| Lignin and its environmental pollutants with similar aromatic chemical structures(such as a large number of azo organic dyes)are one of the main components of biomass waste,and at the same time,they are also the most abundant resources of renewable aromatic compounds in nature and one of the sources of organic waste.The total lignin content of the earth’s biosphere exceeds 300 billion tons,which is an important choice for replacing petrochemical sources of chemical raw materials and transportation energy.However,less than 5%are currently commercially available.Direct depolymerization of lignin biomass waste and synthesis to produce biodiesel is one of the possible ways to realize its high-value transportation energy utilization.However,the complex polymer structure of lignin results in its high hardness and water insolubility,which brings huge challenges to the development of high-efficiency transformation technology,making it difficult to realize its industrial utilization.At present,more than 20%of the discharge of industrial wastewater is from the discharge of organic dye sewage generated by the textile industry.A large amount of chemically synthesized colored dyes has a huge total discharge,which can exceed 1 million tons per year.Some data shows that these organic and colored dyes are not only used in the textile industry,but are also widely used in the food,paper,and cosmetic industries,bringing great benefits to China’s water resources,soil ecology,and vegetation restoration,as well as people’s lives and health.Textile dyes are chemically diverse in nature and are broadly divided into azo,reactive,heterocyclic,polymeric structures,etc.Among these various types,the azo dyes constitute about 70%and are widely used for dyeing purposes.This makes them the largest and most important group of synthetic colorants released into the environment.Azo dyes,or a class of organic compounds with an azo group structure,have the characteristics of high stability of aromatic chemical structures in their molecular structures,and they are very similar to lignin biomass in chemical structure.Similarly,under natural conditions,it is difficult for microorganisms in the environment to achieve rapid biodegradation and complete detoxification.Feedstock is one of the main bottlenecks restricting the development of a biodiesel production system.In terms of the use of raw materials,countries around the world vary according to their national conditions.The United States mainly uses high-yield genetically modified soybeans to develop the biodiesel industry with soybean oil as the raw material;European countries,especially Germany,plant rapeseed oil on a large scale and use rapeseed oil to produce biodiesel;and countries in Southeast Asia use palm oil and waste oil as production raw materials.China has more people than land,and it is impossible to develop biodiesel with oil plants that occupy arable land like in developed countries.In response to such challenges,the development of the third generation of biodiesel,represented by oleaginous yeasts,is considered to be the future direction with the most potential to replace traditional biodiesel feedstocks through integrating bioremediation and biorefinery technology towards some recalcitrant organic wastes,including waste biomass,waste CO2 produced by industrial production,and raw materials such as organic dye wastewater,which greatly expands the selection of raw materials for constructing a biodiesel production system.Clearly,the use of oleaginous yeasts for synthesizing lipids from lignin-based aromatics is a clean,efficient,and competitive path to achieve "sustainable development" towards biodiesel production.In addition,wood-feeding termites have a high capacity for biomass degradation and may thus contribute to global carbon recycling.Therefore,the scientific objectives of this dissertation have been achieved through the development of three research steps.The first step consisted of screening and characterization of novel yeast species or strains from woodfeeding termites for bioremediation and biorefinery of some recalcitrant organic wastes,such as ligninderived monomers and lignin-like dyes,as well as the expression of lignocellulolytic enzymes.The second research step consisted of exploring the performance of selected individual yeast species or consortia towards the degradation and detoxification of lignin-based aromatic wastes such as textile azo dye wastewater and tolerating lignin degradation inhibitors with special emphasis on the metabolic biodegradation mechanisms.The third research step consisted of integrating bioremediation and biorefinery of some recalcitrant organic wastes into high value-added products using a newly constructed manganese peroxidase-producing oleaginous yeast consortium intended for coupling azo dye degradation and constructing a biodiesel production system.The specific results are summarized as follows:(1)Fifteen different yeast genera(27 yeast isolates)were successfully isolated and identified from termite gut symbionts.To the best of our knowledge,to date,there have been no reports documented for the identified yeasts,Sterigmatomyces halophilus,Meyerozyma guilliermondii,Hamamotoa lignophila,Candida silvanorum,Meyerozyma caribbica,Debaryomyces hansenii,Candida stauntonica,Vanrija humicola,Hyphopichia heimii,Barnettozyma californica,Sugiyamaella smithiae,Candida gotoi,Cyberlindnera sp.,Fellozyma inositophila,Wickerhamomyces sp.,Saccharomycete sp.,and Starmera dryadoides.Out of the 27 yeasts,eight isolates showed the highest CMCase,xylanolytic,and ligninolytic activities.According to clustering and ordination analyses,six isolates exhibited unique metabolic activities and physiological capabilities compared with other strains that could make them potentially consortium suitable for a wide variety of applications.(2)The halotolerant yeast strain Sterigmatomyces halophilus SSA-1575,capable of decolorizing and detoxifying the sulfonated diazo dye RB5 effectively,has been successfully identified from a WFT gut system.The growing cells showed optimal growth and decolorization performance at 50 mg/L RB5,5 g/L glucose,0.08 g/L yeast extract,0.05 g/L(NH4)2SO4,30℃,pH 5,and in the presence of up to 50 g/L NaCl.S.halophilus SSA-1575 could efficiently decolorize RB5 due to the unique enzymatic system involved.NADH-DCIP reductase and LiP were determined as the key reductase and oxidase enzymes of S.halophilus SSA-1575,which were induced during RB5 decolorization processing.To understand a possible degradation mechanism well,UV-Vis spectroscopy,FTIR,and Mass Spectrometry analyses were employed to analyze the possible decolorization pathway by SSA-1575.The determination of relatively high NADH-DCIP reductase suggested that the asymmetric cleavage of RB5 azo bond was mainly catalyzed by NADH-DCIP reductase,followed by desulfonation,deamination,and finally resulting in the formation of colorless aromatic amines devoid of any chromophores.(3)The performance of Meyerozyma caribbica SSA1654 as a novel manganese peroxidase-producing yeast for the decolorization and detoxification of Acid Orange 7(AO7)azo dye has been studied.M.caribbica SSA1654 exhibited the highest extracellular MnP activity of 27 U/mL.At a dye concentration of 400 mg/L,the percentage of AO7 decolorization was 93.8%.Based on UV-Vis spectra,FTIR,HPLC,and GC-MS analyses,a plausible AO7 biodegradation mechanism by the MnP-producing M.caribbica strain SSA1654 was proposed.Both 4-aminobenzene sulfonic acid and 1-amino-2-naphthol resulted from the initial cleavage of the AO7 azo bond.The 4-aminobenzene sulfonic acid probably underwent deamination and then desulfonation into 1,4-benzenediol.The toxic aromatic amines were subsequently mineralized completely into CO2 and H2O.(4)MnP-YC4,a newly constructed MnP-producing yeast consortium,has been developed to withstand lignin degradation inhibitors while degrading and detoxifying azo dye.The MnP-YC4 tolerance to major biomass-derived inhibitors was promising.MnP induced by lignin was found to be highly related to dye decolorization by MnP-YC4.Simulated azo dye-containing wastewater supplemented with a lignin cosubstrate(3,5-Dimethoxy-4-hydroxybenzaldehyde)decolorized up to 100,91,and 76%at final concentrations of 20,40,and 60%,respectively.The MnP-YC4 effectively decolorized the real textile wastewater sample,reaching up to 91.4%,and the COD value decreased significantly during the decolorization,reaching 7160 mg/1 within 7 days.A possible dye biodegradation pathway of monosulfonated OII was proposed based on the degradation products identified by UV-vis,FTIR,GC/MS,and HPLC techniques.The ring fission of muconic acid resulted in 3-oxoadipate,which was probably converted into acetyl-CoA.Acetyl CoA is required for triacylglycerol synthesis,which can be transesterified into biodiesel.The azo bond of the dye molecule was first cleaved by MnP-YC4 and eventually mineralized to CO2 and H2O.(5)The construction of a multipurpose yeast consortium suitable for lipid production,textile dye/effluent removal,and lignin valorization is critical for both biorefinery and bioremediation.Therefore,a novel oleaginous consortium,designated as OYC-Y.BC.SH has been developed using three yeast cultures viz.Yarrowia sp.SSA1642,Barnettozyma californica SSA1518,and Sterigmatomyces halophilus SSA1511.The OYC-Y.BC.SH consortium showed impressive potential to utilize phenol,vanillic acid,veratryl alcohol,benzoic acid,vanillin,p-cresol,and 3-chlorobenzoic acid when compared to its individual yeast strains.The OYC-Y.BC.SH was also able to grow on different carbon sources and accumulate lipids,with the highest lipid productivity(1.56 g/L/day)and lipase activity(170.3 U/mL).The total saturated fatty acid content was 36.09%,while the mono-unsaturated and poly-unsaturated fatty acids were 45.44 and 18.30%,respectively,making OYC-Y.BC.SH valuable for biodiesel production.The OYC-Y.BC.SH showed its highest decolorization efficiency of Red HE3B dye(above 82%)in the presence of sorghum husk as an agricultural co-substrate,while it showed the maximum percentages of Red HE3B decolorization with sawdust(77.4%),wheat stalk(59.8%),wheat bran(51.2%)and rice straw(33.5%).The use of agricultural wastes instead of pure carbon and nitrogen substrates for the enhancement of Red HE3B biodegradation indicates an ecofriendly as well as economically feasible process for biomass valorization.The significantly higher performance of OYC-Y.BC.SH on decolorizing the real dyeing effluent sample at pH 8.0 suggests its potential and suitability for degrading most of the wastewater textile effluents.(6)A new MnP-producing and halotolerant oleaginous yeast consortium,designated as NYC-1,was successfully constructed to deal with a salty and various inhibitor-presented fermentation environment.This consortium could well grow in the presence of furfural,5-hydroxymethyl furfural,acetic acid,vanillin,and formic acid in the tested range.It also demonstrated effective decolorization performance to all individual azo dyes tested within 24 h,up to a dye concentration of 250 mg/L.The NYC-1 consortium has been identified as a halotolerant consortium that could potentially be applied prior to the disposal of azo dye wastewater,which is also characterized by high salt concentrations.The values of long-chain saturation factor,cetane number,oxidative stability,and kinematic viscosity of biodiesel were 4.21,53,7.85 h,and 4.38 mm2 s-1,respectively.The produced biodiesel by the NYC-1 consortium had a C18:3 content,which was in accordance with the international biodiesel standards(ES1421).Therefore,the MnP-producing oleaginous yeast consortium NYC-1 could be a novel biological candidate with promising biodiesel production potential.Based on the results discussed above,this investigation indeed proposed a novel approach in exploring and constructing some robust oleaginous yeasts and their consortia discovered from the termite gut for converting various waste lignin biomass(either from paper-industry or agriculture-industry)and various textile wastewater(mainly from textile-industry),a set of recalcitrant aromatic chemicals,into a high value-added biodiesel product.Clearly,integrating bioremediation and biorefinery technology towards such recalcitrant organic wastes is truly considered a new concept for constructing a third-generation biodiesel production system,particularly from a new type of renewable waste and pollutant feedstock.The utilization of oleaginous yeast consortia to construct a biodiesel production system from these organic wastes would potentially pave a new avenue in developing a robust,competitive third-generation biodiesel production system,and simultaneously,benefit our environment from those organic wastes. |
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