Efficacy of White Spot Syndrome Virus Protein VP28-Expressing Chlorella vulgaris as an Oral Vaccine for Shrimp.

The white spot syndrome virus (WSSV) is the causative agent of white spot disease, which kills shrimp within a few days of infection. Although WSSV has a mortality rate of almost 100% and poses a serious threat to the shrimp farming industry, strategies for its prevention and treatment are extremely limited. In this study, we examined the efficacy of VP28, a recombinant WSSV protein expressed in Chlorella vulgaris (C. vulgaris), as an oral shrimp vaccine. When compared with the control group, in which WSSV had a cumulative mortality of 100%, shrimp treated with 5% VP28-expressing C. vulgaris in their feed only had a 20% cumulative mortality rate 12 days after the WSSV challenge. When compared with the nonvaccinated group, the transcription of anti-lipopolysaccharide factor, C-type lectin, and prophenoloxidase genes, which are involved in shrimp defense against WSSV infection, was upregulated 29.6 fold, 15.4 fold, and 11.5 fold, respectively. These findings highlight C. vulgaris as a potential host for industrial shrimp vaccine production.

The establishment of new protein expression system using N starvation inducible promoters in Chlorella.

Chlorella is a unicellular green microalga that has been used in fields such as bioenergy production and food supplementation. In this study, two promoters of N (nitrogen) deficiency-inducible Chlorella vulgaris N Deficiency Inducible (CvNDI) genes were isolated from Chlorella vulgaris UTEX 395. These promoters were used for the production of a recombinant protein, human granulocyte-colony stimulating factor (hG-CSF) in Chlorella vulgaris UTEX 395 and Chlorella sp. ArM0029B. To efficiently secrete the hG-CSF, the protein expression vectors incorporated novel signal peptides obtained from a secretomics analysis of Chlorella spp. After a stable transformation of those vectors with a codon-optimized hG-CSF sequence, hG-CSF polypeptides were successfully produced in the spent media of the transgenic Chlorella. To our knowledge, this is the first report of recombinant protein expression using endogenous gene components of Chlorella.

Augmenting the expression of accD and rbcL genes using optimized iron concentration to achieve higher biomass and biodiesel in Chlorella vulgaris.

Chlorella vulgaris is a form of microalgae commonly employed as a biological source of oil for biodiesel production. Major algal cultivation strategies are focused on stimulating growth rate and lipid content. In the present study, the algal growth media was supplemented with iron (III) chloride (FeCl3), as a stimulating factor for growth and lipid production, in three iron concentrations including 90, 200, and 500 µM. The turbidity of algal cells was measured on different days, to determine the growth rate. In optimum iron concentration, this measurement experienced a 2.1-fold increase. Next, the lipid content was extracted, and the amount of lipid produced in each treatment was calculated, which demonstrated a 4.57-fold increase in lipid productivity. The expression of genes corresponding to the metabolic enzymes (i.e. acetyl-CoA carboxylase (accD) and ribulose bisphosphate carboxylase large chain (rbcL)) was evaluated using real-time PCR under different initial iron feeds. As demonstrated in the results, the initial iron feed of 90 µM was an optimum concentration that obtained the highest growth rate, more cell density, and increased lipid production. In 90 µM initial iron concentration, the expression of accD and rbcL genes showed a 4.8- and 35-fold increase, respectively, compared to that of the control genes. Based on the results, this optimum iron concentration could satisfy the industrial interest in biodiesel production from C. vulgaris as a potential stimulating factor. However, higher levels of iron (e.g. 200 and 500 µM) failed to act as positive stress for increasing biodiesel production. Finally, in this paper, different mechanisms where iron affects acetyl-CoA carboxylase (ACCase) and 1,5-ribulose bisphosphate carboxylase/oxygenase (RuBisCo) are illustrated.

Combined effects of erythromycin and enrofloxacin on antioxidant enzymes and photosynthesis-related gene transcription in Chlorella vulgaris.

Multiple antibiotics are simultaneously detected in aquatic environment, so it is extremely important to study the combined effects of their mixtures. In this study, we investigated the toxic effects of erythromycin (ERY) and enrofloxacin (ENR), added individually or in combination, on Chlorella vulgaris and explored the toxic mechanisms. Results showed that the 96 h-EC50 values of ERY, ENR and ERY-ENR mixture to C. vulgaris were 85.7, 124.5 and 39.9 µg L-1 respectively, and combined toxicity assessment found that joint effect of the two antibiotics was synergism, which was proven by the chlorophyll content in algae. Antioxidant defense system and photosynthesis were involved in toxic mechanisms and the results revealed that both the activities of antioxidant enzymes, and the malondialdehyde (MDA) and glutathione (GSH) contents increased in antibiotic treatments. In addition, the increase was more significant in joint exposure treatment, which implied that the antioxidant defense system was synergistically affected. RT-PCR showed that ERY and ENR upregulated the transcript abundance of psaB, psbC and chlB at low concentrations and the transcription abundance was synergistically increased in combined treatment. Therefore, the risk of the toxicity of antibiotics to aquatic organisms in real environment both at organismal and molecular level increases as a result of their combined presence.

RNA-seq analysis reveals the significant effects of different light conditions on oil degradation by marine Chlorella vulgaris.

Marine Chlorella vulgaris, an efficient hydrocarbon-degrading organism, is easily affected by light. In this study, we investigated the direct effects of different light conditions on crude oil degradation by C. vulgaris and its crude enzyme. Under 12 h illumination, the crude enzyme improved hydrocarbon removal by 39.36%, whereas the addition of the enzyme and C. vulgaris increased the degradation rate by 121.73%. Conversely, the addition of enzyme under heterotrophic condition was negatively related to oil degradation by C. vulgaris, and the degradation rate decreased from 74.32% to 48.65% and further reduced by 34.54%. The results of RNA sequencing analysis suggested that hydrocarbons removal was attributed to C. vulgaris metabolism in heterotrophic physiological state. While enhanced removal efficiency of hydrocarbons was achieved in mixotrophic physiological state due to the coupling of C. vulgaris metabolism with photocatalytic oxidation. Functional enzymes played key roles in photocatalysis and biodegradation processes.

Identification and Functional Analysis of the psaD Promoter of Chlorella vulgaris Using Heterologous Model Strains.

Chlorella has great potential as a bio-factory for production of value-added compounds. To produce the desired chemicals more efficiently in Chlorella, genetic tools for modification of Chlorella need to be developed, especially an endogenous promoter. In this study, the promoter of photosystem I protein D (psaD) from Chlorella vulgaris UTEX395 was identified. Computational analysis revealed the presence of several putative cis-acting elements, including a potential core element, and light-responsive or stress-responsive elements. Gene expression analysis in heterologous expression system in Chlamydomonasreinhardtii and Nicotianabenthamiana showed that CvpsaD promoter can be used to drive the expression of genes. Functional analysis of this promoter suggested that the initiator element (Inr) is important for its function (i.e., TATA-less promoter) and that an additional factor (e.g., downstream of the transcriptional start site) might be needed for light response. We have shown that the CvpsaD promoter is functional, but not sufficiently strong, both in microalgae and higher plant.

Indole-3-acetic acid (IAA) induced changes in oil content, fatty acid profiles and expression of four fatty acid biosynthetic genes in Chlorella vulgaris at early stationary growth phase.

Microalgae lipids and oils are potential candidates for renewable biodiesel. Many microalgae species accumulate a substantial amount of lipids and oils under environmental stresses. However, low growth rate under these adverse conditions account for the decrease in overall biomass productivity which directly influence the oil yield. This study was undertaken to investigate the effect of exogenously added auxin (indole-3-acetic acid; IAA) on the oil content, fatty acid compositions, and the expression of fatty acid biosynthetic genes in Chlorella vulgaris (UMT-M1). Auxin has been shown to regulate growth and metabolite production of several microalgae. Results showed that oil accumulation was highest on days after treatment (DAT)-2 with enriched levels of palmitic (C16:0) and stearic (C18:0) acids, while the linoleic (C18:2) and α-linolenic (C18:3n3) acids levels were markedly reduced by IAA. The elevated levels of saturated fatty acids (C16:0 and C18:0) were consistent with high expression of the ß-ketoacyl ACP synthase I (KAS I) gene, while low expression of omega-6 fatty acid desaturase (ω-6 FAD) gene was consistent with low production of C18:2. However, the increment of stearoyl-ACP desaturase (SAD) gene expression upon IAA induction did not coincide with oleic acid (C18:1) production. The expression of omega-3 fatty acid desaturase (ω-3 FAD) gene showed a positive correlation with the synthesis of PUFA and C18:3n3.

[An assessment of relative contribution of DNA reparation and glutathione-dependent pathway of detoxification in response of Chlorella algae to uranium].

Toxicity of 238U (as uranyl nitrate) in the range of 0.04-84 micromol/L for Chlorella (Chlorella vulgaris Beijerink) was investigated. The best approximation for relationship between the toxic effect in Chlorella and 238U Concentrations is observed using the hormetic Brain-Cousens model. A significant increase in Chlorella biomass, estimated as the optical density of suspension, as well as the level of fluorescence of chlorophyll was observed in the range of 17-29 micromol/L with the maximum at a 23 micromol/L. It was found that 38 micromol/L of 238U induced a significant toxic effect; while at 53 micromol/L inhibition of Chlorella biomass by 50% was observed. According to our observations, the toxic effect of low concentrations of 238U was increased in the presence of 0.02 micromol/L caffeine (used as inhibitor of DNA repair processes) or DL-buthionine-(S, R)-sulfoximine (used as a selective inhibitor of the key glutathione biosynthetic pathway).

Chloroplast NADPH-dependent thioredoxin reductase from Chlorella vulgaris alleviates environmental stresses in yeast together with 2-Cys peroxiredoxin.

Chloroplast NADPH-dependent thioredoxin reductase (NTRC) catalyzes the reduction of 2-Cys peroxiredoxin (2-Cys Prx) and, thus, probably functions as an antioxidant system. The functions of the enzyme in oxidative and salt stresses have been reported previously. We have previously identified and characterized NTRC in Chlorella vulgaris. In the present study, we isolated a full-length cDNA clone encoding 2-Cys Prx from C. vulgaris and investigated the involvement of Chlorella NTRC/2-Cys Prx system in several environmental stress tolerances by using yeast as a eukaryotic model. Deduced Chlorella 2-Cys Prx was homologous to those of chloroplast 2-Cys Prxs from plants, and two conserved cysteine residues were found in the deduced sequence. Enzyme assay showed that recombinant mature C. vulgaris NTRC (mCvNTRC) transferred electrons from NADPH to recombinant mature C. vulgaris 2-Cys Prx (mCvPrx), and mCvPrx decomposed hydrogen peroxide, tert-butyl hydroperoxide, and peroxynitrite by cooperating with mCvNTRC. Based on the results, the mCvNTRC/mCvPrx antioxidant system was identified in Chlorella. The antioxidant system genes were expressed in yeast separately or coordinately. Stress tolerances of yeast against freezing, heat, and menadione-induced oxidative stresses were significantly improved by expression of mCvNTRC, and the elevated tolerances were more significant when both mCvNTRC and mCvPrx were co-expressed. Our results reveal a novel feature of NTRC: it functions as an antioxidant system with 2-Cys Prx in freezing and heat stress tolerances.

Biological hydrogen production by the algal biomass Chlorella vulgaris MSU 01 strain isolated from pond sediment.

Chlorella vulgaris MSU 01 strain isolated from the sediment of the pond is able to produce molecular hydrogen in a clean way. To relate the dynamic coupling between the cultural conditions and biological responses, an original lab scale set up has been developed for hydrogen production. Different sources like mannitol, glucose, alanine, citric acid, aspartic acid, l-alanine, l-cysteine, sodium succinate and sodium pyruvate were used for algal media optimization. Corn stalk, from 1 to 5 g/L was tested for the effective algal growth and hydrogen production. The cell concentration of 1.6-19 g/L dry cell weight (DCW) was found at the 10th day. The kinetic parameters involved in the hydrogen production at 4 g/L corn stalk using the algal inoculum (50 mL) in the bioreactor volume (500 mL) was found to be with the hydrogen production potential (P(s)) of 7.784 mL and production yield of (P(r)) 5.534 mL respectively. The growth profile of the algal biomass at the above mentioned condition expressed the logistic model with R(2) 0.9988. The final pH of the broth was increased from 7.0 to 8.5-8.7. The anaerobic fermentation by C. vulgaris MSU 01 strain involved in the conversion process of complex carbon source has increased the H(2) evolution rate and higher butyrate concentration in the fermentate.

Photoperiod and temperature influence cadmium's effects on photosynthesis-related gene transcription in Chlorella vulgaris.

Routine metal compound toxicity tests are performed at one constant photoperiod and temperature. There is little knowledge about the interactions between metal compound toxicity and photoperiod or temperature. The purpose of this study was to analyze the effects of photoperiod and temperature on cadmium (Cd) toxicity in the fresh alga, Chlorella vulgaris, both singly and in combination. Exposure to 2 or 4 microM Cd alone significantly decreased the transcription of the photosynthesis-related genes psbA, psaB and rbcL. Three-way ANOVA analysis showed that both temperature and photoperiod interacted with the dosage of Cd to influence the abundance of psbA and psaB, but not rbcL. Specifically, psbA transcription was more sensitive to Cd under long photoperiods or high temperatures than under short photoperiods or low temperatures. Because photoperiod and temperature have certain impacts on the toxicity of metal compounds, these two environmental factors should be given more attention in laboratory research.

Strategies to enhance cell growth and achieve high-level oil production of a Chlorella vulgaris isolate.

The autotrophic growth of an oil-rich indigenous microalgal isolate, identified as Chlorella vulgaris C--C, was promoted by using engineering strategies to obtain the microalgal oil for biodiesel synthesis. Illumination with a light/dark cycle of 14/10 (i.e., 14 h light-on and 10 h light-off) resulted in a high overall oil production rate (v(oil)) of 9.78 mg/L/day and a high electricity conversion efficiency (E(c)) of 23.7 mg cell/kw h. When using a NaHCO(3) concentration of 1,500 mg/L as carbon source, the v(oil) and E(c) were maximal at 100 mg/L/day and 128 mg/kw h, respectively. A Monod type model was used to describe the microalgal growth kinetics with an estimated maximum specific growth rate (mu(max)) of 0.605 day(-1) and a half saturation coefficient (K(s)) of 124.9 mg/L. An optimal nitrogen source (KNO(3)) concentration of 625 mg/L could further enhance the microalgal biomass and oil production, leading to a nearly 6.19 fold increase in v(oil) value.

[The mechanisms of 232Th and of Ce (III) toxic action on Chlorella vulgaris Beijer. Estimation of 232Th radiation impact contribution into toxic effect induced].

232Th and Ce (III) toxic effects and its modifications with caffeine and D,L-buthionine-(S,R)-sulphoximine on Chlorella vulgaris Beijer were studied using an optical density measure after 24 hours growth. Concentrations of 232Th or of Ce--toxic effect relationship were shown to be nonlinear. In the first (nontoxic) concentration range (for Ce 0.036-1.642 micromol/L and for 232Th 0.001-1.551 micromol/L) algae biomass production registered by optical density do not significantly differ from the control one. In the second (toxic) concentration range dose-effect relationship for 232Th is characterized with quadratic dependence and in the case of Ce--with exponential dependence. 232Th radiation component contribution into effect observed is appeared as induction of DNA damages additional to spontaneous at the radionuclide concentration (equal to 0.345 micromol/L) that is three times lower than in case of the non-radioactive chemical analog Ce (1.071 micromol/L).