Low-light operation offers clear advantages for energy-efficient treatment of sugar-rich wastewater by alleviating photoinhibition and reducing electricity demand; however, carbon removal efficiency of conventional microalgae remains insufficient under such conditions. Here, we isolated Chlorella sorokiniana NS4-2 (C. sorokiniana NS4-2) from natural waters, a fast-growing strain characterized by a high-respiration phenotype that sustains rapid carbon turnover and growth under low-light conditions. In low-light photobioreactor conditions using simulated glucose-containing wastewater, C. sorokiniana NS4-2 exhibited simultaneous glucose and acetate utilization, consistent with its enhanced respiratory metabolism. Acetate supplementation further increased glucose removal to 2.2 g L-1 d-1, among the highest reported under comparable low-light conditions. Glucose feeding also enhanced lipid accumulation, with eicosapentaenoic acid content increasing nearly tenfold, indicating the potential for value-added lipid co-production during wastewater treatment. When applied to real sugar-rich wastewater under sustained low-light conditions, the system achieved 99% total sugar removal, and the treated effluent met applicable discharge standards. Collectively, these results demonstrate that C. sorokiniana NS4-2 enables efficient carbon removal under low-light operation, with respiratory capacity emerging as a key determinant of this performance, revealing a metabolism-driven mechanism underlying carbon removal under light-limited conditions and providing a rational criterion for selecting low-light-adapted microalgal strains, thereby supporting the development of energy-efficient wastewater treatment strategies with integrated resource recovery.

Adding bisulfite is a significant strategy to enhance H2 photoproduction, but it adversely affects photosystem II (PSII) activity in microalgae, consequently limiting H2 production. Nevertheless, the exact mechanism through which bisulfite disrupts PSII remains unclear. Here, the addition of NaHSO3 to Chlamydomonas reinhardtii cultures suppresses growth by inducing iron poisoning through the Fenton reaction, driven by reduced iron demand and the resultant accumulation of iron. Consequently, the removal of iron from C. reinhardtii cultures eliminates the iron poisoning triggered by the Fenton reaction. This, in turn, mitigates PSII impairment and leads to a significant increase in photosynthetic H2 production in C. reinhardtii cells. Collectively, these discoveries unveil the precise mechanism by which bisulfite disrupts PSII, opening up a new avenue for substantially enhancing green hydrogen production from microalgae through the bisulfite addition strategy.

Green hydrogen, produced during microalgal photosynthesis, is regarded as one of the most promising sustainable energy sources. It utilizes sunlight and water, which are essentially unlimited, and its combustion results in only water as a waste product. In microalgal hydrogen energy production systems, the sensitivity of hydrogenase to O-2 poses a significant challenge, limiting sustained photosynthetic H-2 production in microalgae. Additionally, efficient photosynthetic H-2 production in anaerobic microalgal cells is hindered by impaired electron source (photosystem II) and electron loss through the Calvin-Benson cycle, cyclic electron transfer around photosystem I, and O-2 photoreduction, which are identified as the other key challenges. Over the past eight decades, considerable progress has been made in addressing these challenges and regulating electron transfer to achieve sustainable and efficient photosynthetic H-2 production in microalgae. In this review, we discuss a range of regulatory methods for achieving sustainable and efficient photosynthetic H-2 production in microalgae. Emphasizing the significant progress made over the past eight decades, we also address current challenges and propose potential future solutions.

1School of Ecological and Environmental Sciences, East China Normal University, Shanghai 200241, China. 2College of Life Sciences, Shanghai Normal University, Shanghai 200234, China. 3Key Laboratory of Urbanization and Ecological Restoration of Shanghai, Shanghai 200241, China. 4Institute of Eco-Chongming (IEC), 20 Cuiniao Rd, Chenjia Zhen, Chongming, Shanghai, 202162, China 5Technology Innovation Center for Land Spatial Eco-restoration in Metropolitan Area, Ministry of Natural Resources, 3663 N. Zhongshan Road, Shanghai, 200062, China

The mechanisms of acclimating to a nitrogen-fluctuating environment are necessary for the survival of aquatic cyanobacteria in their natural habitats, but our understanding is still far from complete. Here, the synthesis of phycobiliprotein is confirmed to be much earlier than that of photosystem components during recovery from nitrogen chlorosis and an unknown protein Ssr1698 is discovered to be involved in this synthetic process. The unknown protein is further identified as a c-type heme oxygenase (cHO) in tetrapyrrole biosynthetic pathway and catalyzes the opening of heme ring to form biliverdin IXα, which is required for phycobilin production and ensuing phycobiliprotein synthesis. In addition, the cHO-dependent phycobiliprotein is found to be vital for the growth of cyanobacterial cells during chlorosis and regreening through its nitrogen-storage and light-harvesting functions, respectively. Collectively, the cHO expressed preferentially during recovery from nitrogen chlorosis is identified in photosynthetic organisms and the dual function of this enzyme-dependent phycobiliprotein is proposed to be an important mechanism for acclimation of aquatic cyanobacteria to a nitrogen-fluctuating environment.

NdhO, a regulatory oxygenic photosynthesis-specific subunit, is close to the ferredoxin-binding site of cyanobacterial NDH-1, and its levels are negatively associated with the rates of cyclic electron transfer around PSI mediated by NDH-1 (NDH-CET). However, the effect of NdhO levels on cyanobacterial cell death triggered by high temperature remains elusive. Here, our results uncovered a synergistic effect of NdhO levels on the cell death and reactive oxygen species (ROS) accumulation when cyanobacterial cells grown at 30°C for 1 day were transferred to 45°C for 2 days. Such synergistic effect was found to be closely associated with the activities of NDH-CET and CO2 assimilation during high temperature. Collectively, we propose that the effect of NdhO levels on the cyanobacterial cell bleaching and cell death triggered by high temperature is a result of influencing production of ROS by NDH-CET, which is considered to be vital to balance the ATP/NADPH ratio and improve the Calvin-Benson cycle.

Background Bisulfite addition is an important H-2 photoproduction strategy that removes O-2 and activates hydrogenase. The pH values of cell cultures can change the ratio of bisulfite to sulfite, which may affect H-2 photoproduction. However, little is known regarding the pH effect of bisulfite addition on H-2 photoproduction and relevant underlying mechanism. Results Here, changes in H-2 photoproduction with different initial extracellular pH values showed a parabolic distribution and a pH of 8.0 is an optimal value for H-2 photoproduction in Chlamydomonas reinhardtii cells treated with bisulfite. Compared to the growth pH (pH 7.3), increased photoproduction of H-2 at this optimal pH was primarily caused by a relatively high residual activity of photosystem II (PSII), which provides a relatively plentiful source of electrons for H-2 photoproduction. Such increased H-2 photoproduction was most likely a result of decreased the ratio of bisulfite to sulfite, consistent with the result that the toxicity of bisulfite on PSII was much more than that of sulfite. This possibility was corroborated by the result that treatment with a combination of 7 mM bisulfite and 6 mM sulfite further enhanced H-2 photoproduction compared with 13 mM bisulfite alone. Conclusions Collectively, our findings provide novel mechanistic insights into pH-dependent H-2 photoproduction in C. reinhardtii cells treated with bisulfite, and demonstrate that sulfite addition is another important strategy for H-2 photoproduction, just like bisulfite addition.

Two mutants sensitive to high light for growth and impaired in NDH-1 activity were isolated from a transposon-tagged library of Synechocystis sp. strain PCC 6803. Both mutants were tagged in the ssl3451 gene encoding a hypothetical protein, which shares a significant homology with the Arabidopsis (Arabidopsis thaliana) CHLORORESPIRATORY REDUCTION 42 (CRR42). In Arabidopsis, CRR42 associates only with an NDH-1 hydrophilic arm assembly intermediate (NAI) of about 400 kDa (NAI400), one of total three NAIs (NAI800, NAI500 and NAI400), and its deletion has little, if any, effect on accumulation of any NAIs in the stroma. In comparison, the ssl3451 product was localized mainly in the cytoplasm and associates with two NAIs of about 300 kDa (NAI300) and 130 kDa (NAI130). Deletion of Ssl3451 reduced the abundance of the NAI300 complex to levels no longer visible on gels and of the NAI130 complex to a low level, thereby impeding the assembly process of NDH-1 hydrophilic arm. Further, Ssl3451 interacts with another assembly factor Ssl3829 and they have a similar effect on accumulation of NAIs and NdhI maturation factor Slr1097 in the cytoplasm. We thus propose that Ssl3451 plays an important role in accumulation of the NAI300 and NAI130 complexes in the cytoplasm via its interacting protein Ssl3829.

NaHSO3 addition greatly increases the yield of H2 photoproduction in a unicellular green alga Chlamydomonas reinhardtii through removing O2 and activating hydrogenase but significantly impairs the activity of PSII, an electron source for H2 photoproduction. Here, a stepwise addition mode of total 13 mM NaHSO3, an optimal concentration for H2 photoproduction of C. reinhardtii identified in a previous one step addition method, significantly improved H2 photoproduction. Such improvement was believed to be the result of increased residual PSII activity in an anaerobic background, but was at least independent of two alternative electron sinks for H2 photoproduction, cyclic electron transport around PSI and CO2 assimilation. Based on the above results, we propose that increased residual PSII activity in an anaerobic environment is an efficient strategy to enhance H2 photoproduction in C. reinhardtii, and the stepwise NaHSO3 addition mode is a case study in the strategy.

Chlamydomonas reinhardtii is a unicellular green alga that can use light energy to produce H2 from H2O in the background of NaHSO3 treatment. However, the role of light intensity in such H2 production remains elusive. Here, light intensity significantly affected the yield of H2 production in NaHSO3-treated C. reinhardtii, which was consistent with its effects on the content of O2 and the expression and activity of hydrogenase. Further, NaHSO3 was found to be able to remove O2 via a reaction of bisulfite with superoxide anion produced at the acceptor side of PSI, and light intensity affected the reaction rate significantly. Accordingly, high light and strong light but not low light can create an anaerobic environment, which is important to activate hydrogenase and produce H2. Based on the above results, we conclude that light intensity plays an important role in removing O2 and consequently activating hydrogenase and producing H2 in NaHSO3-treated C. reinhardtii.

NdhV is a subunit of cyanobacterial NADPH dehydrogenase constituting ferredoxin-binding domain essential for cyclic electron flow. Two mutants sensitive to heat stress for growth and impaired in NADPH dehydrogenase (NDH-1)-dependent cyclic electron transport around photosystem I (NDH-CET) were isolated from the cyanobacterium Synechocystis sp. strain PCC 6803 transformed with a transposon-bearing library. Both mutants had a tag in the same sll0272 gene, encoding a protein highly homologous to NdhV identified in Arabidopsis (Arabidopsis thaliana). Deletion of the sll0272 gene (ndhV) did not influence the assembly of NDH-1 complexes and the activities of CO2 uptake and respiration but reduced the activity of NDH-CET. NdhV interacted with NdhS, a ferredoxin-binding subunit of cyanobacterial NDH-1 complex. Deletion of NdhS completely abolished NdhV, but deletion of NdhV had no effect on the amount of NdhS. Reduction of NDH-CET activity was more significant in ΔndhS than in ΔndhV. We therefore propose that NdhV cooperates with NdhS to accept electrons from reduced ferredoxin.

The death and subsequent decomposition of algal blooms is capable of depleting dissolved O2 to anaerobic levels, and this can de-inactivate hydrogenases. Inspired by this fact, a simple method for efficient H2 production from algal bloom biomass was developed. Direct transfer of Taihu Lake Microcystis spp. blooms into dark conditions resulted in H2 evolution, and yield was much greater than compared to Microcystis spp. cultured in the laboratory and reported previously in the literature. Further, efficient H2 production was inhibited significantly by light, which was most likely due to reduced O2 content and the stimulation of hydrogenase activity. Therefore, a simple approach for efficient H2 production from Taihu Lake Microcystis spp. blooms is presented. Furthermore, a post-treatment strategy for dealing with large quantities of refloated algal blooms is proposed.

ndhK is a subunit of cyanobacteria NDH-1 complex,playing an important role in responding to low CO2 stress.Its expression level is induced by low CO2 and high light.To further understand the mechanism of transcriptional regulation of ndhK with light,we find the TSP(transcription start position) with 5′-RACE(Rapid Amplification of cDNA Ends),forecast that ndhK contain four possible promoter,construct a series of promoter probe vectors containing eYFP(enhanced yellow fluorescent protein) and use western blot to detect them.The results showed that the upstream sequence of ndhK in Synechocystis sp.PCC6803 containing four sequences(-374 to-274,-438 to-374,-604 to-543,+1 to +52) which can promote gene expression and one can inhibit(-543 to-440).

Pre-exposure of plants to one form of stress can impact tolerance to other forms of stress. This is called cross-tolerance. However, little is known about whether this type of cross-tolerance also occurs in cyanobacteria. Here, our results clearly indicated that short-time moderate heat pretreatment considerably alleviates the inhibitory effect of high red light but not blue light on the activity of photosystem II in the unicellular cyanobacterium, Synechocystis sp. strain PCC 6803, as determined by a chlorophyll fluorescence parameter, ΦPSII. We therefore conclude that a similar cross-tolerance strategy as identified in higher plants also occurs in cyanobacteria.

发菜是一种陆生蓝藻,分布于一些干旱和半干旱区域.其NADPH脱氢酶(NDH-1)是一种重要的光合膜蛋白复合体,参与CO2吸收、围绕光系统Ⅰ的循环电子传递和细胞呼吸.为研究该物种中ndhK基因的功能,本研究利用特异性引物,通过PCR方法从发菜中扩增ndhK基因并克隆到原核表达载体pET-32a上,得到表达载体pET-32a-ndhK,将其转入大肠杆菌BL21( DE3),经异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达,得到分子量大小为43 kDa的融合蛋白NdhK.随后,采用亲和层析,对融合蛋白进行纯化回收,并以此回收蛋白作为抗原进行免疫,制备NdhK的多克隆抗体.最后,利用Western blot蛋白免疫印迹对所得抗体的特异性进行验证.从而为进一步探索发菜ndhK基因的功能以及发菜中NDH-1复合体各亚基的作用进行前期准备.

Cyanobacterial NADPH:plastoquinone oxidoreductase, or type I NAD(P)H dehydrogenase, or the NDH-1 complex is involved in plastoquinone reduction and cyclic electron transfer (CET) around photosystem I. CET, in turn, produces extra ATP for cell metabolism particularly under stressful conditions. Despite significant achievements in the study of cyanobacterial NDH-1 complexes during the past few years, the entire subunit composition still remains elusive. To identify missing subunits, we screened a transposon-tagged library of Synechocystis 6803 cells grown under high light. Two NDH-1-mediated CET (NDH-CET)-defective mutants were tagged in the same ssl0352 gene encoding a short unknown protein. To clarify the function of Ssl0352, the ssl0352 deletion mutant and another mutant with Ssl0352 fused to yellow fluorescent protein (YFP) and the His(6) tag were constructed. Immunoblotting, mass spectrometry, and confocal microscopy analyses revealed that the Ssl0352 protein resides in the thylakoid membrane and associates with the NDH-1L and NDH-1M complexes. We conclude that Ssl0352 is a novel subunit of cyanobacterial NDH-1 complexes and designate it NdhS. Deletion of the ssl0352 gene considerably impaired the NDH-CET activity and also retarded cell growth under high light conditions, indicating that NdhS is essential for efficient operation of NDH-CET. However, the assembly of the NDH-1L and NDH-1M complexes and their content in the cells were not affected in the mutant. NdhS contains a Src homology 3-like domain and might be involved in interaction of the NDH-1 complex with an electron donor.

Cyanobacterial NADPH dehydrogenase(NDH-1) is an important photosynthetic membrane protein complex,and is essential to CO2 uptake,cyclic electron transport around photosystem I and cellular respiration.This enzyme accepts electrons from NADPH and consists of at least 17 subunits,i.e.,NdhA to NdhQ.Recently,an ndhO gene inactivation mutant,ΔndhO,has also successfully been obtained.However,little is known regarding the functional roles of NdhO subunit in cyanobacteria.Therefore,the encoding gene,ndhO,was PCR amplified from the unicellular cyanobacterium Synechocystis sp.strain PCC 6803,the expression plasmid pET32a(+)-ndhO was constructed and transformed into BL21(DE3)pLysS,and the expression of NdhO protein was induced by IPTG.After purification,the fusion protein pET-NdhO was used to immunize Japanese white rabbit to obtain the polyclonal antibody.The titer of the polyclonal antibody was detected by ELISA and its specificity was analyzed by immunoblotting.The titer of polyclonal antibody was found to be up to 1∶1 025 000,and thus possessed a high specificity.Further,immunoblotting results using the polyclonal antibody showed the presence of NdhO in active NDH-1 mediumcomplex,and not active NDH-1 supercomplex.Therefore,the antibody of NdhO obtained will further help us to reveal the functional roles of cyanobacterial NdhO subunit.

Although many factors that affect the frequency of natural transformation of the unicellular cyanobacterium Synechocystis have been extensively reported, little is known regarding the effects of changes in the spectral quality and intensity of light on its natural transformation. The frequency of natural transformation was significantly enhanced or inhibited by the treatments with low light (LL) or high light (HL) under the incubation condition of cells and DNA before plating and/or on the plates in comparison with that by the treatment with growth light (GL); the changes in the spectral quality of light did not remarkably affect the transformation efficiency of Synechocystis. Further, the lengths of the appearance time of transformants were shortened or retarded by HL or LL illumination when cells and DNA were incubated on the plates relative to that by GL illumination. Further, the transformation efficiency of Synechocystis was closely associated with the permeability of the cell membranes. Treatment with LL significantly enhances the frequency of natural transformation whereas HL illumination remarkably shortens the appearance time of Synechocystis transformants. These phenomena can be extensively applied to future studies according to the specific demands of the transformation experiments. Possible mechanisms underlying these phenomena are discussed.

Treatment with NaHSO3 induces a 10-fold increase in H2 photoproduction in the filamentous N2-fixing cyanobacterium Anabaena sp. strain PCC 7120. However, it is unclear whether this treatment also increases H2 photoproduction in green alga. In this study, treatment with 13 mM NaHSO3 resulted in about a 200-fold increase in H2 production in Chlamydomonas reinhardtii, and this increase was most probably the result of reduced O2 content and enhanced hydrogenase activity. Compared to the conventional strategy of sulfur deprivation, NaHSO3 treatment results in a higher maximum rate of H2 photoproduction, greater efficiency of conversion of light energy into H2, shorter half-time to produce the maximum accumulated H2 levels, and reduced costs because no centrifugation is involved. We therefore conclude that NaHSO3 treatment is an efficient, rapid, and economic strategy for improving photobiological H2 production in the green alga C. reinhardtii.

Cyanobacterial NADPH dehydrogenase (NDH-1) is an important photosynthetic membrane protein complex,and is essential to CO2 uptake,cyclic electron transport around photosystem I and cellular respiration.The enzyme accepts electrons from NADPH and consists of at least 15 subunits,i.e.,NdhA to NdhO.The NdhO is a newly identified subunit,and little is known regarding its roles in cyanobacteria.To obtain the ndhO gene inactivation mutant,the homologous recombination vector,pUC-ΔndhO,was constructed,and then this vector was transferred into wild type Synechocystis sp.strain PCC 6803 by using the natural transformation method.Further,after several subcultures,the transformations were examined by using the PCR and immunoblotting.The experimental results indicated that the kanamycin coding sequence had successfully inserted into the conservative region of ndhO gene,and completely inhibited the expression of ndhO gene.Therefore,an ndhO gene inactivation mutant,ΔndhO,has successfully been obtained,and it will further help us to reveal the roles of NdhO subunit in the NDH-1 complex and in the unicellular cyanobacterium Synechocystis sp.strain PCC 6803.