The effect of PK gene overexpression on content and antioxidant properties of carotenoids in marine microalga Dunaliella parva.

Dunaliella parva can extensively accumulate carotenoids, which is a promising raw material for carotenoids production. Carotenoids have important medicinal value. D. parva is an ideal organism for studying the mechanism of carotenoid synthesis. Our previous study identified a transcription factor DpAP2 which could regulate carotenoid synthesis in D. parva. In addition, DpAP2 could interact with three proteins with different activities (DNA binding transcription factor activity, protein kinase activity, and alpha-D-phosphohexomutase). To investigate the function of PK gene encoding interacting protein of DpAP2 with protein kinase activity in D. parva, PK gene was cloned into vector pBI221-GFP-UbiΩ-CAT and transformed into D. parva in this study. The results showed that overexpression of PK gene enhanced the contents of carotenoids, total sugars, proteins, and antioxidant activities of carotenoid extract such as superoxide radical scavenging activity, reducing power, hydroxyl radical scavenging activity in transgenic D. parva with overexpression of PK gene. This study explored the function of PK gene, and improved the medicinal value of D. parva.

Physiological changes of microalga Dunaliella parva under the treatment of PEG, CaCl2.

Carotenoids are antioxidants, which reduce various chronic diseases of human, and have many industrial applications. The halophilic Dunaliella parva (D. parva) is rich in carotenoids. The compounds CaCl2 and PEG are the popular metabolic enhancers. To further enhance carotenogenesis, D. parva was treated with two compounds polyethylene glycol (PEG) and CaCl2. Application of CaCl2 and PEG enhanced the carotenoids contents and the antioxidant activities of carotenoids compared to control group (no treatment of CaCl2 or PEG). The highest carotenoids contents were obtained by treating D. parva with 40 ppm CaCl2 (3.11 mg/g dry weight, DW) and 80 ppm PEG (2.78 mg/g DW) compared with control group (1.96 mg/g DW). When D. parva was treated with 40 ppm CaCl2 and 80 ppm PEG, protein contents reached the highest values (90.28 mg/g DW and 89.57 mg/g DW) compared to that of control group (73.42 mg/g DW). The antioxidant activities of carotenoids samples were determined. Generally, the antioxidant activities of carotenoids from D. parva treated with PEG and CaCl2 were superior to that of control group. The antioxidant activities of carotenoids mainly contained reducing power, hydroxyl radical scavenging activity and superoxide radical scavenging activity. The reducing powers of carotenoids extracts from 20 ppm CaCl2 group (2.07%/mg carotenoids) and 120 ppm PEG group (1.59%/mg carotenoids) were significantly higher than that of control group (<1.25%/mg carotenoids). The superoxide radical scavenging activities of carotenoids extracts from 40 ppm CaCl2 group (70.33%/mg carotenoids) and 80 ppm PEG group (65.94%/mg carotenoids) were significantly higher than that of control group (<55%/mg carotenoids). This paper laid a foundation for massive accumulation of carotenoids in microalga D. parva.

Optimization of Molasses and Soybean Meal Content to Enhance Tetramethylpyrazine Yield by Bacillus sp. TTMP20.

Microbial fermentation for the production of tetramethylpyrazine (TTMP) is considered to be the most promising method, and the development of a cheap fermentation substrate is of great importance for large-scale TTMP production. In this study, inexpensive by-products from the food industry, i.e., molasses and soybean meal (instead of glucose and tryptone), were used as substrates for TTMP fermentation. The pretreatment of soybean meal was explored in order to achieve a better fermentation effect. The contents of each component in the fermentation medium were optimized by central composite design (CCD). The optimum contents were as follows: 72.5 g/L of molasses, 37.4 g/L of diammonium hydrogen phosphate (DAP), 53.4 g/L of soybean meal, and 5 g/L of yeast powder. The software predicted a maximum TTMP yield of 1469.03 mg/L, and the actual TTMP yield was 1328.95 mg/L for the validation experiment in the optimum medium. Under the optimum conditions (72.5 g/L of molasses, 37.4 g/L of DAP, 53.4 g/L of soybean meal, and 5 g/L of yeast powder), the actual maximum TTMP yield (1328.95 mg/L) in this study was much higher than the TTMP yield (895.13 mg/L) under the conditions (150 g/L of molasses, 30 g/L of DAP, 30 g/L of tryptone, and 10 g/L of yeast powder) of our previous study published in Molecules. In this study, the TTMP yield improved by 48.46%, with decreased molasses (more than half), decreased yeast powder (half) and by-product soybean meal instead of tryptone compared to our previous study. In summary, the cheaper fermentation medium had a higher TTMP yield in this study, which improves the application potential of Bacillus sp. TTMP20.

Production of Tetramethylpyrazine from Cane Molasses by Bacillus sp. TTMP20.

2,3,5,6-Tetramethylpyrazine (TTMP) is an active ingredient of Ligusticum wallichii Franch. It can be used in medicine and food fields. In this study, Bacillus sp. TTMP20 was applied to produce TTMP using cane molasses as a carbon source. After pretreatment with phosphoric acid, 170 mL/L treated molasses, combined with 10 g/L yeast powder, 30 g/L tryptone and 30 g/L (NH4)2HPO4 were used for fermentation. After 36 h, TTMP output reached the highest value of 208.8 mg/L. The yield of TTMP using phosphoric acid-treated molasses as carbon source was 145.59% higher than control. Under the sulfuric acid treatment process of molasses (150 g), the maximum yield of TTMP was 895.13 mg/L, which was 183.18% higher than that of untreated molasses (316.1 mg/L). This study demonstrated that molasses is a high-quality and inexpensive carbon source for the manufacture of TTMP, laying the groundwork for the future industrial production of TTMP.

Identification of differentially expressed genes for Pseudomonas sp. Cr13 stimulated by hexavalent chromium.

Over exploitation of mineral resources has increasingly caused serious heavy metal contamination such as chromium (Cr). Cr(VI), the pathogenicity factor, is one of common environmental contaminants and widely known health hazards to living organisms. Therefore, it is urgent to control the polluted soil. Up to now, little is known about the regulatory mechanisms of Cr response in Pseudomonas sp. Cr13. In this study, transcriptome and differentially expressed genes in Pseudomonas sp. Cr13 strain was characterized by a comparison between Cr(VI)-treated sample and control sample using transcriptome sequencing approach. In total, 2974 genes were annotated, including 1245 (1154 down-regulated genes and 91 up-regulated genes) differentially expressed genes (DEGs). All DEGs could be assigned to 29 pathways, of which pathways related to amino acid metabolism, carbohydrate metabolism, energy metabolism and signal transduction mechanism were significantly enriched in Pseudomonas sp. Cr13. A possible mechanism for Cr toxicity response might be an active efflux which utilized a heavy metal translocating P-type ATPase to lower the intracellular Cr concentration. The down-regulated genes related to the antioxidant defense system had a key role in Cr reduction, such as SodA, Gst, osmC, BtuE, KatE, csdA and AhpC. The proteins that were visibly up-regulated, were likely to involve in alleviating Cr(VI) stress, and the significantly down-regulated genes such as MarR, Lrp, FhlA, GntR, HrcA, LysR family genes, were likely to reduce Cr(VI) induced oxidative stress. In addition, real-time quantitative PCR was used to analyze the expression patterns of some Cr responsive genes. This study reported the first identification of Cr responsive genes, and inferred the underlying regulatory mechanisms of response to Cr(VI) stress in Pseudomonas sp. Cr13.

Optimization of carotenoid extraction of a halophilic microalgae.

Dunaliella parva can produce abundant carotenoids under certain conditions. This paper optimized the extraction efficiency of carotenoids from D. parva. Different organic solvents were examined to determine the most suitable solvent for the extraction. After the determination of the solvent (dimethylsulfoxide, DMSO), the extraction conditions including time, temperature, and volume were then optimized to maximize the extraction efficiency of carotenoids from D. parva using response surface methodology. DMSO was identified as the most suitable solvent. The optimal extraction conditions were as follows: temperature of 57.2°C, time of 11.35 min, the volume of 410 µl, and the optimal extraction efficiency reached 0.517‰. The results showed that the optimal extraction efficiency (0.517‰) improved 31.69% in comparison to the initial extraction efficiency (0.3926‰). In addition, The optimal levels of three influence factors (temperature of 57.2°C, time of 11.35 min, volume of 410 µl) decreased compared with the initial levels (temperature of 60°C, time of 20 min, volume of 1000 µl). In this paper, Central Composite Design (CCD) was used to optimize the extraction efficiency of carotenoids from D. parva, which would lay the groundwork for the extraction and utilization of carotenoids from D. parva in the future.

Analysis of Bacterial Diversity in Different Types of Daqu and Fermented Grains From Danquan Distillery.

Bacterial communities in high-temperature Daqu and fermented grains are important for brewing Jiang-flavor Baijiu such as Danquan Baijiu. Daqu is a saccharifying and fermenting agent, which has a significant impact on the flavor of Baijiu. However, bacterial communities in three different types of samples from the Danquan distillery (dqjq_ck, dqjqcp, and dqjp3) were still unclear, which limited further development of Danquan Baijiu. "dqjq_ck" and "dqjqcp" indicate high-temperature Daqu at days 45 and 135, respectively. "dqjp3" indicates fermented grains. In this study, the bacterial communities of three samples were analyzed by Illumina Miseq high-throughput sequencing. The bacterial communities of three samples primarily composed of thermophilic bacteria and bacteria with stress resistance. The most abundant species in dqjq_ck, dqjqcp, and dqjp3 were Comamonas, Bacillus, and unclassified Lactobacillales, respectively. The main bacteria included Bacillus, Comamonas, Myroides, Paenibacillus, Acetobacter, Kroppenstedtia, Staphylococcus, Saccharopolyspora, Planifilum, Lactobacillus, Acinetobacter, Oceanobacillus, Enterococcus, Thermoactinomyces, Lactococcus, Streptomyces, Saccharomonospora, Tepidimicrobium, Anaerosalibacter, unclassified_Lactobacillales, unclassified_Thermoactinomycetaceae_1, unclassified_Bacillaceae_2, unclassified_Bacillales, unclassified_Microbacteriaceae, unclassified_Rhodobacteraceae, unclassified_Actinopolysporineae, and unclassified_Flavobacteriaceae in three samples (percentage was more than 1% in one of three samples). In our study, the succession of microbiota in three samples representing three important stages of Danquan Baijiu brewing was revealed. This article lays a good foundation for understanding the fermentation mechanism and screening some excellent indigenous bacteria to improve the quality of Danquan Baijiu in future.

Optimization of Extraction Conditions of Carotenoids from Dunaliella parva by Response Surface Methodology.

Extraction conditions can exert a remarkable influence on extraction efficiency. The aim of this study was to improve the extraction efficiency of carotenoids from Dunaliella parva (D. parva). Dimethyl sulfoxide (DMSO) and 95% ethanol were used as the extraction solvents. The extraction time, extraction temperature and the proportions of mixed solvent were taken as influencing factors, and the experimental scheme was determined by Central Composite Design (CCD) of Design Expert 10.0.4.0 to optimize the extraction process of carotenoids from D. parva. The absorbance values of the extract at 665 nm, 649 nm and 480 nm were determined by a microplate spectrophotometer, and the extraction efficiency of carotenoids was calculated. Analyses of the model fitting degree, variance and interaction term 3D surface were performed by response surface analysis. The optimal extraction conditions were as follows: extraction time of 20 min, extraction temperature of 40 °C, and a mixed solvent ratio (DMSO: 95% ethanol) of 3.64:1. Under the optimal conditions, the actual extraction efficiency of carotenoids was 0.0464%, which was increased by 18.19% (the initial extraction efficiency of 0.03926%) with a lower extraction temperature (i.e., lower energy consumption) compared to the standard protocol.

Anti-Inflammatory Effects of an Extract from Pseudomonas aeruginosa and Its Purified Product 1-Hydroxyphenazine on RAW264.7 Cells.

The purpose of this study was to discuss the effects of an extract from the culture medium of Pseudomonas aeruginosa (P. aeruginosa) 2016NX1 (chloroform extract of P. aeruginosa, CEPA) and its purified product 1-hydroxyphenazine on RAW264.7 cell inflammation. Cell viability was evaluated by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) method. TNF-α production was determined by an ELISA method. The effects of CEPA and its purified product 1-hydroxyphenazine on cell morphology were investigated using an inverted microscope. Quantitative real-time PCR was performed to determine mRNA expression levels. CEPA and 1-hydroxyphenazine had no obvious toxicity to cells when their concentrations were no more than 20 µg ml-1 and 5 µg ml-1, respectively. Both CEPA and 1-hydroxyphenazine suppressed the secretion of TNF-α and significantly reduced the mRNA expression levels of TNF-α, IL-1ß, and IL-6. Both CEPA and 1-hydroxyphenazine inhibited M1 cell polarization after lipopolysaccharide (LPS) stimulation. The results in this article lay a good foundation for the biopharmaceutical applications of CEPA and 1-hydroxyphenazine in the future. CEPA and 1-hydroxyphenazine had certain anti-inflammatory activity, and inhibited LPS-induced RAW264.7 cell inflammation. Our findings suggest that CEPA and 1-hydroxyphenazine are potential chemicals with anti-inflammatory activity.

Cloning and Characterization of phzR Gene from Pseudomonas aeruginosa.

A gene encoding phzR was isolated from a phenazine-producing bacterium Pseudomonas aeruginosa 2016NX1. This paper provided the full-length cDNA encoding phzR (GenBank Accession no., MW143078). The cDNA of the phzR contained an open reading frame (ORF) of 714 bp. The potential regulatory elements were predicted in the phzR promoter region. The deduced amino acid sequence of P. aeruginosa phzR showed significant homology to the known phzRs from different organisms. Gene overexpression analysis showed that the phenazine content was improved (44.39%) in comparison to wild-type 2016NX1.

Optimization of Growth Conditions of Acinetobacter sp. Cr1 for Removal of Heavy Metal Cr Using Central Composite Design.

Growth conditions can significantly affect the removal efficiency of heavy metals by microorganisms. The goal of this study was enhancing the removal efficiency of Cr(VI) and improving the application of Acinetobacter sp. Cr1 (GenBank accession number of 16S rDNA sequence, MN900681). This study focused on pH, Cr(VI) concentration and culture time, which were the major influence factors for removal efficiency of Cr(VI). A central composite design was employed to optimize the removal efficiency by optimizing three variables. The optimum growth conditions were as: pH of 9.52, Cr(VI) concentration of 128.55 mg l-1, culture time of 43.30 h, and the predicted and actual maxima were 65.13% and 67.26%, respectively. Therefore, it is suggested that the strain Acinetobacter sp. Cr1 had a promising potential to be used for bioremediation of Cr(VI).

Isolation and Characterization of Pseudomonas sp. Cr13 and Its Application in Removal of Heavy Metal Chromium.

The purpose of this study was to elaborate the characteristics of Pseudomonas sp. Cr13, including physiological and biochemical characteristics, optimization of growth conditions, minimum inhibitory concentration of Cr6+ and resistance to other heavy metals, removal efficiency of Cr6+, and antibiotics sensitivity. A strain Pseudomonas sp. Cr13 was screened from mine-contaminated soils, which could tolerate high concentration of Cr6+ (up to 250 mg l-1) and Cd2+ (50 mg l-1). The optimum pH, NaCl concentration, and temperature for growth were 6, 10% NaCl, and 30 °C, respectively. The removal efficiency of Cr6+ by strain Pseudomonas sp. Cr13 was studied. The removal efficiency of Cr6+ decreased with the increased concentration of Cr6+. Under the optimal conditions, the maximum of the removal rate can reach up to 94.26% in contaminated soils. In addition, antibiotics sensitivity of this strain was investigated. It was found that this strain was sensitive to nine types of antibiotics, which would lay a good foundation for the choice of selective marker in genetic engineering modification of this strain. The results in this article would lay a good foundation for the bioremediation of heavy metals pollution in the future. Pseudomonas sp. Cr13 can tolerate high concentration of Cr6+ and partially remove Cr6+, which make Cr13 an attractive option for the bioremediation of heavy metal chromium (Cr). Our findings suggest that Pseudomonas sp. Cr13 is a potential bacterium with the ability of bioremediation of heavy metal Cr.

Analysis of Fungal Composition in Mine-Contaminated Soils in Hechi City.

Fungi play an important role in bioremediation of contaminated soil. However, the diversity of fungal populations in four mine-contaminated soils located in Hechi City has remained unexplored. In this study, high-throughput sequencing of ITS was performed to investigate the diversity and abundance of fungal communities in four mine-contaminated soils in Hechi city. Phylogenetic taxonomy showed that the fungal communities included five phyla. Ascomycota and Basidiomycota were the most abundant phyla in four samples. The most abundant fungi included Agaricomycetes, Nectriaceae, Eurotiomycetes, Mortierellaceae, Incertae sedis, Trichocomaceae, Sordariomycetes, and Fusarium. Various fungi with the potential of bioremediation and industrial application were discussed. The results of fungal composition will provide a clue for isolation of new fungi with the potential of bioremediation and industrial application. Furthermore, this study will lay a good foundation for modifying the indigenous fungi by genetic engineering in the future.

Performance of a microalgal-bacterial consortium system for the treatment of dairy-derived liquid digestate and biomass production.

To enhance the treatment performance of dairy-derived liquid digestate (DLD) using microalgal-bacterial consortium system composed of Chlorella vulgaris and indigenous bacteria (CV), activated sludge was introduced to form a new microalgal-bacterial consortium system (Co-culture). The activated sludge shortened the lag phase and increased the specific growth rate of C. vulgaris (0.56 d-1). The biomass yield in the Co-culture was 2.72 g L-1, which was lower than that in the CV (3.24 g L-1), but the Co-culture had an improved COD (chemical oxygen demand) removal (25.26%) compared to the CV (13.59%). Quantitative PCR and metagenomic analyses demonstrated that microalgae also promoted bacterial growth, but influenced differently on the bacterial communities of indigenous bacteria and activated sludge. Compared with indigenous bacteria, activated sludge was more prone to forming a favorable symbiosis with C. vulgaris. These findings contribute to the construction of efficient microalgal-bacterial consortium system in wastewater treatment.

Cultivation of Chlorella vulgaris on unsterilized dairy-derived liquid digestate for simultaneous biofuels feedstock production and pollutant removal.

In order to assess viability of microalgae cultivation using unsterilized dairy-derived liquid digestate (DLD) for simultaneous biofuels feedstock production and contaminant removal, four DLD concentrations (25%, 50%, 75% and 100%) were used to grow Chlorella vulgaris in batch photobioreactors (PBRs). The 25% DLD was an ideal alternative medium in that high growth rate (0.69 d-1), high lipid productivity (112.9 mg L-1 d-1) as well as high nutrient removal were attained. The high DLD concentration caused inhibition of microalgal growth, where COD was more inhibitive than ammonium. The presence of bacteria did not influence microalgae production because of limited growth. Microalgal growth reduced the richness and diversity of bacterial community. Furthermore, the species of Bacteroidetes, Candidatus Saccharibacteria, and Chlamydiae rather than Proteobacteria benefited microalgal-bacterial symbiosis. These findings contribute to better application of microalgal-bacterial system for large-scale microalgae cultivation as well as environmental sustainability.

Treatment of low C/N ratio wastewater and biomass production using co-culture of Chlorella vulgaris and activated sludge in a batch photobioreactor.

The aim of this work was to study the performance of pollutants removal and biomass production by co-culture of Chlorella vulgaris and activated sludge in a batch photobioreactor (PBR), compared with their single system to treat a low C/N ratio (COD/N = 4.3) wastewater. The co-culture system surpassed activated sludge system in terms of nutrients removal and outperformed microalgae alone system in regard to COD removal. Biomass productivity of the co-culture system was 343.3 mg L-1 d-1, and the harvested biomass could be developed as biofuels, animal feeds or soil conditioners due to the improved calorific value and cellular composition compared with activated sludge. The low C/N ratio wastewater enabled bacteria to maintain a relatively low level, hence in favor of microalgae enrichment and nutrient recovery.

Outdoor Growth Characterization of an Unknown Microalga Screened from Contaminated Chlorella Culture.

Outdoor microalgae cultivation process is threatened by many issues, such as pest pollution and complex, changeable weather. Therefore, it is difficult to have identical growth rate for the microalgae cells and to keep their continuous growth. Outdoor cultivation requires the algae strains not only to have a strong ability to accumulate oil, but also to adapt to the complicated external environment. Using 18S rRNA technology, one wild strain Scenedesmus sp. FS was isolated and identified from the culture of Chlorella zofingiensis. Upon contamination by Scenedesmus sp., the species could quickly replace Chlorella zofingiensis G1 and occupy ecological niche in the outdoor column photobioreactors. The results indicated that Scenedesmus sp. FS showed high alkali resistance. It also showed that even under the condition of a low inoculum rate (OD680, 0.08), Scenedesmus sp. FS could still grow in the outdoor raceway pond under a high alkaline environment. Even under unoptimized conditions, the oil content of Scenedesmus sp. FS could reach more than 22% and C16-C18 content could reach up to 79.68%, showing that this species has the potential for the biodiesel production in the near future.

Luxury uptake of phosphorus changes the accumulation of starch and lipid in Chlorella sp. under nitrogen depletion.

The aim of this research was to study the effect of phosphorus supply on starch and lipid production under nitrogen starvation using Chlorella sp. as a model. High phosphate level had marginal effect on cell density but increased biomass growth. Massive phosphorus was assimilated quickly and mainly stored in the form of polyphosphate. The algal cells ceased phosphorus uptake when intracellular phosphorus reached a certain level. 5mM phosphate in the culture rendered a 16.7% decrease of starch synthesis and a 22.4% increase of lipid synthesis relative to low phosphate (0.17 mM). It is plausible that phosphate can regulate carbon partitioning between starch and lipid synthesis pathway by influencing ADP-glucose pyrophosphorylase activity. Moreover, high phosphate concentration enhanced the abundance of oleic acid, improving oil quality for biodiesel production. It is a promising cultivation strategy by integration of phosphorus removal from wastewater with biodiesel production for this alga.

Characterization of lipid and fatty acids composition of Chlorella zofingiensis in response to nitrogen starvation.

Cellular biochemical composition of the microalga Chlorella zofingiensis was studied under favorable and nitrogen starvation conditions, with special emphasis on lipid classes and fatty acids distribution. When algal cells were grown in nitrogen-free medium (N stress), the increase in the contents of lipid and carbohydrate while a decrease in protein content was detected. Glycolipids were the major lipid fraction (50.7% of total lipids) under control condition, while neutral lipids increased to be predominant (86.7% of total lipids) under N stress condition. Triacylglycerol (TAG) content in N stressed cells was 27.3% dw, which was over three times higher than that obtained under control condition. Within neutral lipids fraction, monounsaturated fatty acids (MUFA) were the main group (40.6%) upon N stress, in which oleic acid was the most representative fatty acids (34.5%). Contrarily, glycolipids and phospholipids showed a higher percentage of polyunsaturated fatty acids (PUFA). Lipid quality assessment indicated the potential of this alga as a biodiesel feedstock when its neutral lipids were a principal lipid fraction. The results demonstrate that the neutral lipids content is key to determine the suitability of the microalga for biodiesel, and the stress cultivation is essential for lipid quality.

Growth and lipid accumulation characteristics of Scenedesmus obliquus in semi-continuous cultivation outdoors for biodiesel feedstock production.

In an effort to identify suitable microalgal species for biodiesel production, seven species were isolated from various habitats and their growth characteristics were compared. The results demonstrated that a green alga Scenedesmus obliquus could grow more rapidly and synthesize more lipids than other six microalgal strains. S. obliquus grew well both indoors and outdoors, and reached higher µmax indoors than that outdoors. However, the cells achieved higher dry weight (4.36 g L(-1)), lipid content (49.6%) and productivity (183 mg L(-1) day(-1)) outdoors than in indoor cultures. During the 61 days semi-continuous cultivation outdoors, high biomass productivities (450-550 mg L(-1) day(-1)) and µmax (1.05-1.44 day(-1)) were obtained. The cells could also achieve high lipid productivities (151-193 mg L(-1) day(-1)). These results indicated that S. obliquus was promising for lipids production in semi-continuous cultivation outdoors.