Phytohormones Enhance the Viability and Biomass Accumulation of Microalgae Cultivated in Wastewater

Author:Jiang Li Qun

Supervisor:pei hai yan


Degree Year:2019





Microalgae as photosynthetic autotrophs can take advantage of the solar energy,inorganic carbon and nutrients for the synthesis of required self-materials,with the characteristics of high growth rate,rich lipid,strong environment adaptability and high productivity.As a result,microalgae are treated as the third generation of biofuel and have attracted extensive attention and research.However,the large-scale development and application of microalgae are limited by its higher cost than traditional fuel and huge demand on water.One of the main ways to solve this problem is to replace regular medium with wastewater and simultaneously keep or even promote the algal lipid productivity.However,wastewater usually contains adverse factors for algal viability,leading to retarded growth and decrease biomass production.Therefore,how to achieve the rapid and efficient accumulation of algal biomass in wastewater without weakening the growth is the key to economic and scale up algal cultivation.Aiming to lower the cost of algal cultivation and holding the lipid productivity,this research started from growing algae in wastewater with analyzing the inhibitors to microalgae,then observing the responses of algal growth and activities to phytohormones,eventually applying phytohormones on algae cultured in wastewater.An efficient method was expected to obtain high algal biomass and lipid generation from low-cost wastewater.The main studies and conclusions reached in the present study are as follows:1.We observed algal growth and lipid accumulation in different wastewaters in this part and analyzed the factors in wastewater relevant to algal activities and their mechanisms.Spirulina subsalsa only grew in monosodium glutamate residue(MSGR)diluted with seawater or freshwater and obtained chlorophyll and biomass accumulation with same or higher level than that in regular medium.MSGR diluted with freshwater decreased algal lipid production,while MSGR diluted with seawater increased lipid content and productivity by 1.8 and 2.5 times,respectively,compared to regular medium.Through observing the growth and biomass accumulation of S.subsalsa in modified Zarrouk medium supplemented with complex wastewater(CW,from a monosodium glutamate factory)in different concentrations,we found that high ammonia in 75%and 100%CW inhibited algal growth,while algae could grow with low addition ratios of CW.These results suggested that CW did not have sufficient nutrients and contained adverse factors for algal growth,which requested dilution or nutrient supplement before cultivating algae.Another strain isolated from local lake,Monoraphidium sp.SDEC-17,was explored to grow in pure CW.The final biomass densities of SDEC-17 in CW(1.29± 0.09 g·L-1)and BG11 medium(1.31±0.08 g·L-1)did not show a statistically significant difference(p>0.05),while lipid productivity decreased in CW.Effluent from anaerobic digestion of kitchen waste(ADE-KW)disrupted the membranes of nuclear and chloroplast in the cells of Chlorella ellipsoidea SDEC-11,and reduced the room between chloroplast and cell membrane and increased the starch size of Scenedesmus quadricauda SDEC-13.As a result,algae obtained a reduced biomass and biocompound accumu ation in ADE-KW.Through above results,we found that directly cultivating microalgae in wastewater might be possible only after some pretreatment,like dilution,nutrient supplement or using appropriate species.Those methods alleviated the inhibiting effects from high-level toxicants,extreme inappropriate nutrient ratios,wastewater-borne bacteria and solid particles in wastewater.However,in comparison with regular medium,algal growth and lipid production in wastewater were still lower and needed to be promoted further.2.For integrating algal cultivation into wastewater production process and taking full advantages of anaerobic digestion process,ADE-KW and two algae species that could grow in ADE-KW were considered as subjects in follow-up research about phytohormones.Kinetin(KT)immediately stimulated cells into dividing phase,but not followed by increase biomass production.High-level addition of KT inhibited the growth of C.ellipsoidea SDEC-11 and S.quadricauda SDEC-13.Low-level KT enhanced the growth of SDEC-11 and utilizing efficiency of phosphate in SDEC-I 1 and SDEC-13.The growth and chlorophyll synthesis of SDEC-11 and SDEC-13 differently responded to KT,which indicated that the effect of phytohormone on microalgae was a species depended behavior.At a very low concentration(10-7 M)of diethyl aminoethyl hexanoate(DA-6),SDEC-11 and SDEC-13 obtained enlarged cell size,higher biomass and lipid productivities.Second hormone dose in 10-6 M DA-6 medium resulted in increased biomass productivity(1 06 mg L-1d-1)and long exponential growth of SDEC-13.DA-6 also ensured the property of microalgae biodiesel to meet the EN 14214 standard.The current investigation demonstrated that DA-6 accelerated the microalgae growth and simultaneously improved the quality and quantity of lipid for biodiesel production.The agricultural phytohormones(GIB,combination of gibberellin acid,indole-3-acetic acid and brassinolide)were also applied on C.ellipsoidea SDEC-11 and S.quadricauda SDEC-13 at different growth stage of algae.Biomass concentration and photosynthetic pigments of two algal strains was improved by GIB,accompanied by the promotion of percentage of cells in division,number of daughter cells.The dose of hormones would be better at the beginning of batch cultivation for SDEC-11 and at seed preparation phase for SDEC-13 for biomass production.For the two algae species treated with GIB,the mass ratio of carbohydrate to protein decreased due to lower carbohydrate content or higher protein accumulation.The noting increment was 14 mg·L-1d-1 in lipid productivity of SDEC-13 with GIB dose at seed preparation phase,compared to 9 mg·L-1d-1 in control.The current investigation suggested that the agricultural phytohormones could also be a strategy for promoting biomass and biocompound accumulation of algae.3.The cheap and stable GIB was employed to C.ellipsoidea SDEC-11 and S.quadricauda SDEC-13 cultivated in ADE-KW at different stages of algal growth.Inoculation with an algal seed pretreated with GIB(S+ADE-KW)aided the adaptability and viability of algae in ADE-KW,which for SDEC-13 was even promoted to the level in BG11.GIB mitigated the inhibition of ADE-KW on algal cell division and photosynthetic pigments and apparatus,and increased lipid droplets,which might result from the change in the synthesis and fate of nicotinamide adenine dinucleotide phosphate.GIB addition significantly promoted lipid productivity of the two algal species,following 13 mg L-1d-1 of SDEC-11 in B+ADE-KW(adding GIB at the begining of batch cultivation)and especially 13 mg· L-1d-1 of SDEC-13 when priming of algal seed with the hormones that is 139%higher than 5 mg·L-1d-1 in ADE-KW control.Agricultural phytohormones could be applied as a strategy for promoting biomass and biocompound accumulation of algae in ADE-KW,in which pretreatment of the algal inoculum with hormones is a unique way to help algae survive under stress.Since our target wastewater ADE-KW was characterized as high N/P ratio and phosphate deficiency,we also compared algal viabilities under P supplement and phytohormone addition to find a better way for producing biomass from wastewater.Phosphate supplement slightly enhanced biomass production,while GA3 made a big promotion on algal growth and lipid accumulation.These results suggest that phytohormone could stimulate algal activities and be more suitable for algal cultivation in wastewater.Reutilization of wastewater via microalgae was enabled greatly by phytohormone based on its promotion on algal activity,photosynthesis and metabolism.An eco-friendly system could be established by coupling enhancing algal growth with phytohormone and anaerobically digesting kitchen waste.This system was set to comprise of algae cultivation,algae-based biofuel production,algal residue treatemnt and kitchen waste treatment where almost no nitrogen or phosphorus is lost.