Studies On Surfactants Controlling Biological Contaminations In Microalgae Cultivation And Its Safety Evaluation

Microalgae are a kind of lower plants,tiny in size and can carry out photosynthesis.Their cells are rich in proteins,polysaccharides,lipids,pigments and bioactive substances.Microalgae are widely used in the fields of food,feed,bioenergy,and environment remediation.However,the problem of biological contamination is widespread in the process of large-scale culture of microalgae,which has become one of the key technical problems restricting the development of microalgae industrialization.The contaminations of zooplankton and parasitic fungi in microalgae mass culture usually develop rapidly and impact the culture seriously,even lead to culture failure.At present,efficient and economical control technology has not been developed.In the previous study of our research team,sodium dodecyl benzene sulfonate(SDBS)was found to be effective in controlling fungal infection without negatively affecting biomass and lipid contents of oleaginous Graesiella sp.WBG-1for the first time.Therefore,this paper takes microalgae species Haematococcus pluvialis,Chlorella pyrenoidosa and Graesiella sp.WBG-1 as the research objects,on the basis of identifying the organisms which causing the biological contaminations,explores the control effect of surfactant on the Paraphysoderma sedebokerense contamination in Haematococcus pluvialis culture and rotifer contamination in Chlorella pyrenoidosa culture,determines the safe concentration of surfactant on the growth of microalgae,and optimizes the control technology of surfactant and verifies it in large scale culture.At the same time,the safety of surfactants controlling biological contaminations in large-scale culture of microalgae was evaluated preliminarily,and an efficient,economical and safe biological contamination control technology in microalgae mass culture was established.The research results provide sufficient scientific evidence and safety guarantee for the industrial application of surfactant technology to control microalgae diseases and pests.The main results and conclusions of this paper are as follows:1.The pathogenic fungus EPG02,was successfully isolated and purified from the infected H.pluvialis K-0084 raceway pond,and identified as Paraphysoderma sedebokerense.On this basis,five kinds of surfactants were selected to control EPG02.The laboratory results showed that 7-10 mg/L sodium dodecylbenzene sulfonate(SDBS),60 mg/L sodium dodecyl sulfate(SDS)and 30-40 mg/L primary alcohol ethoxylate(AEO-7)could effectively control the infection of P.sedebokerense.Among them,7 mg/L SDBS treatment was the best,which could not only completely control the infection of P.sedebokerense,but also had no significant effect on the growth and astaxanthin accumulation of H.pluvialis.It has been suggested that surfactants do not prevent infection by destroying the recognition sites on the surface of P.sedebokerense and H.pluvialis cells,but cut off the infection pathway by killing zoospores of P.sedebokerense.Further study showed that the surfactant SDBS could cause the rupture of cell membrane and the degradation of cell contents,but had no effect on the cell structure of the alga H.pluvialis.It may be due to the fact that the non-motile cyst of H.pluvialis and zoospores of P.sedebokerense have different cell surface coverings and structures.Therefore H.pluvialis may prevent SDBS from penetrating into the algal cell membrane,while the zoospores of P.sedebokerense lack protective cell walls,which makes SDBS can directly destroy the cell membrane of zoospores,resulting in the disintegration of zoospores.2.In the experiment of 5 m² open raceway pond,7 mg/L SDBS could effectively control the infection of P.sedebokerense and the infection control effect lasted until the end of culture.The average biomass yield and astaxanthin yield of H.pluvialis reached55.3 g/m² and 1.41 g/m²,respectively.In the 20 m² open raceway pond experiment,7mg/L SDBS could also effectively control the infection of P.sedebokerense and the infection control effect lasted until the end of culture.The biomass yield and astaxanthin yield of H.pluvialis reached 53.9 g/m² and 1.32 g/m²,respectively.It was proved that the application of surfactant SDBS can completely control the infection of P.sedebokerense and maintain the long-term stable growth of H.pluvialis.The cost of SDBS for the treatment of 1000 L algal culture is only?0.07 RMB(0.01 US dollar),which is economical and easy to operate.3.In the process of C.pyrenoidosa XQ-20044 cultivation in open raceway pond,it was found that Brachionus calyciflorus was the most harmful to C.pyrenoidosa,which could quickly and massively prey on C.pyrenoidosa and cause the culture to collapse within 72 hours.A stable co-culture system of C.pyrenoidosa and B.calyciflorus was established in the laboratory.On this basis,five kinds of surfactants were selected to control the B.calyciflorus,and the effects on the growth of C.pyrenoidosa were investigated.The laboratory results showed that SDBS,SDS,AES,AEO-7 and CDEA could effectively eliminate B.calyciflorus and its eggs in C.pyrenoidosa culture,and the effective concentrations were?10 mg/L,?15 mg/L,?10mg/L,?7.5 mg/L and?10 mg/L,respectively.Among them,10 mg/L SDBS and 7.5-10 mg/L AEO-7 had the best effect,which not only killed B.calyciflorus completely,but also had no significant effect on the biomass of C.pyrenoidosa.Considering the dosage,control effect,cost and environmental impact of the five surfactants,SDBS is most appropriate for controlling B.calyciflorus in large-scale culture of C.pyrenoidosa.4.In the 5 m² open raceway pond test,10 mg/L SDBS treatment can killed rotifer B.calyciflorus quickly and had no significant effect on the growth of alga,and the dry biomass of C.pyrenoidosa reached 0.74 g/L.Compared with other reported chemicals,SDBS has the advantages of wide application range and low application cost.Using 10mg/L SDBS to treat a 5 m² raceway pond(1000 L culture),the cost of single treatment is only?0.09 RMB(0.014 US dollar).Therefore,SDBS is appropriate for the control of rotifer contamination in large-scale cultivation of economic microalgae and energy microalgae.5.The determination method of SDBS in algal solution was established and the operation process was optimized.The optimized methylene blue spectrophotometry can detect SDBS in the range of 0.04-1 mg/L,with the relative standard deviation of 4.6%,and the average recovery rate of blank standard was 96.4%.The extraction time and reagent dosage were significantly reduced.The optimized HPLC detection range of SDBS was 0.5-100 mg/L,with the relative standard deviation of 0.3%,and the average recovery of blank standard was 101.3%.The above two SDBS determination methods meet the requirements of Environmental Water Quality Monitoring Quality Assurance Manual(second edition),and the SDBS detection ranges of the above two method are complementary.6.Using C.pyrenoidosa,Graesiella sp.and H.pluvialis as materials,the degradation of SDBS in algal solution and the residue of SDBS in algal biomass were investigated.The results showed that SDBS were degraded rapidly in the aerated cultures of H.pluvialis,C.pyrenoidosa and Graesiella sp under non-aseptic condition,and the concentration of SDBS at the end of culture were 0.38 mg/L,0.3 mg/L and 0.36mg/L,respectively,which were lower than the national sewage discharge standard(GB18918-2002,0.5 mg/L),and will not cause negative impact on the environment.At the same time,SDBS residues were not detected in the biomass of these three microalgae,indicating that the use of SDBS(?10 mg/L)to control biological contamination in microalgae culture will not lead to the enrichment of SDSB in biomass,and will not cause safety risks to the production of microalgae.In addition,the effects of algal cell,light intensity,culture temperature and air aereation on the degradation of SDBS in algal medium were investigated under aseptic condition.It was found that the air aeration greatly promoted the degradation of SDBS,while the three microalgae,C.pyrenoidosa,Graesiella sp.and H.pluvialis,could not degrade SDBS in the culture,and the changes in light intensity(0,100,200,300?mol·m^-2·s^-1)and culture temperature(15?,25?,35?)had no significant effect on the degradation of SDBS.