this study suggest that compared to current leading chemical pretreatment processes（Fenton）,fungal pretreatment with the specialized lignocellulolytic fungal consortium is a feasible method to accelerate RS degradation and humification and it is an environmentally friendly and energy-efficient process.