Treatment Of Slaughtering Wastewater By Chlorella Sp. DT025 And Chloroplast Genome Analysis

The discharge of slaughtering wastewater is massively increasing with the large-scale development of slaughtering industry.The high content of organic matter and pollutants in slaughterhouse wastewater makes effluent treatment difficult and costly.Therefore,the development of efficient and low-cost treatment technology of slaughterhouse wastewater has attracted much attention.Using microalgae to treat slaughtering wastewater can not only purify the wastewater,but also harvest the microalgal biomass and additional products at the same time,,so as to achieve the purpose of wastewater reuse.In this study,three microalgal strains,which could effectively purify slaughtering wastewater and accumulate oil,were screened from laboratory preserved microalgae.Chlorella sp.DT025,which had the best effect among the three strains,was further studied to exploit the influence of external carbon source and nano-Fe₃O₄ additons on slaughtering wastewater treatment and intracellular oil production.Meanwhile,the expression levels of key genes related to growth and oil accumulation of Chlorella sp.DT025 were analyzed.In addition,the chloroplast genome of Chlorella sp.DT025 had also been analyzed in order to provide the basis for later genetic modification of this strain.The specific research results were as follows:1.The ability of microalgal growth and oil accumulation under different concentrations of slaughterhouse wastewater were rapidly detected using 24-well plate based on a series of microalgal species preserved in our laboratory,amongst,three strains,DT025,DT0259 and DT0264,which could effectively grow in the concentration range of 0-75%wastewater,and obtain the highest intracellular lipid content of（41.44±0.7）%,were screened.Further research results showed that Chlorella sp.DT025 had the best treatment effect on original concentration（100%）of slaughtering wastewater.The removal rates of TN,TP and COD reached to 79.00%,44.01%and 31.60%,respectively.And the microalgae biomass and lipid yield were（4.37±0.43）×10⁷ cells/m L and（190±0.02）mg/L,respectively.Based on the above results,Chlorella sp.DT025 was selected for subsequent experiments.2.The effects of external carbon source and magnetic Fe₃O₄NPs additive on Chlorella sp.DT025 growth and treatment of slaughtering wastewater were investigated..The results showed that the addition of 2-8 g/L glucose significantly improved the microalgal biomass and lipid yield,and the addition of 4 g/L glucose exhibited the best effect with 1.42times enhancedbiomass and oil yield,which reached（36.49±0.75）×10⁷cells/m L and（270±4.08）mg/L,respectively,after 15 days of culture.Meanwhile,the removal rates of TN,TP and COD of slaughter wastewater were 82.51%,65.54%and 81.94%,respectively.On the basis,the addition of appropriate amount（0～100 mg/L）of Fe₃O₄ NPs could effectively promote the microalgal growth and improve the wastewater treatment effect.Specially,the addition of 80 mg/L Fe₃O₄ NPs enhanced 22%of the microalgal biomass to（40.65±0.75）×10⁷ cells/m L,and 23.33%of the lipid yield to（455±5.6）mg/L,and the TP removal rate reached 94%.3.In the treatment of adding 4 g/L glucose and 80 mg/L Fe₃O₄ NPs,the expression levels of key enzyme genes related to growth and oil accumulation of Chlorella sp.DT025 were analyzed using q PCR technology.The results showed that,compared with the control treatment without adding external carbon source and NPs,the expression levels of phosphoenolpyruvate carboxylase（PEPC）and ribulose bisphosphate carboxylase large subunit（rbc L）genes,which related to carbon fixation in microalgae,and ADP-glucose pyrophosphorylase（AGP）and starch synthase（SS1,SS2）genes,which related to starch synthesis,were significantly upregulated.However,there was no significant change in the expression of acetyl-Co A carboxylase（ACC）gene,which was the key gene in oil synthesis,indicating that the microalgal strain increased biomass mainly through promoting carbon fixation,starch synthesis and other vegetative growth and metabolic activities,thus improving oil production.4.Chlorella sp.DT025 chloroplast genome was sequenced based on Illumina Nova Seq 6000 high-throughput sequencing platformand its chloroplast genome characteristics were analyzed.The chloroplast genome of Chlorella sp.DT025 was 118106 bp,and GC content was 33.95%.Seventy-six protein coding genes（PCG）,32 t RNA genes,3 r RNA genes and 1 pseudogene were identified.A total of 233 SSR（simple repeat sequence）loci were identified,including 179 mononucleotide,1dinucleotide,49 trinucleotide,2 tetranucleotide and 2 pentanucleotide,respectively,and most of them were composed of A and T.There are 20567codons in chloroplast genome,and 29 of them have codon bias（RSCU）>1.The frequency of codon endings A and U is higher,and the amino acid that codes most is leucine（Leu）.Phylogenetic analysis showed that Chlorella sp.DT025 was the most closely related to Chlorella Vulgaris.The results provide basic data and theoretical reference for subsequent genetic analysis and modification of Chlorella sp.DT025.