Characteristics of mixotrophic growth of Synechocystis sp. in an enclosed photobioreactor

Synechocystis sp. PCC 6803 was grown in a 2.5 l enclosed photobioreactor on medium with or without glucose. The incident light intensities ranged from 1.5 klux to 7 klux. The highest average specific growth rates of mixotrophic culture and photoautotrophic culture were, respectively, 1.3 h^–1 at a light intensity of 7 klux on 3.2 g l^–1 glucose and 0.3 h^–1 at both light intensities of 5 klux and 7 klux. The highest cell density 2.5 g l ^–1 was obtained at both of light intensities 5 klux and 7 klux on 3.2 g glucose l^–1. Glucose consumption decreased with decreasing light intensity. The energy yields of mixotrophic cultures were 4 to 6 times higher than that of photoautotrophic cultures. Light favored mixotrophic growth of Synechocystis sp. PCC 6803, especially at higher light intensities (5–7 klux).     

Photoautotrophic cultivating options of freshwater green microalgal Chlorococcum humicola for biomass and carotenoid production

Photoautotrophic cultivation of Chlorococcum humicola was performed in batch and continuous modes in different cultivating system arrangements to compare biomass and carotenoids’ concentration and their productivities. Batch result from stirred tank and airlift photobioreactors indicated the positive effect of increasing light intensity on growth and carotenoid production, whereas the finding from continuous cultivation indicated that carotenoid enhancement preferred high light intensity and nitrogen-deficient environment. The highest biomass (1.31?±?0.04?g?L^?1) and carotenoid (4.59?±?0.06?mg?L^?1) concentration as well as the highest productivities, 0.46?g?L^?1 d^?1 for biomass and 1.61?mg?L^?1 d^?1 for carotenoids, were obtained when maintaining high light intensity of 10 klx, BG-11 medium and 2% (v/v) CO₂ simultaneously, while the highest carotenoid content (4.84?mg?g^?1) was associated with high light intensity and nitrogen-deficient environment, which was induced by feed-modified BG-11 growth medium containing nitrate 20 folds lower than the original medium. Finally, the cultivating system arranged into smaller stirred tank photobioreactors in series yielded approximately 2.5 folds increase in both biomass and carotenoid productivities relative to using single airlift photobioreactor with equivalent working volume and similar operating condition.     

Evaluation of Arthrospira platensis extracellular polymeric substances production in photoautotrophic, heterotrophic and mixotrophic conditions

The kinetic study of Arthrospira platensis extracellular polymeric substances (EPS) production under different trophic modes??photoautotrophy (100???mol photons m^?2?s^?1), heterotrophy (1.5?g/L glucose), and mixotrophy (100???mol photons m^?2?s^?1 and 1.5?g/L glucose)??was investigated. Under photoautotrophic and heterotrophic conditions, the maximum EPS production 219.61?±?4.73 and 30.30?±?1.97?mg/L, respectively, occurred during the stationary phase. Under a mixotrophic condition, the maximum EPS production (290.50?±?2.21?mg/L) was observed during the early stationary phase. The highest specific EPS productivity (433.62?mg/g per day) was obtained under a photoautotrophic culture. The lowest specific EPS productivity (38.33?mg/g per day) was observed for the heterotrophic culture. The effects of glucose concentration, light intensity, and their interaction in mixotrophic culture on A. platensis EPS production were evaluated by means of 32 factorial design and response surface methodology. This design was carried out with a glucose concentration of 0.5, 1.5, and 2.5?g/L and at light levels of 50, 100, and 150???mol photons m^?2?s^?1. Statistical analysis of the model demonstrated that EPS concentration and EPS yield were mainly influenced by glucose concentration and that conditions optimizing EPS concentration were dissimilar from those optimizing EPS yield. The highest maximum predicted EPS concentration (369.3?mg/L) was found at 150???mol photons m^?2?s^?1 light intensity and 2.4?g/L glucose concentration, while the highest maximum predicted EPS yield (364.3?mg/g) was recorded at 115???mol photons m^?2?s^?1 light intensity and 1.8?g/L glucose concentration.     

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.     

Pigment production in Spirulina fussiformis in different photophysical conditions

The present investigation makes a comparative investigation of individual light source on the different commercially important pigments in Spirulina fussiformis in photobioreactor culture condition. Continuous culture system was carried out throughout the experimental condition. Initially, seed culture, corresponding to 0.2 g/L on dry weight basis was cultivated in Zarrouks medium with different colored light source in reactor. Maximum daily biomass productivity, 0.8 g/L, 0.75 g/L and 0.69 g/L in white light (WL), blue light (BL) and green light (GL), respectively, conditions was noticed. Pigment content during WL treatment showed the highest accumulation (5.5 microg/mL) of chlorophyll whereas, other pigments roughly remained constant without much change, implying WL intensity is better for chlorophyll synthesis. On the other hand, chlorophyll and phycocyanin content gradually increased up to 7 microg/mL and 2 mg/mL, respectively, at BL intensity. The response to GL was negative to all pigments studied except for phycocyanin; in this case a highest production (2.5 mg/mL) was seen during 18 days experimental period. Additionally, when yellow light (YL) treatment experiments were conducted, the rate of production gradually decreased from 6th day onward in all pigments demonstrating the photobleaching effect of YL. The average rate of pigments production did not show significant accumulation in red light (RL) light treatment except phycoerythrin which showed an increasing trend of production. It is worth to mention here that higher light intensity is better for production of phycocyanin and phycoerythrin in Spirulina.     

Effects of intensity and quality of light on phycocyanin production by a marine cyanobacterium Synechococcus sp. NKBG 042902

Among 150 strains, including marine cyanobacteria isolated from coastal areas of Japan and a freshwater cyanobacterium from the IAM collection, Spirulina platensis IAM M-135, the marine cyanobacterium Synechococcus sp. NKBG 042902 contained the highest amount of phycocyanin (102 mg/g dry cell weight). We have proposed that the cyanobacterium could be an alternative producer for phycocyanin. The effects of light intensity and light quality on the phycocyanin content in cells of Synechococcus sp. NKBG 042902 were investigated. When the cyanobacterium was cultured under illumination of 25 mol m^–2 s^–1 using a cool-white fluorescent lamp, the phycocyanin content was highest, and the phycocyanin and biomass productivities were 21 mg 1^–1 day^–1 and 100 mg 1^–1 day^–1 respectively. Red light was essential for phycocyanin production by this cyanobacterium. Phycocyanin and biomass production were carried out by the cyanobacterium cultures grown under only red light (peak wavelength at 660 nm) supplied from light-emitting diodes (LED). Maximum phycocyanin and biomass productivities were 24 mg 1^–1 day^–1 and 130 mg 1^–1 day^–1 when the light intensity of the LED was 55 mol m^–2 s^–1.     

Influence of carbon source on growth,biochemical composition and pigmentation of Ankistrodesmus convolutus

The unicellular chlorophyte Ankistrodesmus convolutus Corda was grown in the light using inorganic medium (Bold's Basal Medium, BBM) and BBM enriched with 0.1% w/v of glucose, sodium acetate, sodium citrate or sodium bicarbonate. Glucose supported the highest specific growth rate (μ = 0.93 d^-1) and gave the highest biomass (453 mg dry weight L^-1) at the time of harvest. Of four glucose concentrations (0.05, 0.1, 0.25, 0.5% w/v), best growth was attained at 0.1% w/v. At 0.5% w/v glucose, the cells had high carbohydrates but low lipids and proteins. The relative amounts of 16:0, 18:0, 18:1 and 18:2 increased at the expense of 18:3(n-3) in the carbon-supplemented cultures and at glucose concentrations higher than 0.1% w/v. Cultures grown on glucose had less chlorophyll and carotenoid contents than cultures grown on other carbon sources. Chlorophyll and carotenoid contents decreased with increasing glucose concentrations in the medium.     

Bioenergetic analysis of mixotrophic growth in Chlorella vulgaris and Scenedesmus acutus

The specific growth rate of Chlorella vulgaris in the presence of glucose (mixotroph) was always larger than that in autotroph when the light intensity was less than 10 klux. However, Scenedesmus acutus behaved differently, i.e., when the light intensity was more than 6 klux, the specific growth rate in mixotroph became smaller than that in autotroph. Cellular contents of chlorophyll a and b in Scenedesmus acutus that deteriorated more markedly in mixotroph than those of Chlorella vulgaris could account for these different growth behaviors. In other words, mechanisms relevant to the photosynthesis and the oxidative phosphorylation of glucose seem to function independently with respect to Chlorella Vulugaris.     

Accumulation of phycocyanin in heterotrophic and mixotrophic cultures of the acidophilic red alga Galdieria sulphuraria

The relationship between light intensity, nitrogen availability and pigmentation was investigated in mixotrophic and heterotrophic cultures of the unicellular red alga Galdieria sulphuraria 074G, a potential host for production of the blue pigment, phycocyanin (PC). During the exponential growth phase of batch cultures, G. sulphuraria 074G contained 2–4 mg phycocyanin per g dry weight. In carbon-limited and nitrogen-sufficient batch cultures grown in darkness, this value increased to 8–12 mg g⁻¹ dry weight during the stationary phase, whereas the phycocyanin content in nitrogen-deficient cells decreased to values below 1 mg g⁻¹ dry weight during stationary phase. Light intensities between 0 and 100 μmol photons m⁻² s⁻¹ had no influence on phycocyanin accumulation in mixotrophic cultures grown on glucose or fructose, while light stimulated phycocyanin synthesis in cultures grown on glycerol, in which the phycocyanin content in stationary phase was increased from 10 mg g⁻¹ dry weight in darkness to 20 mg g⁻¹ dry weight at a light intensity of 80 μmol photons m⁻² s⁻¹. At higher light intensities, less phycocyanin accumulated than at lower intensities, irrespective of the carbon substrate used. In carbon-limited continuous flow cultures grown on glucose or glycerol at a dilution rate of 0.63 day⁻¹, corresponding to 50% of the maximum specific growth rate, the highest steady-state phycocyanin content of 15–28 mg g⁻¹ dry weight was found at 65 μmol photons m⁻² s⁻¹. In contrast to the apparent glucose repression of light-induced PC synthesis observed in batch cultures, no glucose repression of the light stimulation was observed in continuous flow cultures because the glucose concentration in the culture supernatant always remained at limiting levels. Despite the fact that G. sulphuraria 074G contains less phycocyanin than some other microalgae and cyanobacteria, the ability of G. sulphuraria 074G to grow and synthesize phycocyanin in heterotrophic or mixotrophic cultures makes it an interesting alternative to the cyanobacterium, Spirulina platensis presently used for synthesis of phycocyanin.     

The effects of light intensity, inoculum size, and cell immobilisation on the treatment of sago effluent withRhodopseudomonas palustris strain B1

A study was carried out to determine a suitable light intensity and inoculum size for the growth ofRhodopseudomonas palustris strain B1. The pollution reduction of sago effluent using free and immobilisedR. palustris cells was also evaluated. The growth rate in glutamatemalate medium was highest at 4 klux compared to 2.5 and 3 klux. The optimal inoculum size was 10% (v/v). Both the COD and BOD of the sago effluent were reduced by 67% after three days of treatment. The difference in biomass production or BOD and COD removal with higher inoculum sizes of 15 and 20% was minimal. This could be attributed to limited nutrient availabillity in the substrate. The use of immobilised cells ofR. palustris reduced the pollution load 10% less compared to pollution reduction by free cells. Hence, there was no significant difference in using free or immobilised cells for the treatment of sago effluent.     

Modeling growth and photosynthetic response in <Emphasis Type="Italic">Arthrospira platensis</Emphasis> as function of light intensity and glucose concentration using factorial design

Combined effect of light intensity and glucose concentration on Arthrospira platensis growth and photosynthetic response was evaluated using a 3² factorial design. This design was carried out with light levels of 50, 100, and 150 μmol photons m⁻² s⁻¹ and glucose concentrations of 0.5, 1.5, and 2.5 g L⁻¹. Results from the response surface methodology were that the highest level of light intensity and glucose concentration improved biomass (1.33 g L⁻¹), maximum specific growth rate (0.49 day⁻¹), and net photosynthetic rate (139.89 μmol O₂ mg Chl⁻¹ h⁻¹). Furthermore, the interaction of both factors showed that at low light, glucose had a low effect on maximum biomass and maximal net photosynthetic rate. However, at the highest light levels, the effect of glucose was more sensitive and the increase of glucose concentration increased the levels of all responses. The rates of the instantaneous relative growth, net photosynthesis, and dark respiration of growth cultures showed two different phases in mixotrophic condition. The first was distinguished by the preponderance of the photoautotrophic mode; the second was based mainly on photoheterotrophy.     

Growth,vitamin B12, and photopigment formations of Rhodopseudomonas sphaeroides S growing on propionate media under dark and light conditions

Summary The effects of light illumination and dissolved oxygen concentration (DO) on the growth characteristics of Rhodopseudomonas sphaeroides S and intracellular accumulations of vitamin B₁₂ and photopigments were studied in continuous cultures of aerobic-dark (S-1, DO>5 mg l^–1), aerobic-light (S-2, DO>5 mg l^–1, 4.5 klux), microaerobic-light (S-3, DO0,4.5 klux) and anaerobic-light (S-4, 4.5 klux) conditions using propionate media. Growth yields from propionic acid determined in S-3 and 4 were 1.5 to twofold greater than in S-1 and 2. Carbon dioxide evolution observed in S-3 and 4 was 0.05 to 0.1 times that in S-1 and 2. Overall carboxylase activity was maximal in S-4. Intracellular accumulations of bacteriochlorophyll and carotenoid were maximal in S-4, followed by S-3 and almost nil in S-1 and 2. The high growth yields observed in S-3 and 4 could be accounted for the high level of activity of carbon dioxide fixation and by the additional or effective utilization of light energy.Intracellular accumulation of vitamin B₁₂ in S-2, 3 and 4 was 1.5 to 1.8 times that in S-1. The maximum content of the vitamin was 74 g-B₁₂ g-cell^–1. The maximum productivity of vitamin B₁₂, g-B₁₂ l^–1 h^–1, was 6.6 in S-3 since specific growth rate, growth yield and the vitamin content of the cells were maximal in S-3.     

Comparison of photobioreactors for cultivation of Undaria pinnatifida gametophytes

Undaria pinnatifida gametophytes were grown in 2.5 l bubble column and airlift reactor at 25 °C and light intensity of 40 mol m^–2 s^–1 for 6 days. With aeration at 1 l min^–1, the airlift reactor yielded higher growth rate (0.12 mg DW ml^–1 d^–1) than a bubble column (0.08 mg DW ml^–1 d^–1). The advantages were related to the more homogeneous fluid dynamic characteristics of the airlift reactor.     

Use of different carbon sources in cultivation of recombinant Pichia pastoris for angiostatin production

To improve the growth of recombinant Pichia pastoris with a phenotype of Mut^S and expression of angiostatin, the effects of glycerol, sorbitol, acetate and lactic acid which were, respectively, added together with methanol in the expression phase, were studied in a 5-l fermentor. Methanol concentration was automatically controlled at 5 g/l by a methanol monitor and control system, while the feeding of the other carbon source was manually adjusted. The angiostatin production level was 108 mg/l when glycerol was added at an initial rate of 2.3 g/h and gradually increased to 9.9 g/h within an induction period of 96 h. The angiostatin concentration was 141 mg/l as sorbitol was used, while only 52 mg/l were obtained on acetate. The highest angiostatin production of 191 mg/l was achieved as lactic acid was used; whose feeding rate was gradually increased from 2.6 to 11.3 g/h. Lactic acid accumulated during the induction phase and reached 6.3 g/l at the end of fermentation. However, the accumulation of lactic acid did not interfere with angiostatin production, indicating that lactic acid to be a non-repressive carbon source. The average productivity and specific productivity of angiostatin obtained on lactic acid and methanol were, respectively, 2.96 and 0.044 mg/(g h), 1.7- and 2.5-fold of those obtained in the fermentation fed with glycerol and methanol.     

Optimization of glucose feeding approaches for enhanced glucosamine and N-acetylglucosamine production by an engineered Escherichia coli

In this work, a recombinant Escherichia coli was constructed by overexpressing glucosamine (GlcN) synthase and GlcN-6-P N-acetyltransferase for highly efficient production of GlcN and N-acetylglucosamine (GlcNAc). For further enhancement of GlcN and GlcNAc production, the effects of different glucose feeding strategies including constant-rate feeding, interval feeding, and exponential feeding on GlcN and GlcNAc production were investigated. The results indicated that exponential feeding resulted in relatively high cell growth rate and low acetate formation rate, while constant feeding contributed to the highest specific GlcN and GlcNAc production rate. Based on this, a multistage glucose supply approach was proposed to enhance GlcN and GlcNAc production. In the first stage (0–2 h), batch culture with initial glucose concentration of 27 g/l was conducted, whereas the second culture stage (2–10 h) was performed with exponential feeding at μ _set = 0.20 h⁻¹, followed by feeding concentrated glucose (300 g/l) at constant rate of 32 ml/h in the third stage (10–16 h). With this time-variant glucose feeding strategy, the total GlcN and GlcNAc yield reached 69.66 g/l, which was enhanced by 1.59-fold in comparison with that of batch culture with the same total glucose concentration. The time-dependent glucose feeding approach developed here may be useful for production of other fine chemicals by recombinant E. coli.     

Study of docosahexaenoic acid production by the heterotrophic microalga <Emphasis Type="Italic">Crypthecodinium cohnii</Emphasis> CCMP 316 using carob pulp as a promising carbon source

In this work, carob pulp syrup was used as carbon source in C. cohnii fermentations for docosahexaenoic acid production. In preliminary experiments different carob pulp dilutions supplemented with sea salt were tested. The highest biomass productivity (4 mg/lh) and specific growth rate (0.04/h) were observed at the highest carob pulp dilution (1:10.5 (v/v), corresponding to 8.8 g/l glucose). Ammonium chloride and yeast extract were tested as nitrogen sources using different carob pulp syrup dilutions, supplemented with sea salt as growth medium. The best results were observed for yeast extract as nitrogen source. A C. cohnii fed-batch fermentation was carried out using diluted carob pulp syrup (1:10.5 v/v) supplemented with yeast extract and sea salt. The biomass productivity was 420 mg/lh, and the specific growth rate 0.05/h. Under these conditions the DHA concentration and DHA production volumetric rate attained 1.9 g/l and 18.5 mg/lh respectively after 100.4 h. The easy, clean and safe handling of carob pulp syrup makes this feedstock a promising carbon source for large-scale DHA production from C. cohnii. In this way, this carob industry by-product could be usefully disposed of through microbial production of a high value fermentation product.     

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.     

Continuous citric acid fermentation by <Emphasis Type="Italic">Candida oleophila</Emphasis> under nitrogen limitation at constant C/N ratio

Summary The central aspect of this work was to investigate the influence of nitrogen feed rate at constant C/N ratio on continuous citric acid fermentation by Candida oleophila ATCC 20177. Medium ammonia nitrogen and glucose concentrations influenced growth and production. Space-time yield (STY) meaning volumetric productivity, biomass specific productivity (BSP), product concentration, product selectivity and citrate/isocitrate ratio increased with increasing residence time (RT). BSP increased in an exponential mode lowering nitrogen feed rates. Highest BSP for citric acid of 0.13 g/(g h) was achieved at lowest NH₄Cl concentration of 1.5 g/l and highest STY (1.2 g/l h) with 3 g NH₄Cl/l at a RT of 25 h. Citric acid 74.2 g/l were produced at 58 h RT and 6 g NH₄Cl/l. Glucose uptake rate seems to be strictly controlled by growth rate of the yeast cells. Optimum nitrogen concentration and adapted C/N ratio are essential for successful continuous citric acid fermentation. The biomass-specific nitrogen feed rate is the most important factor influencing continuous citric acid production by yeasts. Numerous chemostat experiments showed the feasibility of continuous citrate production by yeasts.     

Exploration of the hydrogen producing potential of Rhodobacter capsulatus chemostat cultures: The application of deceleration‐stat and gradient‐stat methodology

In this work, the dependency of the volumetric hydrogen production rate of ammonium‐limited Rhodobacter capsulatus chemostat cultures on their imposed biomass concentration and dilution rate was investigated. A deceleration‐stat experiment was performed by lowering the dilution rate from 1.0 d^?1 to zero aimed at a constant biomass concentration of 4.0 g L^?1 at constant incident light intensity. The results displayed a maximal volumetric hydrogen production rate of 0.6 mmol m^?3 s^?1, well below model predictions. Possibly the high cell density limited the average light availability, resulting in a sub‐optimal specific hydrogen production rate. To investigate this hypothesis, a gradient‐stat experiment was conducted at constant dilution rate of 0.4 d^?1 at constant incident light intensity. The biomass concentration was increased from 0.7 to 4.0 g L^?1 by increasing the influent ammonium concentration. Up to a biomass concentration of 1.5 g L^?1, the volumetric hydrogen production rate of the system increased according to model predictions, after which it started to decline. The results obtained provide strong evidence that the observed decline in volumetric hydrogen production rate at higher biomass concentrations was at least partly caused by a decrease in light availability. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Kinetic models for phycocyanin production by high cell density mixotrophic culture of the microalga Spirulina platensis

Phycocyanin production by high cell density cultivation of Spirulina platensis in batch and fed-batch modes in 3.7-L bioreactors with a programmed stepwise increase in light intensity program was investigated. The results showed that the cell density in fed-batch culture (10.2 g L⁻¹) was 4.29-fold that in batch culture (2.38 g L⁻¹), and the total phycocyanin production in the fed-batch culture (0.795 g L⁻¹) was 3.05-fold that in the batch culture (0.261 g L⁻¹). An unstructured kinetic model to describe the microalga culture system including cell growth, phycocyanin formation, as well as glucose consumption was proposed. The data fitted the models well (r ² > 0.99). Furthermore, based on the kinetic models, the potential effects of light limitation and photoinhibition on cell growth and phycocyanin formation can be examined in depth. The models demonstrated that the optimal light intensity for mixotrophic growth of Spirulina platensis in batch or fed-batch cultures using a 3.7-L bioreactor was 80160 μE m⁻² s⁻¹, and the stepwise increase in light intensity can be replaced by a constant light intensity mode. Received 28 July 1998/ Accepted in revised form 8 October 1998