Metagenomic analysis and nitrogen removal performance evaluation of activated sludge from a sequencing batch reactor under different salinities.

The effect of salinity on the nitrogen removal performance and microbial community of activated sludge was investigated in a sequencing batch reactor. The NH4+-N removal efficiency was over 95% at 0-4% salinity, indicating that the nitrification performance of activated sludge was slightly affected by lower salinity. The obvious nitrite accumulation was observed with the increment of the salinity to 5%, followed by a notable decline in the nitrogen removal performance at 6% salinity. The salinity inhibited the microbial activity, and the specific rate of nitrification and denitrification was decreased by the increasing salinity obviously. Additionally, the lower activity of superoxide dismutase and peroxidase and higher reactive oxygen species content in activated sludge might account for the deteriorative nitrogen removal performance at 6% salinity. Metagenomics analysis revealed that the genes encoding the ABC-type quaternary amine transporter in the ABC transporter pathway were abundant in the activated sludge at 2% and 4% salinity, and the higher salinity of 6% led to the loss of the genes encoding the p-type Na+ transporter in the ABC transporter pathway. These results indicated that the salinity could weaken the ABC transporter pathway for the balance of osmotic pressure in activated sludge. The microbial activity and nitrogen removal performance of activated sludge were decreased due to the unbalanced osmotic pressure at higher salinity.

Effect of external carbon addition and enrofloxacin on the denitrification and microbial community of sequencing batch membrane reactor treating synthetic mariculture wastewater.

The effect of sequencing batch membrane bioreactor (SMBR) on external carbon addition and enrofloxacin was investigated to treat synthetic mariculture wastewater. Anoxic/anaerobic and low COD/TN can improve the ammonia oxidation of the system, and the NH4+-N removal efficiency above 99%. External carbon was added and an anoxic environment was set to provide a suitable environment for denitrifying bacteria. When the external carbon source was 50-207 mg/L, the TN removal efficiency (31.82%-37.73%) and the COD of the effluent (28.85-36.58 mg/L) had little change. The partition resistance model showed that cake deposition resistance (RC,irr) and irreversible resistance (RPB) were the main components. And with the increase in cleaning times, the fouling rate of membrane components accelerated. Enrofloxacin can promote the TN removal efficiency (45.66%-93.74%) and had a significant effect on TM7a, Cohaesibacter, Vibrio and Phaeobacter.

Mixotrophic cultivation of Chlorella pyrenoidosa under sulfadiazine stress: High-value product recovery and toxicity tolerance evaluation.

Sulfadiazine (SDZ) as a common sulfonamide antibiotic is frequently detected in wastewater, but there is little information on the high-value product recovery and toxicity tolerance evaluation of mixotrophic microalgae under SDZ stress. In this study, effects of SDZ on growth, photosynthesis, cellular damage, antioxidant capacity and intracellular biochemical components of Chlorella pyrenoidosa were investigated. Results showed that the growth of C. pyrenoidosa was inhibited by about 20% under high SDZ stress, but there was little impact on photosynthesis. Cellular damage and antioxidant capacity were evaluated using malondialdehyde (MDA) content and superoxide dismutase (SOD) activity to further explain the toxicity tolerance of mixotrophic microalgae. The SDZ stress not only increased lipid and carbohydrate content, respectively attaining to the maximum of 390.0 and 65.4 mg/L, but also improved the biodiesel quality of C. pyrenoidosa. The findings show the potential of mixotrophic microalgae for biodiesel production and wastewater treatment.

Removal effect of enrofloxacin from mariculture sediments by bioelectrochemical system and analysis of microbial community structure.

Based on the application of sediment microbial fuel cell (SMFC) in the bioremediation of sediment, this study used the sediment microbial fuel cell technology as the leading reactor. Modification of anode carbon felts (CF) by synthesis of PANI/MnO2 composited to improve the electrical performance of the sediment microbial fuel cell. This study investigated the degradation effects, degradation pathways of the specific contaminant enrofloxacin and microbial community structure in sediment microbial fuel cell systems. The results showed that the sediment microbial fuel cell system with modified anode carbon felt (PANI-MnO2/CF) prepared by in-situ chemical polymerization had the best power production performance. The maximum output voltage was 602 mV and the maximum power density was 165.09 mW m-2. The low concentrations of enrofloxacin (12.81 ng g-1) were effectively degraded by the sediment microbial fuel cell system with a removal rate of 59.52%.

Nitrogen and sulfamethoxazole removal in a partially saturated vertical flow constructed wetland treating synthetic mariculture wastewater.

This study investigated the removal of nitrogen and sulfamethoxazole (SMX), and the microbial communities in a partially saturated vertical flow constructed wetland (PS-VFCW) fed with synthetic mariculture wastewater operated at different saturated zone depths (SZDs), i.e. 51, 70, and 60 cm. Removal efficiencies were 99.8%-100.0% for COD, 34.1%-100.0% for NH4+-N, 67.8%-97.3% for total inorganic nitrogen (TIN), and 29.8%-57.2% for SMX. Excellent nitrification performance was achieved at the SZDs of 51 and 60 cm. Denitrification performed well at 70 and 60 cm SZDs. The highest TIN removal efficiency (97.3%) was achieved as the SZD was 60 cm. SMX removal was significantly influenced by SZD and was promoted by higher SZD. The removal of organics, nitrogen, and SMX mainly occurred in the unsaturated zone. Ammonia-oxidizing bacteria, nitrite-oxidizing bacteria, denitrifying bacteria, and SMX-degrading bacteria were detected in the unsaturated and saturated zones, and showed an increasing trend in abundance along the depth.

Insights into the response of anammox sludge to the combined stress of nickel and salinity.

Anaerobic ammonium oxidation (anammox) is a promising technology applied to treat industrial wastewater, while the commonly coexistent heavy metals and salinity usually become a challenging issue to be addressed. In this study, the responses of anammox sludge in terms of performance, activity, functional enzyme and extracellular polymeric substance (EPS) to the combined stress of Ni(II) and salinity (20 ‰) were investigated holistically. It turned out that low Ni(II) concentration (0.2 mg·L-1) together with salinity (20 ‰) showed an insignificant effect on the anammox performance, while a decreased nitrogen removal by 46.96 % was observed with the increased Ni(II) concentration to 1 mg·L-1. It should be pointed out that the anammox system exhibited good robustness evidenced by rapid recovery to achieve 89.13 % of nitrogen removal efficiency and 1.21 kg·m-3·d-1 of nitrogen removal rate after the elimination of stress factors within 40 days. Ni(II) concentration was revealed to play a more important role in the specific activity of anammox sludge. The functional enzymes related to nitrogen removal, e.g. nitrite reductase (NIR), hydrazine oxidase (HZO) and heme c were found to be inhibited by the combined stress of Ni(II) and salinity, with decreased activity by 49.54 %, 39.39 % and 45.88 %, respectively. However, the enzyme related to assimilation, e.g. alkaline phosphatase (AKP) and nitrate reductase (NAR) appeared to be enhanced. The EPS content was found to decrease by 55.19 % under the combined stress. Detailed analysis of 3D-EEM and FTIR spectra further revealed that the combined stress of Ni(II) and salinity could change both the quantity and composition of EPS in anammox sludge. These results are expected to offer insights into the combined effect of nickel and salinity on the anammox system, and benefit the application of anammox technology for industrial metal-rich saline wastewater treatment.

Electrochemical degradation of tetracycline hydrochloride in sulfate solutions on boron-doped diamond electrode: The accumulation and transformation of persulfate.

In this study, a novel electrifying mode (divided power-on and power-off stage) was applied in the system of BDD activate sulfate to degrade tetracycline hydrochloride (TCH). The BDD electrode could activate sulfate and H2O to generate sulfate radicals (SO4•-) and hydroxyl radicals (•OH) to remove TCH, and SO4•- could dimerize to form S2O82-. Then, the S2O82- was activated by heat and quinones to generate SO4•- for the continuous degradation of TCH during the power-off stage. In addition, the intermittent time has a significant effect on the degradation of TCH. Factors, affecting the accumulation of S2O82-, were analyzed using a full factorial design, and the accumulation of S2O82- could reach 16.2 mM in 120 min. The results of electron spin resonance and radical quenching test showed that SO4•-, •OH, direct electron transfer (DET), and non-radical in the system could effectively degrade TCH, and SO4•- was dominated. The intermediate products of TCH were analyzed by HPLC-QTOF-MS/MS, and the TCH mainly underwent hydroxylation, demethylation and ring opening reactions to form small molecules, and finally mineralized. The results of the feasibility analysis revealed that some intermediates have high toxicity, but the system could improve the toxicity. The results of energy consumption indicated that the intermittent electrifying mode could make full use of the persulfate generated during the power-on stage and reduce about 30% energy consumption. In conclusion, this work demonstrated that it was economically feasible to degrade TCH in wastewater by activating sulfate with BDD electrodes with an intermittent electrifying mode.

Roles of illumination on distribution of phosphorus in Chlorella vulgaris under mixotrophic cultivation.

Phosphorus (P) is a non-substitutable resource and global reserves of phosphate rock are limited. In this study, phosphorus recovery by Chlorella vulgaris, and the effects of different light intensities (2000 Lux, 5000 Lux, 8000 Lux, 12,000 Lux) on the phosphorus distribution in the soluble microbial product (SMP), extracellular polymeric substance (EPS) and intracellular polymeric substance (IPS) were analyzed. The results showed that the 5000 Lux was the optimum light intensity for P uptake and transformation by Chlorella vulgaris under mixotrophic cultivation. At the light intensity of 5000 Lux, the P uptake rate was 100% after 32 days of cultivation, and the concentration of intracellular organic phosphorus (OP) was 5.77 mg P/L. Moreover, EPS was the main P pool when inorganic phosphorus (IP) was depleted in bulk solution. Phosphorus recovery by microalgae is an important solution to treat P-containing wastewater.

Comparison of primary and secondary sludge carbon sources derived from hydrolysis or acidogenesis for nitrate reduction and denitrification kinetics: Organics utilization and microbial community shift.

Seeking available and economical carbon sources for denitrification process is an intractable issue for wastewater treatment. However, no study compared different types of waste sludge as carbon source from denitrification mechanism, organics utilization and microbial community aspects. In this study, primary and secondary sludge were pretreated by thermophilic bacteria (TB), and its hydrolysis or acidogenic liquid were prepared as carbon sources for denitrification. At C/N of 8-3, the variations of NO3--N and NO2--N were profiled in typical cycles and denitrification kinetics was analyzed. Primary sludge achieved a competitive NOX-N removal efficiency with less dosage than secondary sludge. Fourier transform infrared (FTIR) spectroscopy was introduced to analyze organic composition from functional-group perspective and the utilization of organic matters in different sludge carbon sources was investigated. To further analyze the microbial community shift in different denitrification systems, high-throughput sequencing technology was applied. Results showed that denitrifier Thauera, belonging to Proteobacteria, was predominant, and primary sludge acidogenic liquid enriched Thauera most intensively with relative abundance of 47.3%.

Metagenomics and network analysis elucidating the coordination between fermentative bacteria and microalgae in a novel bacterial-algal coupling reactor (BACR) for mariculture wastewater treatment.

To achieve the goal of treating mariculture wastewater economically and efficiently, a novel bacterial-algal coupling reactor (BACR) integrating acidogenic fermentation and microalgae cultivation was firstly investigated for mariculture wastewater treatment. Volatile fatty acids (VFAs) generated in the dark chamber migrated into the photo chamber for microalgal utilization, which alleviated the pH drop and feedback inhibition of the acidogenic fermentation. The maximum dry cell weight (DCW) of microalgae was 1.46 g/L, and pollutants such as chemical oxygen demand (COD), ammonium (NH4+-N) and total phosphorus (TP) in the BACR were effectively removed under the mixotrophic culture condition. Furthermore, bacterial community profiles and functional genes in the BACR and single acidogenic fermentation reactor were identified. Compared with the single acidogenic fermentation reactor, most of the fermentative bacteria (e.g., Ruminococcus, Christensenellaceae R-7 group, Exiguobacterium, Pseudomonas and Levilinea) were enriched by the BACR. From the genetic perspective, the abundances of dominant genes (ackA, acs and atoD) associated with acetic, propionic and butyric acid production were greatly enhanced in the BACR. In the fatty acid biosynthesis pathway (ko00061), three kinds of high-abundance acetyl-CoA carboxylase genes and eight kinds of downstream functional genes were up-regulated in the BACR. Finally, based on co-occurrence network analysis, the coordination between fermentative bacteria and microalgae in the BACR was revealed. This study provided a deep insight into the advantage and potential of the BACR in mariculture wastewater treatment.

Enhanced aerobic granular sludge by static magnetic field to treat saline wastewater via simultaneous partial nitrification and denitrification (SPND) process.

Saline wastewater poses a threat to biological nitrogen removal. This study investigated whether and how static magnetic field (SMF) can improve the salt-tolerance of aerobic granular sludge (AGS) in two simultaneous partial nitrification and denitrification (SPND) reactors. Results confirmed that the SMF improved the mean size and settleability of granules, stimulated secretion of extracellular polymeric substances with high protein content, in turn enhancing the aerobic granulation. Although high salt stress inhibited functional microorganisms, the SMF maintained better SPND performance with average COD removal, TN removal and nitrite accumulation ratio finally recovering to 100%, 72.9% and 91.1% respectively. High throughput sequencing revealed that functional bacteria evolved from Paracoccus to halotolerant genera Xanthomarina, Thauera, Pseudofulvimonas and Azoarcus with stepwise increasing salinity. The enhanced salt-tolerance may be because the SMF promoted the activity of these halotolerant bacteria. Therefore, this study proposes an economic, effective and environmental biotechnology for saline wastewater treatment.

Comparison on anaerobic phosphorus release and recovery from waste activated sludge by different chemical pretreatment methods: Focus on struvite quality and benefit analysis.

Phosphorus recovery from waste activated sludge (WAS) is expected to alleviate the shortage of phosphate rock and reduce eutrophication. In this study, acid, alkali and sodium polyacrylate (PAAS) were compared to enhance phosphorus release and recovery from WAS. During anaerobic fermentation (AF) stage, the optimal pretreated conditions for ortho-phosphate release were the pH of 4 (AF 12 h), 13 (AF 12 h) and 22.4 g PAAS/L (AF 24 h) with the phosphorus release efficiencies of 40.9%, 62.6% and 31.7%, respectively. Acid, alkali and PAAS addition were beneficial for apatite phosphorus (AP), non-apatite inorganic phosphorus (NAIP) and organic phosphorus (OP) release from WAS, respectively. Strong acidic (pH = 4) and alkaline (pH = 12 and 13) conditions inhibited the release of soluble ammonia, while PAAS would not have a negative impact on the release of soluble ammonia. By means of precipitation crystallization, the ortho-phosphate could be almost recovered after acid/alkali pretreatment compared with PAAS (88.9%) at optimal Mg/P molar ratio of 1.5:1. The XRD, FT-IR and SEM-EDX analyses confirmed the main component in the product was struvite. The purity of the struvite in the product recovered from acid (named PreAC, 78.9%) and alkali (named PreAL, 89.6%) pretreated sludge were higher than that of the PAAS (named PrePA, 72.3%) by elemental analysis. The mercury and chromium content existed in PreAC were above the Control Standards of Pollutants in Sludge for Agricultural Use, whereas detected heavy metal elements level of the PreAL and PrePA were below the standard. By means of cost analysis, acid/alkali pretreatment could obtain economic benefits compared with PAAS. Thus, those discoveries would broaden the phosphorus recovery way to serve in practice.

Regulation of carbon source metabolism in mixotrophic microalgae cultivation in response to light intensity variation.

Microalgae are one of the promising sources for renewable energy production, and the light intensity variation can affect the biofuel generation and carbon assimilation of mixotrophic microalgae. To reveal the response of carbon assimilation to light intensity, the effect of light intensity on the carbon source metabolism of Chlorella vulgaris under mixotrophic cultivation was investigated in this study. Moreover, the optimal carbon source composition for mixotrophic microalgae cultivation was evaluated using bicarbonate (HCO3-) and carbonate (CO32-) as inorganic carbon sources, and glucose and acetate as organic carbon sources. The optimal light intensity for Chlorella vulgaris growth was at the range of 8000-12000 lux. For the accumulation of biochemical components, low light intensity was beneficial to protein accumulation, and high light intensity was advantageous for carbohydrate and lipid accumulation. With HCO3- and glucose, the maximum lipid content reached 37.0% at a light intensity of 12000 lux. The citrate synthase activity was negatively correlated with light intensity, showing an opposite trend to biomass production. High light intensity had a positive impact on Rubisco expression, which promoted the microalgae growth and carbon fixing. The energy produced by heterotrophic metabolic activities increased at low light intensity, and the enhancement of biomass production with high light intensity was mainly caused by the improved photoreaction efficiency during the mixotrophic cultivation.

Effect of mixed primary and secondary sludge for two-stage anaerobic digestion (AD).

As an energy-efficient and eco-friendly sludge treatment process, two-stage anaerobic digestion (AD) is widely employed to recovery biomass energy from waste sludge. However, the effect of primary and secondary sludge for two-stage AD was not clear. In this study, two-stage AD of mixed sludge in different volume ratio was investigated. The maximum cumulative H2 yield (100.5 ml) and CH4 yield (2643.6 ml) were obtained in volume ratio of 1:3 (primary sludge: secondary sludge). In two-phase AD, mixed sludge could induce positive effect on both organics releasing in extracellular polymeric substances (EPS) and the utilization of volatile fatty acids (VFAs). By investigating the compositional characteristics of dissolved organic matters (DOM) through excitation-emission matrix (EEM) coupling with fluorescence regional integration (FRI), it revealed more degradable substances utilization in mixture of sludge. Results from this work suggest that two-phase AD with mixed sludge is efficient for renewable energy recovery.

Impact of carbon/nitrogen ratio on the performance and microbial community of sequencing batch biofilm reactor treating synthetic mariculture wastewater.

The differences of cultured organism species, aquaculture model and supervisor mode lead to different carbon/nitrogen ratios in mariculture wastewater. Therefore, the performance, microbial community and enzymatic activity of sequencing batch biofilm reactor were compared in treating synthetic mariculture wastewater at different chemical oxygen demand/nitrogen (COD/N) ratios. Compared with COD/N ratio of 6, the ammonia-oxidizing rate and nitrite-oxidizing rate at COD/N ratio of 5, 4 and 3 increased by 3.66 % and 3.08 %, 11.19 % and 14.95 %, and 24.50 % and 32.54 %, respectively. Similarly, the ammonia monooxygenase and nitrite oxidoreductase activities increased by 3.50 % and 6.76 %, 11.09 % and 16.22 %, and 25.43 % and 39.19 % at COD/N ratio at 5, 4 and 3, respectively. However, the denitrifying rate and denitrification enzymatic activity declined with the decrease of C/N ratio from 6 to 3. The production, protein content and polysaccharide content of loosely bound extracellular polymeric substances (LB-EPS) and tightly bound EPS (TB-EPS) reduced with the decrease of COD/N ratio from 6 to 3. The abundance of nitrifying genera increased with the decrease of COD/N ratio from 6 to 3, whereas most of denitrification genera displayed a decreasing trend. The microbial co-occurrence pattern, keystone taxa and significant difference were altered with the decrease of COD/N ratio. Among the keystone taxa, Thauera, Denitromonas, Nitrosomonas and Denitratisoma had a close link with nitrogen transformation. The present results can provide some theoretical basis for evaluating the effect of carbon/nitrogen ratio on the nitrogen removal of biological wastewater treatment systems.

Bacterial-algal coupling system for high strength mariculture wastewater treatment: Effect of temperature on nutrient recovery and microalgae cultivation.

In the present study, bacterial-algal coupling system, an integration process of acidogenic fermentation and microalgae cultivation was used for high strength mariculture wastewater (HSMW) treatment, resource recovery and low-cost biomass production. The effect of temperature on Chlorella vulgaris (C. vulgaris) cultivation was investigated with culture medium of acidogenic liquid. The results showed that acidogenic liquid could be used as culture medium for C. vulgaris and higher biomass was obtained compared to control. The acidogenic liquid obtained at initial pH of 8 was the most suitable culture medium for C. vulgaris growth due to befitting C/N and considerable volatile fatty acids. Moreover, the optimum temperature for C. vulgaris cultivation was 25 °C and the removal efficiency of chemical oxygen demand (COD) and NH4+-N from acidogenic liquid could reach 94.4% and 68.8%, respectively. The outcome could create an innovative value chain with environmental sustainability and economic feasibility in aquaculture industry.

Mariculture wastewater treatment with Bacterial-Algal Coupling System (BACS): Effect of light intensity on microalgal biomass production and nutrient removal.

Mariculture wastewater generated from the mariculture industry has increased public concern due to its impact on the sustainability of aquatic environments and aquaculture practices. Herein, the Bacterial-Algal Coupling System was applied for mariculture wastewater treatment. Microalgae growth in heterotrophy and mixotrophy (2000-8000 lux) was first compared. The best microalgal growth and nutrient removal were obtained at 5000 lux, where biomass productivity of microalgae was 0.465 g L-1 d-1, and 98.1% of chemical oxygen demand, 70.7% of ammonia-nitrogen, and 90.0% of total phosphorus were removed. To further understand the nutrient removal through microalgae cultivation, the enzyme activities involved in the Calvin cycle and the Tricarboxylic Acid cycle at different light intensities were determined. Under mixotrophic cultivation, there was a coordination between photosynthesis and heterotrophic metabolism in the agal cell, which resulted in a high algal biomass production and removal efficiency of nutrients. This study provided a novel insight into the bioremediation of mariculture wastewater and microalgae cultivation.

Phosphorus uptake, distribution and transformation with Chlorella vulgaris under different trophic modes.

Phosphorus (P) uptake, distribution and transformation are important processes associated with the growth and metabolism of microalgae. In this study, the fate of P in soluble microbial products (SMP), extracellular polymeric substances (EPS), and intracellular polymeric substances (IPS) of Chlorella vulgaris C7 in the form of inorganic P (IP) and organic P (OP) was studied under different trophic modes, including photoautotrophy, heterotrophy and mixotrophy. The results showed that mixotrophic cultivation of microalgae brought highest biomass yield, which was 2.09 times and 11.10 times higher than that of the photoautotrophic and heterotrophic conditions. Regarding P distribution and transformation, the trophic modes affected the transformation trends and rates of P in the form of IP and OP among SMP, EPS and IPS. Under photoautotrophic condition, EPS was the main P pool, and most of P in C. vulgaris was IP. While, under mixotrophic condition, IPS was the main P pool, and most of P was transformed to OP. The addition of glucose promoted the uptake of P by algal cells and the transformation of IP to OP, which accounted for 79.32% of total phosphorus at the end of cultivation. Collectively, mixotrophic cultivation was regarded as the optimum approach for microalgae cultivation and nutrient recovery.

Thermophilic bacteria combined with alkyl polyglucose pretreated mariculture solid wastes using as denitrification carbon source for marine recirculating aquaculture wastewater treatment.

In marine recirculating aquaculture systems (RAS), efficient nitrogen removal is challenging due to the high NO3--N concentration, low organic matters content, and high salinity. In this study, mariculture solid wastes (MSW) acidogenic liquid pretreated by thermophilic bacteria (TB) combined with alkyl polyglucose (APG) was first used as carbon source for denitrification to remove NO3--N. TB + APG pretreatment could accelerate the hydrolysis of MSW, and the highest volatile fatty acids (VFAs) yield (40.3%) was obtained with TB + 0.2 g/g VSS APG pretreatment. MSW acidogenic liquid pretreated by TB + 0.2 g/g VSS APG was a reliable carbon source for denitrification, and the optimum COD/NO3--N ratio (C/N) was 8 with no residue of NOx--N. VFAs were more effectively utilized by denitrifiers than carbohydrate and protein. The high denitrification potential (PDN) and denitrification rate (VDN) indicated the higher denitrification ability at C/N of 8 using MSW acidogenic liquid as carbon source. The outcomes of this work could provide useful information for promoting technological innovation in marine RAS wastewater treatment.

Enhancing microalgae growth and product accumulation with carbon source regulation: New perspective for the coordination between photosynthesis and aerobic respiration.

The coordination between photosynthesis and aerobic respiration under mixotrophic cultivation can make a difference to the growth and biochemical composition of microalgae. However, the response of carbon metabolism to carbon source composition under mixotrophic microalgae cultivation has not been well studied. In this study, the synergistic effects of inorganic carbon (IC) and organic carbon (OC) supply on the growth and carbon metabolism of Chlorella vulgaris under mixotrophic cultivation were investigated. The increase of the proportion of HCO3- had a positive effect on the expression of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO), which promoted the biomass production and carbon fixing. The activity of citrate synthase was attenuated with the increase of IC/OC ratio, indicating that the energy needed for the biomass production in groups with high IC/OC ratio was contributed by photoreaction. Biochemical analysis showed that CO32- was more efficient than HCO3- for carbohydrate and lipid accumulation of Chlorella vulgaris, and the highest amount of carbohydrate (30.2%) and lipid (35.8%) was recorded with the combined use of CO32- and glucose. The results could provide a new perspective on carbon metabolism and enzyme regulation in mixotrophic microalgae cultivation.