Application of mathematical models to the determination optimal glucose concentration and light intensity for mixotrophic culture of Spirulina platensis

The effects of initial glucose concentration and light intensity on specific growth rate, phycocyanin concentration and cell dry weight concentration in mixotrophic batch cultivation of Spirulina platensis using both shake flask and fermenter were investigated. Based on experimental results in shake flask culture, a number of mathematical models were constructed, and the optimal initial glucose concentration and the optimal light intensity were calculated to be S_opt=2.4471 g liter⁻¹ and L_opt=3.8632 klx. Finally, a time-dependent kinetic model for mixotrophic batch cultivation of Spirulina platensis in fermenter was also proposed. This was in good agreement with the experimental results and could be employed to predict the production of biomass and phycocyanin, and the consumption of glucose in fermenter culture.     

Simultaneous cultivation of Spirulina platensis and the toxigenic cyanobacteria Microcystis aeruginosa

Mangueira Lagoon, located in the extreme south of Brazil, has water with physicochemical characteristics such as alkaline pH and carbonate levels propitious for the growth of the cyanobacterium Spirulina platensis. Previously published studies have shown that Mangueira Lagoon water supplemented with small quantities of carbon and nitrogen is suitable for S. platensis cultivation and can significantly reduce production costs. We studied mixed cultures of Spirulina platensis and the toxic cyanobacterium Microcystis aeruginosa using a 2(3) factorial design in which the three factors were the initial biomass concentration of S. platensis and M. aeruginosa and the type of culture medium (100% Zarrouk's medium or 80% Mangueira Lagoon water plus 20% Zarrouk's medium). The highest S. platensis maximum specific growth rate (mu(max)) occurred in the culture with the highest M. aeruginosa biomass concentration and when undiluted culture medium was used (micro(max) = 0.283 d(-1)). The highest M. aeruginosa specific death rate (k) was obtained in the presence of S. platensis (k = 0.555 d(-1)) and was independent of the initial M. aeruginosa biomass concentration and culture medium, demonstrating that S. platensis cultures are not susceptible to contamination by M. aeruginosa. The culture medium had no significant influence (p > 0.05) on S. platensis micro(max) values, indicating that production costs could be reduced by using a medium consisting of 80% Mangueira Lagoon water plus 20% Zarrouk's medium.     

Bioeffects of selenite on the growth of Spirulina platensis and its biotransformation

The bioeffects of selenium on the growth of Spirulina platensis and the selenium distribution were investigated. S. platensis was batch cultured in Zarrouk medium containing increasing concentrations of sodium selenite. The biotransformation characteristic of selenium was analysed by the determination of the detailed selenium distribution forms. At 35 degrees C, 315.2 microEm(-2) x s(-1), sodium selenite concentrations below 400 mg x l(-1) were found to stimulate algal growth, especially in the range of 0.5-40 mg x l(-1). However, above 500 mg x l(-1) sodium selenite was toxic to this alga with the toxicity being related to the sulfite level in the medium. S. platensis was found to resist higher selenite by reducing toxic Se(IV) to nonsoluble Se(0). Selenium was accumulated efficiently in S. platensis during cultivation with accumulated selenium increasing with selenite concentration in the medium. It was demonstrated that inorganic selenite could be transformed into organic forms through binding with protein, lipids and polysaccharides and other cell components. The organic selenium accounted for 85.1% of the total accumulated selenium and was comprised of 25.2% water-soluble protein-bound, 10.6% lipids-bound and 2.1% polysaccharides-bound selenium. Among the organic fractions lipid possessed the strongest ability to accumulate Se (6.47 mg x kg(-1)). The 14.9% inorganic selenium in S. platensis was composed of Se(IV) (13.7%) and Se(VI) (1.2%).     

The production of biomass and phycobiliprotein pigments in filamentous cyanobacteria: the impact of light and carbon sources

Cyanobacteria are recognized as producers of bioactive substances and phycobiliproteins, whose medicinal and functional food properties have led to increased interest in recent years. In the present study, the biomass production and phycobiliprotein content in cyanobacterial strains belonging to Anabaena, Nostoc and Spirulina genera were investigated under the conditions of continuous illumination and mixotrophic nutrition. The results showed that biomass production was strongly stimulated by continuous light in Spirulina strains (4.5-fold), and by organic carbon sources in N₂-fixing strains (2.1–2.8-fold). The strategy of cells to accumulate primarily blue pigment phycocyanin and bluish green allophycocyanin was revealed under tested conditions. Furthermore, in the case of Spirulina S1 grown with glycerol, the culture medium became dense and changed its colour to pink, which may indicate the release of compounds including pigment(s) outside the cell, the phenomenon that seem to be rare among cyanobacteria. Moreover, under continuous light, in this strain the highest biomass level of 4.0 mg/mL was achieved, wherein phycocyanin and allophycocyanin content was increased 12- and 16-fold, respectively, which indicates the high potential of this strain for further investigation.     

Optimization of phycocyanin extraction from Spirulina platensis using factorial design

Phycocyanin extraction from cyanobacteria Spirulina platensis was optimized using factorial design and response surface techniques. The effects of temperature and biomass-solvent ratio on phycocyanin concentration and extract purity were evaluated to determine the optimum conditions for phycocyanin extraction. The optimum conditions for the extraction of phycocyanin from S. platensis were the highest biomass-solvent ratio, 0.08 gmL(-1), and 25 degrees C. Under these conditions it's possible to obtain an extract of phycocyanin with a concentration of 3.68 mgmL(-1) and purity ratio (A(615)/A(280)) of 0.46.     

Heterotrophic high cell-density fed-batch cultures of the phycocyanin-producing red alga Galdieria sulphuraria

Growth and phycocyanin production in batch and fed-batch cultures of the microalga Galdieria sulphuraria 074G, which was grown heterotrophically in darkness on glucose, fructose, sucrose, and sugar beet molasses, was investigated. In batch cultures, specific growth rates and yields of biomass dry weight on the pure sugars were 1.08-1.15 day-1 and 0.48-0.50 g g-1, respectively. They were slightly higher when molasses was the carbon source. Cellular phycocyanin contents during the exponential growth phase were 3-4 mg g-1 in dry weight. G. sulphuraria was able to tolerate concentrations of glucose and fructose of up to 166 g L-1 (0.9 M) and an ammonium sulfate concentration of 22 g L-1 (0.17 M) without negative effects on the specific growth rate. When the total concentration of dissolved substances in the growth medium exceeded 1-2 M, growth was completely inhibited. In carbon-limited fed-batch cultures, biomass dry weight concentrations of 80-120 g L-1 were obtained while phycocyanin accumulated to concentrations between 250 and 400 mg L-1. These results demonstrate that G. sulphuraria is well suited for growth in heterotrophic cultures at very high cell densities, and that such cultures produce significant amounts of phycocyanin. Furthermore, the productivity of phycocyanin in the heterotrophic fed-batch cultures of G. sulphuraria was higher than is attained in outdoor cultures of Spirulina platensis, where phycocyanin is presently obtained.     

响应面法优化普通小球藻混合营养培养基组成生产生物质

利用响应面法优化了混合营养培养普通小球藻生产生物质的培养基组成.首先采用Plackett-Burman设计对11个相关营养因素的效应进行了评价,并筛选出影响小球藻细胞生长的3个主要因素为KNO3、葡萄糖和NaC1;然后结合Box-Behnken设计建立了以小球藻浓度为响应值的二次回归方程模型,获得优化的培养基组成为KNO31.64g/L、葡萄糖45g/L、NaC1 1.57g/L;模型预测的最大浓度为5.28g/L,验证值为5.68g/L;验证结果表明,所建立模型预测精度较好,可用于优化小球藻的混养培养基组成.优化条件下混养小球藻细胞的蛋白质和色素含量较优化前降低,而可溶性糖和油脂含量提高,脂肪酸以棕榈酸和油酸为主;细胞组分分析结果显示,混养培养所得小球藻生物质具有作为生产微藻生物能源原料的潜力.     

A two-stage cultivation process for the growth enhancement of Chlorella vulgaris

This study proposes a two-stage cultivation process with an autotrophic growth followed by a mixotrophic process. The results indicated that a two-stage cultivation process using a daily dose of 3 g/L of glucose could achieve 7.4 g/L of biomass, which was about a 64 % increase over simple autotrophic cultivation. In the second stage of mixotrophic cultivation, glucose was regarded as a better carbon source for cell growth, than was glycerol. Linoleic acid (C18:2) would be the primary component in the two-stage cultivation as in the autotrophic cultivation. Even carbon source was provided in the second stage of mixotrophic cultivation; lower light intensity limited the mixotrophic growth, which indicated that photosynthesis still plays an important role in the second stage of mixotrophical cultivation. The final biomass was higher after this two-stage cultivation process, which made it suitable for application in the production scale-up of algal biomass.     

Growth characteristics of the cyanobacterium Nostoc flagelliforme in photoautotrophic, mixotrophic and heterotrophic cultivation

Nostoc flagelliforme is a terrestrial cyanobacterium with high economic value. Dissociated cells separated from a natural colony of N. flagelliforme were cultivated for 7 days under either phototrophic, mixotrophic or heterotrophic culture conditions. The highest biomass, 1.67 g L⁻¹ cell concentration, was obtained under mixotrophic culture, representing 4.98 and 2.28 times the biomass obtained in phototrophic and heterotrophic cultures, respectively. The biomass in mixotrophic culture was not the sum as that in photoautotrophic and heterotrophic cultures. During the first 4 days of culture, the cell concentration in mixotrophic culture was lower than the sum of those in photoautotrophic and heterotrophic cultures. However, from the 5th day, the cell concentration in mixotrophic culture surpassed the sum of those obtained from the other two trophic modes. Although the inhibitor of photosynthetic electron transport DCMU [3-(3,4-dichlorophenyl)-1,1-dimethylurea] efficiently inhibited autotrophic growth of N. flagelliforme cells, under mixotrophic culture they could grow by using glucose. The addition of glucose changed the response of N.flagelliforme cells to light. The maximal photosynthetic rate, dark respiration rate and light compensation point in mixotrophic culture were higher than those in photoautotrophic cultures. These results suggest that photoautotrophic (photosynthesis) and heterotrophic (oxidative metabolism of glucose) growth interact in mixotrophic growth of N. flagelliforme cells.     

Dimethylsulfoniopropionate turnover is linked to the composition and dynamics of the bacterioplankton assemblage during a microcosm phytoplankton bloom

Processing of the phytoplankton-derived organic sulfur compound dimethylsulfoniopropionate (DMSP) by bacteria was studied in seawater microcosms in the coastal Gulf of Mexico (Alabama). Modest phytoplankton blooms (peak chlorophyll a [Chl a] concentrations of approximately 2.5 microg liter(-1)) were induced in nutrient-enriched microcosms, while phytoplankton biomass remained low in unamended controls (Chl a concentrations of approximately 0.34 microg liter(-1)). Particulate DMSP concentrations reached 96 nM in the enriched microcosms but remained approximately 14 nM in the controls. Bacterial biomass production increased in parallel with the increase in particulate DMSP, and nutrient limitation bioassays in the initial water showed that enrichment with DMSP or glucose caused a similar stimulation of bacterial growth. Concomitantly, increased bacterial consumption rate constants of dissolved DMSP (up to 20 day(-1)) and dimethylsulfide (DMS) (up to 6.5 day(-1)) were observed. Nevertheless, higher DMSP S assimilation efficiencies and higher contribution of DMSP to bacterial S demand were found in the controls compared to the enriched microcosms. This indicated that marine bacterioplankton may rely more on DMSP as a source of S under oligotrophic conditions than under the senescence phase of phytoplankton blooms. Phylogenetic analysis of the bacterial assemblages in all microcosms showed that the DMSP-rich algal bloom favored the occurrence of various Roseobacter members, flavobacteria (Bacteroidetes phylum), and oligotrophic marine Gammaproteobacteria. Our observations suggest that the composition of the bacterial assemblage and the relative contribution of DMSP to the overall dissolved organic sulfur/organic matter pool control how efficiently bacteria assimilate DMSP S and thereby potentially divert it from DMS production.     

Growth and phycocyanin formation of Spirulina platensis in photoheterotrophic culture

Summary Glucose and acetate enhanced cell growth and phycocyanin production of S. platensis. The highest specific growth rate, cell concentration and phycocyanin production were respectively 0.62 d^-1, 2.66 g/l and 322 mg/l on glucose and 0.52 d^-1, 1.81 g/l and 246 mg/l on acetate. The specific growth rate of the alga on 2.5 g glucose/l was markedly increased with increasing light intensity up to 2 klux. Further increasing light intensity to 4 klux only resulted in a very slight increase in specific growth rate. At a light intensity above 4 klux, photoinhibition occurred. Light favoured phycocyanin formation. The highest phycocyanin content was obtained at a light intensity of 4 klux. When the light intensity decreased to 2 klux or less, the optimal glucose concentration for biomass production shifted from 2.5 g/l to 5.0 g/l.     

Computer control of carbon-nitrogen ratio in Spirulina platensis

An on-line computer was used to control the ratio of carbon to nitrogen in algal biomass. An indirect method of growth and biomass estimation was utilized. This was based on balancing the amount of CO(2) carbon in and out of the algal bioreactor. It was shown that growth conditions govern the morphology and composition of Spirulina platensis. Cells grown under light limitation were narrower, had high levels of phycocyanin pigments, and were packed full of small lipid granules. Whereas cells grown under nitrogen limitation lost their characteristic blue-green color, had reduced levels of phycocyanin, were fatter, and were packed full of larger lipid granules.     

Enhanced biomass and gamma-linolenic acid production of mutant strain Arthrospira platensis

A mutant of Arthrospira platensis PCC 9108, strain M9108, obtained by mutagenesis with UV treatment, was able to mixotrophically grow in an SOT medium containing 40 g of glucose/l. The biomass and specific growth rate of strain M9108 (4.10 g/l and 0.70/d) were 1.9-fold and 1.4-fold higher, respectively, than those of the wild type (2.21 g/l and 0.58/d) under mixotrophic culture condition. In addition, when compared with the wild type, the content of gamma- linolenic acid (GLA) in the mutant was increased when glucose concentration was increased. Compared with the wild type, the GLA content of the mutant was 2-fold higher in autotrophic culture and about 3-fold higher in mixotrophic culture. Thus, the mutant appears to possess more efficient facility to assimilate and metabolize glucose and to produce more GLA than its wild-type strain.     

Temperature affects the response of heterotrophic bacteria and mixotrophic algae to enhanced concentrations of soil extract

To investigate the consequences of increased temperature and enhanced input of dissolved organic matter (DOM) into lakes for heterotrophicic bacteria and for mixotrophic algae which use DOM in addition to photosynthesis, the hypotheses were tested whether (1) both bacteria and mixotrophic algae benefit from increased input of DOM, or (2) increased DOM input enhances bacterial biomass and thereby decreases algal biomass. Growth experiments in batch cultures, exudation measurements, and competition experiments in chemostats were performed at two temperature levels. Increased temperature stimulated the autotrophic growth rate of Chlorella protothecoides. Bacteria and Chlorella increased their heterotrophic growth rates at higher DOM concentration at lower temperature whereas enhanced DOM concentration hardly stimulated their growth at higher temperature. In chemostats, enhanced input of soil extract increased both bacterial and algal biomass at lower temperature whereas bacterial biomass increased only slightly and algal biomass decreased at higher temperature. Thus, the temperature determines the response of microorganisms to enhanced DOM concentration.     

Chemostat study of citric acid production from glycerol by Yarrowia lipolytica

The aim of the study was to examine how the dilution rate and the chemical composition of the production medium impacts on the synthesis of citric acid by the Yarrowia lipolytica strain Wratislavia AWG7 from glycerol in a chemostat culture. The yeast Y. lipolytica Wratislavia AWG7, an acetate (acet(-)) and morphological (fil(-)) mutant, was cultured in a nitrogen- and phosphorus-limited medium at the dilution rate of 0.009-0.031h(-1) in the chemostat. Under steady-state conditions, the increase in the dilution rate was paralleled by the decrease in citric acid concentration (from 86.5 to 51.2gL(-1)), as well as by the increase in the volumetric rate (from 0.78 to 1.59gL(-1)h(-1)) and specific rate (from 0.05 to 0.18gg(-1)h(-1)) of citric acid production. The yield of the production process varied from 0.59 to 0.67gg(-1). In a 550-h continuous culture of the yeast test, at a dilution rate of 0.01h(-1), in a medium with enhanced concentrations of carbon, nitrogen and phosphorus sources, the concentration of citric acid, the concentration of biomass and the volumetric rate of citric acid production were 97.8gL(-1), 22.2gL(-1) and 0.98gL(-1)h(-1), respectively. The yield of the process decreased to 0.49gg(-1). The number of dead cells did not exceed 1% while that of the budding cells accounted for about 20%. Owing to the low content of isocitric acid and polyols, the fermentation process was characterized by a high purity. This study has produced the following finding: the double mutant Y. lipolytica AWG7 is an effective citric acid producer, with the ability to preserve its properties unchanged during the long run of the continuous chemostat process. This is a valued technological feature of such mutants.     

MIXOTROPHIC GROWTH MODIFIES THE RESPONSE OF SPIRULINA (ARTHROSPIRA) PLATENSIS (CYANOBACTERIA) CELLS TO LIGHT

Spirulina (Arthrospira) platensis (Nordstedt) Geitler cells grown under mixotrophic conditions exhibit a modified response to light. The maximal photosynthetic rate and the light saturation value of mixotrophic cultures were higher than those of the photoautotrophic cultures. Dark respiration and light compensation point were also significantly higher in the mixotrophically grown cells. As expected, the mixotrophic cultures grew faster and achieved a higher biomass concentration than the photoautotrophic cultures. In contrast, the growth rate of the photoautotrophic cultures was more sensitive to light. The differences between the two cultures were also apparent in their responses to exposure to high photon flux density of 3000 μmol·m ^{− 2}·s ^{− 1}. The light-dependent O₂ evolution rate and the maximal efficiency of photosystem II photochemistry declined more rapidly in photoautotrophically grown than in mixotrophically grown cells as a result of exposure to high photon flux density. Although both cultures recovered from the high photon flux density stress, the mixotrophic culture recovered faster and to a higher extent. Based on the above results, growth of S. platensis with a fixed carbon source has a significant effect on photosynthetic activity.     

Alternative medium for production of Pleurotus ostreatus biomass and potential antitumor polysaccharides

Pleurotus species are recognized for producing beta-glucans with important medicinal properties as a constituent of the cellular wall of the fruiting body or of the mycelium. The aims of this work were to select a culture medium that maximized the production of biomass and polysaccharides produced by Pleurotus ostreatus DSM 1833 and to evaluate the selected medium in two values of initial oxygen transfer rate -K(L)a (10.2 and 19.3 h(-1)). A 2* *4 factorial design was constructed to evaluate the supplementation of wheat extract with corn steep liquor--CSL (10 or 20 g L(-1)), yeast extract--YE (2 or 5gL(-1)), ammonium sulfate--AS (0 or 5 g L(-1)) and glucose (20 or 40 g L(-1)). In terms of maximum productivity in biomass and global productivity in polysaccharides, the best values were obtained when 5 g L(-1) of YE and 40 g L(-1) of glucose were used. In terms of maximum concentration of biomass, the best results were obtained when 20 g L(-1) of CSL and 40 g L(-1) of glucose were used. The best results in terms of production of biomass and polysaccharides were achieved when lower initial K(L)a (10.2 h(-1)) was used.     

Selenium-induced changes in activities of antioxidant enzymes and content of photosynthetic pigments in Spirulina platensis

Spirulina platensis exposed to various selenium （Se） concentrations （0, 10, 20, 40, 80, 150, 175, 200, 250 mg/L） accumulated high amounts of Se in a dose- and time-dependent manner. Under low Se concentrations （〈150 mg/L）, Se induced increases in biomass concentration, content of photosynthetic pigments, and activities of glutathione peroxidase （GPX）, superoxide dismutase （SOD）, catalase （CAT） and Gua-dep peroxidases （POD）, which indicates that antioxidant enzymes play an important role in protecting cells from Se stress. Higher Se concentrations （≥175 mg/L） led to higher Se accumulation and increases in activities of GPX, SOD, CAT and POD, but also induced lipid peroxidation （LPO） coupled with potassium leakage and decreases in biomass concentration and contents of photosynthetic pigment. The results indicate that increases in activities of the antioxidant enzymes were not sufficient to protect cell membranes against Se stress. Time-dependent variations in the activities of antioxidant enzymes, contents of chlorophyll a and carotenoid and the LPO level were also investigated under representative Se concentrations of 40 and 200mg/L. Opposite variation trends between SOD-CAT activities, and GPX-POD-APX activities were observed during the growth cycles. The results showed that the prevention of damage to cell membranes of S. platensis cells could be achieved by cooperative effects of SOD-CAT and GPX-POD-APX enzymes. This study concludes that S. platensis possessed tolerance to Se and could protect itself from phytotoxicity induced by Se by altering various metabolic processes.     

有机碳源对三角褐指藻生长、胞内物质和脂肪酸组分的影响

研究了3种有机碳对三角褐指藻生长、胞内物质和脂肪酸组分的影响。结果表明, 三角褐指藻具有利用有机碳进行兼养生长的能力, 生长速率加快, 倍增时间缩短, 生物量显著提高, 100 mmol/L甘油兼养的生物量最高(713 mg/L), 是自养(460 mg/L)的1.60倍, 乙酸钠和葡萄糖兼养的生物量分别是自养的1.28倍和1.21倍。兼养下蛋白质含量较自养明显下降, 碳水化合物和总脂含量高于自养, 乙酸钠和甘油兼养的总脂含量分别是自养的1.43倍和1.20倍, 葡萄糖兼养的总脂含量与自养无明显差异。3种有机碳兼养的饱和脂肪酸和单不饱和脂肪酸占总脂肪酸的比例增大, 多不饱和脂肪酸比例降低, EPA(eicosapentaenoic acid)比例降低, 乙酸钠兼养的胞内EPA含量(6.23%)和产量(36.59 mg/L)均高于自养, 分别是自养的1.10倍和1.40倍, 甘油和葡萄糖兼养的EPA含量和产量均低于自养。     

Effect of mixed carbon substrate on exopolysaccharide production of cyanobacterium Nostoc flagelliforme in mixotrophic cultures

The effects of organic carbon sources on cell growth and exopolysaccharide (EPS) production of dissociated Nostoc flagelliforme cells under mixotrophic batch culture were investigated. After 7?days of cultivation, glycerol, acetate, sucrose, and glucose increased the final cell density and final EPS concentrations, and mixotrophic growth achieved higher biomass concentrations. The increase in cell growth was particularly high when glucose was added as the sole carbon source. On the other hand, EPS production per dry cell weight was significantly enhanced by adding acetate. For more effective EPS production, the effects of the mixture of glucose and acetate were investigated. Increasing the ratio of glucose to acetate resulted in higher growth rate with BG-11 medium and higher EPS productivity with BG-11₀ medium (without NaNO₃). When the medium was supplemented with a mixture of glucose (4.0?g?L^?1) and acetate (2.0?g?L^?1), 1.79?g?L^?1 biomass with BG-11 medium and 879.6?mg?L^?1 of EPS production with BG-11₀ medium were achieved. Adopting this optimal ratio of glucose to acetate established in flask culture, the culture was also conducted in a 20-L photobioreactor with BG-11 medium for 7?days. A maximum biomass of 2.32?g?L^?1 was achieved, and the EPS production was 634.6?mg?L^?1.