Photosynthesizing carbonate/nitrate into Chlorococcum humicola biomass for biodiesel and Bacillus coagulans-based biohydrogen production.

Biofuel can be generated by different organisms using various substrates. The green alga Chlorococcum humicola OQ934050 exhibited the capability to photosynthesize carbonate carbon, maybe via the activity of carbonic anhydrase enzymes. The optimum treatment is C:N ratio of 1:1 (0.2 mmoles sodium carbonate and 0.2 mmoles sodium nitrate) as it induced the highest dry mass (more than 0.5 mg.mL-1). At this combination, biomass were about 0.2 mg/mL-1 carbohydrates, 0.085 mg/mL-1 proteins, and 0.16 mg/mL-1 oil of this dry weight. The C/N ratios of 1:1 or 10:1 induced up to 30% of the Chlorococcum humicola dry mass as oils. Growth and dry matter content were hindered at 50:1 C/N and oil content was reduced as a result. The fatty acid profile was strongly altered by the applied C.N ratios. The defatted leftovers of the grown alga, after oil extraction, were fermented by a newly isolated heterotrophic bacterium, identified as Bacillus coagulans OQ053202, to evolve hydrogen content as gas. The highest cumulative hydrogen production and reducing sugar (70 ml H2/g biomass and 0.128 mg/ml; respectively) were found at the C/N ratio of 10:1 with the highest hydrogen evolution efficiency (HEE) of 22.8 ml H2/ mg reducing sugar. The optimum treatment applied to the Chlorococcum humicola is C:N ratio of 1:1 for the highest dry mass, up to 30% dry mass as oils. Some fatty acids were induced while others disappeared, depending on the C/N ratios. The highest cumulative hydrogen production and reducing sugar were found at the C/N ratio of 10:1.

Proteomics reveals toxin tolerance and polysaccharide accumulation in Chlorococcum humicola under high CO2 concentration.

Algae have great application prospects in excess sludge reclamation and recovery of high-value biomass. Chlorococcum humicola was cultivated in this research, using sludge extract (mixed with SE medium) with additions of 10%, 20%, and 30% CO2 (v/v). Results showed that under 20% CO2, the dry weight and polysaccharide yield reached 1.389 ± 0.070 g/L and 313.49 ± 10.77 mg/L, respectively. 10% and 20% CO2 promoted the production of cellular antioxidant molecules to resist the toxic stress and the toxicity of 20% CO2 group decreased from 62.16 ± 3.11% to 33.02 ± 3.76%. 10% and 20% CO2 accelerated the electron transfer, enhanced carbon assimilation, and promoted the photosynthetic efficiency, while 30% CO2 led to photosystem damage and disorder of antioxidant system. Proteomic analysis showed that 20% CO2 mainly affected energy metabolism and the oxidative stress level on the early stage (10 d), while affected photosynthesis and organic substance metabolism on the stable stage (30 d). The up-regulation of PSII photosynthetic protein subunit 8 (PsbA, PsbO), A0A383W1S5 and A0A383VRI4 promoted the efficiency of PSII and chlorophyll synthesis, and the up-regulation of A0A383WH74 and A0A2Z4THB7 led to the accumulation of polysaccharides. The up-regulation of A0A383VDH1, A0A383VX37 and A0A383VA86 promoted respiration. Collectively, this work discloses the regulatory mechanism of high-concentration CO2 on Chlorococcum humicola to overcome toxicity and accumulate polysaccharides.

Growth kinetics of Chlorococcum humicola - A potential feedstock for biomass with biofuel properties.

Economically viable production facilities for microalgae depend on the optimization of growth parameters with regard to nutrient requirements. Using microalgae to treat industrial effluents containing heavy metals presents an alternative to the current practice of using physical and chemical methods. Present work focuses on the statistical optimization of growth of Chlorococcum humicola to ascertain the maximum production of biomass. Plackett Burman design was carried out to screen the significant variables influencing biomass production. Further, Response Surface Methodology was employed to optimize the effect of inoculum, light intensity and pH on net biomass yield. Optimum conditions for maximum biomass yield were identified to be inoculum at 15%, light intensity to be 1500lx and pH 8.5. Theoretical and predicted values were in agreement and thus the model was found to be significant. Gas chromatography analyses of the FAME derivatives showed a high percentage of saturated fatty acids thereby confirming the biofuel properties of the oil derived from algal biomass.

Analysis of the regulatory mechanism of exogenous IAA-mediated tryptophan accumulation and synthesis of endogenous IAA in Chlorococcum humicola.

The activated sludge method is widely used for the treatment of phenol-containing wastewater, which gives rise to the problem of toxic residual sludge accumulation. Indole-3-acetic acid (IAA), a typical phytohormone, facilitates the microalgal resistance to toxic inhibition while promoting biomass accumulation. In this study, Chlorococcum humicola (C. humicola) was cultured in toxic sludge extract and different concentrations of IAA were used to regulate its physiological properties and enrichment of high value-added products. Ultimately, proteomics analysis was used to reveal the response mechanism of C. humicola to exogenous IAA. The results showed that the IAA concentration of 5 × 10-6 mol/L (M) was most beneficial for C. humicola to cope with the toxic stress in the sludge extract medium, to promote the activity of rubisco enzyme, to enhance the efficiency of photosynthesis, and, finally, to accumulate protein as a percentage of specific dry weight 1.57 times more than that of the control group. Exogenous IAA altered the relative abundance of various amino acids in C. humicola cells, and proteomic analyses showed that exogenous IAA stimulated the algal cells to produce more indole-3-glycerol phosphate (IGP), indole, and serine by up-regulating the enzymes. These precursors are converted to tryptophan under the regulation of tryptophan synthase (A0A383V983), and tryptophan can be metabolized to endogenous IAA to promote the growth of C. humicola. These findings have important implications for the treatment of toxic residual sludge while enriching for high-value amino acids.

Analysis of the mechanism of exogenous indole-3-acetic acid on the enrichment of d-glucose in Chlorococcum humicola cultured by sludge extracts.

Addressing problems of high organic toxicity in the wastewater treatment process, microalgae have been used to reduce the toxicity in sludge and to synthesize non-toxic and recoverable biomass of resources. Phytohormone is a core regulator of plant growth and current research has generally focused on their promotion of cell division and cell expansion. Effects of phytohormone on the enrichment mechanism of microalgae directional polysaccharides accumulation remain poorly elucidated. This study was carried out to investigate the effects of exogenous indole-3-acetic acid (IAA) on growth characteristics, biomass accumulation, and photosynthesis capacity of Chlorococcum humicola cultured in sludge extract and further find the d-glucose enrichment mechanism of it through proteomic. The results indicated that the optimal culture conditions were the 75 % sludge extract and 25 % selenite enrichment (SE) medium with 5 × 10-6 mol/L indole-3-acetic acid. Polysaccharides increased significantly from day 20 and accumulated to (326.59 ± 13.06) mg/L on day 30, in which the d-glucose proportion increased to 61.53 %. Most notably, proteomic tests were performed and found that the photosynthesis-related proteins including the differential proteins of photosystem electron transport, ATP and NADPH catalytic synthesis were significantly up-regulated. At the end of the path, three pathways of d-glucose enrichment with α-d-Glucose-1P as a precursor were summarized through indole-3-acetic acid activation on amylase, endoglucanase and Beta-glucosidase, etc. These results provide insights to explore the directed enrichment of biomass in Chlorococcum humicola by indole-3-acetic acid.

Enhancing starch accumulation/production in Chlorococcum humicola through sulphur limitation and 2,4- D treatment for butanol production.

Depleting fuel resources is a global concern worldwide due to the unstable and cost of fuel resources. Increased transportation has gradually depleted the fossil-based fuel resources leading to find a cost-effective, readily available, and renewable source. Considering these issues, various private and government organizations have focussed on producing bio-based fuels from natural sources. In this scenario, algae are a potential emerging source of feedstock or biomass for biobutanol production, which can effectively replace fossil fuels and their environmental drawbacks. The present study focussed on evaluating the potential of freshwater microalga Chlorococcum humicola isolated from temple pond as feedstock for biobutanol production using Clostridium acetobutylicum. The results indicated that C. humicola produced 846.33 µgmg-1of starch under full strength Chu10 medium. While under sulphur and phosphorus limitation, the accumulation of starch was 947.33 µg mg-1 and 766.67 µgmg-1, respectively. Also, C. humicola was exposed to different concentrations of 2,4-Dichlorophenoxyacetic acid (2,4-D). At 10µgml-1 of 2,4-D, the highest starch concentration of 989µgmg-1was achieved in C. humicola. Finally, starch in C. humicola were hydrolysed and ABE fermentation was performed using C. acetobutylicum under anaerobic condition in a 5 L automated fermenter. After 72 h of fermentation, the fermented broth is analyed in Gas Chromatography showing the fermented product containing Acetone: Butanol: Ethanol. The present study is the first report on the production of biobutanol from C. humicola isolated from Temple pond. This study emphasizes the importance of local isolates of microalgae as a third-generation substrate to produce butanol to replace fossil-based fuels.

Evaluation of antigenotoxic effects of carotenoids from green algae Chlorococcum humicola using human lymphocytes.

OBJECTIVE: To identify the available phytochemicals and carotenoids in the selected green algae and evaluate the potential genotoxic/antigenotoxic effect using lymphocytes. METHODS: Organic solvent extracts of Chlorococcum humicola (C. humicola) were used for the phytochemical analysis. The available carotenoids were assessed by HPLC, and LC-MS analysis. The genotoxicity was induced by the benzo(a)pyrene in the lymphocyte culture, the genotoxic and antigenotoxic effects of algal carotenoids with and without genotoxic inducer were evaluated by chromosomal aberration (CA), sister chromatid exchange (SCE) and micronucleus assay (MN). RESULTS: The results of the analysis showed that the algae were rich in carotenoids and fatty acids. In the total carotenoids lutein, ß-carotene and α-carotene were found to be present in higher concentration. The frequency of CA and SCE increased by benzo(a)pyrene were significantly decreased by the carotenoids (P<0.05 for CA, P<0.001 for SCE). The MN frequencies of the cells were significantly decreased by the treatment with carotenoids when compared with the positive controls (P<0.05). CONCLUSIONS: The findings of the present study demonstrate that, the green algae C. humicola is a rich source of bioactive compounds especially carotenoids which effectively fight against environmental genotoxic agents, the carotenoids itself is not a genotoxic substance and should be further considered for its beneficial effects.