Conversion Of Bamboo Shoot Shell Into Mciroalgae Biomass Through Multi-stage Fermentation

Bamboo shoot shell（BSS）is a type of by-product found abundantly in nature but harbors low value.It can cause serious environmental pollution and result into affecting human life if it is not treated properly.It is possible to convert BSS into some bioproducts only through biorefinery because BSS contains more than 70% of cellulose and hemicellulose.Chlamydomonas is a kind of unicellular plant and its metabolites such as polysaccharides,triglycerides,proteins and pigments can be applied in the fields of food process,pharmaceuticals industry,gene engineering and biofuels production.Chlamydomonas prefer to utilize acetic acid as carbon source.However,higher cost hampers the application of Chlamydomonas on commercial scale if pure acetic acid is employed to compose the medium.To couple BSS utilization with the development of economical carbon source for Chlamydomonas cultivation,a set of studies were carried out in the present research work including（1）isolation,identification and characterization of cellulase producing microorganism to degrade BSS cellulose,（2）screening of laccase producing white-rot fungus to pretreat BSS,（3）constructing fungal consortium to decompose BSS and the hydrolysate was further subjected to sacharification and acidification to culture Chlamydomonas reihardtii.,（4）investigating the differential expression genes and their regulation when C.reinhardtii grew in the acidic solution of BSS hydrolysate,based on transcriptome technology.Above mentions objective could benefit the environment,economy and human society in various aspects.The following are the detailed statements about these objectives:1.The screening and characterization of cellulase-producing microorganism.One isolate was obtained after two rounds of screening.It was further classified as Aspergillus niger and named as Aspergillus niger N3 based on morphological and molecular tests.Subsequently,single factor tests and response surface methods（RSM）were performed to optimize the conditions of cellulase production.The results suggested that A.niger N3 could produce maximum carboxymethylcellulase（CMCase）in the medium consisted of（g/L）: BSS powder 10.0,yeast extract 2.0,KH₂PO₄ 1.0,MgSO4·7H2O 0.5.The maximum production was achieved at the initial pH,the culturing temperature and duration time of 6.42,33.8 ℃ and 96 h,respectively.The result of verification test indicated that the maximum CMCase activity was 5.92 U/mL,which increased 7.4% compared to the activity under original conditions.2.The enhancement mechanism of Pleurotus ostreatus bio-pretreatment.A bio-pretreatment strain,P.ostreatus,was obtained after a successful isolation.Thereafter,the effects of carbon,nitrogen source and inducer on the production of laccase were investigated.The results indicated that（1）BSS supplemented with a small amount of organic nitrogen could benefit the mycelia growth of P.ostreatus,（2）wheat bran could be employed in the BSS pretreatment as auxiliary carbon source and laccase inducer.Subsequently,BSS was pretreated by the isolated P.ostreatus strain.The following analysis suggested that the P.ostreatus mycelia could break down the recalcitrant BSS fiber.Furthermore,the degraded BSS could significantly induce the synthesis of cellulase in A.niger N3.After a series of tests,the water-soluble inducer molecule was proposed as a lignin derivative without aromatic ring,this discovery had not been reported before our research.3.Subsequently,process optimization of BSS converted into C.reinhardtii biomass was targeted.For this,firstly,the co-culture model of P.ostreatus and A.niger was constructed.Secondly,PB and Box-Behnken design were applied to predict the contents of wheat bran,yeast extract and KH₂PO₄ in the medium for BSS decomposition,the predicted values were 2.26%,0.47% and 0.46%,respectively.The result of confirmatory experiment suggested that the maximus decomposition rate was 34.14% when BSS was fermented under the optimized conditions.RSM design was employed to optimize the consecutive saccharification of BSS.The result predicted that under the optimal conditions of 61.99 ℃,pH 5.35,supplemental BSS 7.55 g/L,the concentration of reducing sugar could reach 3.517mg/m L.The result of confirmatory experiment revealed that the final concentration of reducing sugar was 3.77mg/m L.In order to meet the requirements of C.reinhardtii growth,the saccharification solution of BSS was acidified by E.coli DH5α following the twice concentration.The acidulated solution contained acetic acid 0.74 mg/m L and reducing sugar 3.41mg/mL.Finally,C.reinhardtii was grown in the acidification solution under mixotrophic conditions with the specific growth rate of 2.71±0.06 d^-1 and the biomass concentration of 201.9±9.3 mg L^-1.The contents of carbohydrate and total lipids were 44.3% and 18.7% in dry cells,respectively.4.The transcriptome analysis of C.reinhardtii 21gr（cc1690）grown in the BSS hydrolysate.Total 126 new genes were discovered based on the sequencing.Among them,10,28,7,11 and 83 were annotated by COG,GO,KEGG,Swiss-Prot and NR.Based on the analysis of transcriptome data,the 606 genes were identified as differentially expressed.Among them,230 were up-regulated and 376 were observed to be down-regulated.The results of COG annotation indicated that the differentially expressed genes were distributed in the pathways including energy metabolism,amino acid and carbohydrate transportation,DNA replication,signal transduction and ion transportation.The results of KEGG annotation revealed 47 differentially expressed genes which were involved into 27 metabolic pathways.Based on the results of transcriptome analysis,15 genes related with energy and glucose metabolism were selected for further confirmatory experiments by qPCR.And the results displayed that the genes involved in glucose synthesis and energy-rich macromolecules decomposition were down-regulated significantly when C.reinhardtii was grown in the BSS hydrolysate.This change could benefit the accumulation of polysaccharides in C.reinhardtii cells.