The Mechanism Of Algal-bacterial Cooperation Improves Hydrogen Production Of Chlamydomonas Reinhardtii

The utilization of fossil fuels has prompted the development of human society and economy,but also causes serious environmental pollution.Therefore,finding a clean and renewable energy is a trend of eneney development in the future.Hydrogen is considered as a potential renewable energy because of its low density,light weight,high combustion value,clean and renewable,and easy transportation.Establishing efficient,clean,low-cost and renewable hydrogen production method is a great significance to economic development in the future.At present,hydrogen production mainly come from fossil materials,followed by electrolytic water and solar energy.Biological hydrogen production is still in the research stage.But biological hydrogen production is an ideal way of hydrogen production because of its low energy consumption and low pollution.At present,there are mainly photo-fermentation hydrogen production by photosynthetic bacteria,dark fermentation hydrogen production by heterotrophic bacteria and photo split water for hydrogen production by microalgae.Photofermentation bacteria use organic acids as substrates to grow and produce CO₂.Electrons produced by metabolism are transferred to nitrogenase to produce hydrogen through photosynthetic electron transfer chain and Fd.However,the efficiency of hydrogen production is low and it is difficult to wipe off CO₂.Dark fermentation bacteria degrade organic matter to produce electron to hydrogenase for hydrogen production.It does not need light and has high hydrogen production efficiency,but the substrate utilization rate is low and the fermentation broth pollutes the environment.Microalgae can produce hydrogen by light energy and water.The process of hydrogen production is clean and the theoretical hydrogen production efficiency is high.However,the efficiency of hydrogen production by Chlamydomonas is low because of the low efficiency of light conversion and the sensitivity of hydrogenase to oxygen.The coculture of microalgae and bacteria to produce hydrogen is a method to increase hydrogen production by bacteria consuming intracellular oxygen of Chlamydomonas.Rhizobia and Pseudomonas respectively cultured with Chlamydomonas have increased hydrogen production,but the mechanism of hydrogen production is unclear.In this paper,the mechanism of hydrogen production by co-culture of bacteria and Chlamydomonas reinhardtii was investigated.The hydrogen production was enhanced by adding Ca²⁺ to protect photosynthetic system,and the efficiency of light energy conversion was improved by ARTP mutation for high hydrogen production.The details are as follows:1)Under sulphur deprivation,the natural inoculation of microorganisms increased the hydrogen production of Chlamydomonas reinhardtii.Six bacteria isolated from algal sample.Each bacterium co-cultured respectively with Chlamydomonas reinhardtii for hydrogen production showed that Pseudomonas sp.S2 was the main contributors for hydrogen production of co-culture.Pseudomonas sp.S2 consumed the fastest oxygen and delayed the degradation of chlorophyll.When the co-culture of Pseudomonas sp.S2,E.coli and Chlamydomonas reinhardtii or Pseudomonas sp.S2,B.subtilis and Chlamydomonas reinhardtii just enhanced hydrogen production.Moreover,the co-culture of Pseudomonas sp.S2 and other green algaes producing hydrogen promoted hydrogen production.2)To investigate the mechanism of hydrogen production by co-culture between bacteria and Chlamydomonas,DCMU was used to inhibit electron transfer in photosynthetic transfer chain,starch,protein,chlorophyll,dissolved oxygen and comparative transcriptome analysis showed that co-culture of bacteria and Chlamydomonas delayed chlorophyll degradation,promoted starch accumulation,maintained protein content and increased transcriptional level of Chlamydomonas.It was also found that hydrogen production by photolysis was about 60 %.In addition,the maximum hydrogen production was 170 mL L^-1 after optimization of light density,sulfur concentration,ammonium concentration,acetic acid concentration and bacteriaalgae ratio.3)As to photocatalytic water splitting of Clamydomonas reinhardtii was the main contributor of hydrogen production.Therefore used Ca²⁺ to protect the photosynthetic system of Clamydomonas reinhardtii,reduced the PS? activity as to sulfer deprivation,to hydrogen production of algae-bacteria co-culture,and 5 mM Ca²⁺ imporved the highest hydrogen production,up to 310 mL L^-1.It was found that Ca²⁺ could induce antioxidant enzymes to scavenge ROS of intracellular algae,and was less 4 times than the control.In addition,Ca²⁺ reduced chlorophyll degradation and maintained PS? activity to provide more electronic for hydrogen production.Ca²⁺ also promoted the utilization of acetic acid,increased starch accumulation,and promoted starch degradation in Chlamydomonas reinhardtii to provide hydrogen production.Moreover,Ca²⁺ promoted the utilization of formic acid and ethanol to reduce the toxicity for hydrogenase to improve the hydrogen production and prolong the hydrogen production time.4)The water splitting of Chlamydomonas reinhardtii is main electron source for hydrogen production.In order to improve solar conversion efficiency,Chlamydomonas reinhardtii was mutagenized by ARTP to screen mutants with low chlorophyll content to increase hydrogen production.An algal mutant was observed to have 1.8-5.2 times(28.5-84.1 mL L^-1)and 2.7-3.1 times(356.5-405.2 mL L^-1)higher H2 production than wild-type in pure and co-cultures.Compared with wild-type algae,the mutant grew as lighter green colonies on TAP agar plate,about 2 times larger cell diameter and 5.3-6.1 times lower chlorophyll content per unit cell volume.Results from the comparative transcriptomic analysis indicated that the genes relating to algal photosynthesis had higher expressions in mutant cells,suggested the improvement of solar conversion efficiency improved algal hydrogen production.And results showed that the electrons of hydrogen production was provided from photolysis of water.