Synergistic carbon metabolism in a fast growing mixotrophic freshwater microalgal species Micractinium inermum

In recent years microalgae have attracted significant interest as a potential source of sustainable biofuel. Mixotrophic microalgae are able to simultaneously photosynthesise while assimilating and metabolising organic carbon. By combining autotrophic and heterotrophic metabolic pathways biomass productivity can be significantly increased. In this study, acetate-fed mixotrophic Micractinium inermum cultures were found to have a specific growth rate 1.74 times the sum of autotrophic and heterotrophic growth. It was hypothesised that gas exchange between the two metabolic pathways within mixotrophic cultures may have prevented growth limitation and enhanced growth. To determine the extent of synergistic gas exchange and its influence on metabolic activity, dissolved inorganic carbon (DIC), dissolved oxygen (DO) and photosynthesis and respiration rates were measured under different trophic conditions. A 32.7 fold and 2.4 fold increase in DIC and DO concentrations, relative to autotrophic and heterotrophic cultures respectively, were coupled with significant increases in rates of photosynthesis and respiration. These data strongly support the hypothesis of mixotrophic gas exchange within M. inermum cultures. In addition to enhanced growth, this phenomenon may provide reductions in aeration and oxygen stripping costs related to microalgae production.     

Mixotrophic cultivation,a preferable microalgae cultivation mode for biomass/bioenergy production,and bioremediation,advances and prospect

Microalgae have received much attention in recent years as a feedstock for producing renewable fuels. Microalgae cultivation technology is one of the main factors restricting biomass production as well as energy fuel production and bioremediation. There are four types of cultivation conditions for microalgae: photoautotrophic, heterotrophic, mixotrophic and photoheterotrophic cultivation. Though photoautotrophic and heterotrophic cultivation are two common growth modes of microalgae, some microalgae can also grow better under mixotrophic condition, which may combine the advantages of autotrophic and heterotrophic and overcome the disadvantages. This review compared these growth modes of microalgae and discussed the advantages of mixotrophic mode in bioenergy production by considering the difference in growth, photosynthesis characteristic and bioenergy production. Also, the influence factors of mixotrophic cultivation and the application of mixotrophic microalgae in bioremediation are discussed, laying theoretical foundation for large scale microalgae cultivating for biomass production, bioenergy production and environmental protection.     

Sequential dark–photo fermentation and autotrophic microalgal growth for high-yield and CO2-free biohydrogen production

Dark fermentation, photo fermentation, and autotrophic microalgae cultivation were integrated to establish a high-yield and CO₂-free biohydrogen production system by using different feedstock. Among the four carbon sources examined, sucrose was the most effective for the sequential dark (with Clostridium butyricum CGS5) and photo (with Rhodopseudomonas palutris WP3-5) fermentation process. The sequential dark–photo fermentation was stably operated for nearly 80 days, giving a maximum H₂ yield of 11.61 mol H₂/mol sucrose and a H₂ production rate of 673.93 ml/h/l. The biogas produced from the sequential dark–photo fermentation (containing ca. 40.0% CO₂) was directly fed into a microalga culture (Chlorella vulgaris C–C) cultivated at 30 °C under 60 μmol/m²/s illumination. The CO₂ produced from the fermentation processes was completely consumed during the autotrophic growth of C. vulgaris C–C, resulting in a microalgal biomass concentration of 1999 mg/l composed mainly of 48.0% protein, 23.0% carbohydrate and 12.3% lipid.     

Investigation of the combined effects of acetate and photobioreactor illuminated fraction in the induction of anoxia for hydrogen production by Chlamydomonas reinhardtii

In the context of hydrogen production by microalgae, the growth of Chlamydomonas reinhardtii was characterized under autotrophic and mixotrophic conditions in a fully controlled photobioreactor (PBR). The combined effect of light transfer conditions, as represented by the illuminated fraction γ, with acetate consumption was observed upon establishment of anoxia. Anoxia was reached in batch cultures when γ was close to 1 (almost fully illuminated culture) in mixotrophic conditions while a value of γ ≈ 0.46 in autotrophic conditions was not sufficient. Based on these results, continuous hydrogen production was established in a cylindrical PBR operated in luminostat with constant illumination and in mixotrophic conditions. Maximum hydrogen gas production was equal to 1.4 ± 0.1 ml_H2 l⁻¹ h⁻¹ for photon flux density of 110 μmol m⁻² s⁻¹ and reactor illuminated fraction of γ = 0.5. Carbon mass balance was realized, emphasizing the necessity to work in strictly autotrophic conditions for hydrogen production with no concomitant CO₂ release.     

Enhancement of lipid productivity with a novel two-stage heterotrophic fed-batch culture of Chlorella protothecoides and a trial of CO2 recycling by coupling with autotrophic process

In this study, the influences of oxygen supply and nitrogen starvation on cell growth and lipid production by heterotrophic culture of Chlorella protothecoides were investigated in a 5-L stirred-tank bioreactor. The results demonstrated that both a low oxygen supply and nitrogen starvation are favorable for lipid accumulation, but limit cell growth and may therefore reduce the overall lipid production. Hence, a novel two-stage strategy for oxygen supply and nitrogen starvation was proposed in a batch culture, increasing the lipid content and yield on glucose by a factor 2.14 and 2.03 compared to the one-stage batch culture with high oxygen supply and sufficient nitrogen availability, respectively. Applying the two-stage process in a fed-batch culture further increased the lipid productivity to 175.2 mg/(L·h), giving a lipid yield of 195.6 mg/g. Furthermore, a dual-mode culture, coupling a heterotrophic process with autotrophic process, was implemented to allow for CO₂ recycling. This improved the lipid productivity and carbon utilization efficiency, further contributing towards cost-effective and environment-friendly algal lipid production.     

Mixotrophic cultivation of Botryococcus braunii

Cultivation of Botryococcus braunii has been mainly reported in photoautotrophic mode which has some disadvantages such as low cell growth rate and low cell density. For high density and high productivity, this study attempts to cultivate B. braunii in mixotrophic mode. Effects of different organic carbon sources (including maltose, glucose, saccharose, lactose, glycerol and starch), glucose and KNO₃ concentrations as well as inoculation amount on the growth of B. braunii were investigated in 250 mL shake flasks, and a fed-batch mixotrophic cultivation technique was developed in a 10 L enclosed automatic air-lift photobioreactor.B. braunii grew faster in mixotrophic cultivation with all the six organic carbon sources than that in photoautotrophic mode, and glucose was the optimum. The optimal concentrations of glucose and KNO₃ were 2.5 g L⁻¹ and 0.4 g L⁻¹ respectively. Within the inoculums of 46-200 g m⁻³, the lag phase of cell growth was very short, and cells grew fast into exponential phase after inoculation, the average cell growth rate of B. braunii increased with the increment of inoculation amount. With a 10 L air-lift photobioreactor, B. braunii was cultured by feeding glucose under mixotrophic condition for 19 days, and the cell density and hydrocarbon content in dry cell reached 4.55 g L⁻¹ and 29.7%, respectively.     

Production of microbial oil with high oleic acid content by Trichosporon capitatum

Microbial oils with high unsaturated fatty acids content, especially oleic acid content, are good feedstock for high quality biodiesel production. Trichosporon capitatum was found to accumulate lipid with around 80% oleic acid and 89% total unsaturated fatty acids content on nitrogen-limited medium. In order to improve its lipid yield, effects of medium components and culture conditions on cell growth and lipid accumulation were investigated. Optimization of media resulted in a 61% increase in the lipid yield of T. capitatum after cultivation at 28 °C and 160 rpm for 6 days. In addition, T. capitatum could grow well on cane molasses and afford a lipid yield comparable to that on synthetic nitrogen-limited medium. The biodiesel from the microbial oil produced by T. capitatum on cane molasses displayed a low cold filter plugging point (−15 °C), and so T. capitatum might be a promising strain to provide lipid suitable for high quality biodiesel production.     

Lipid production in mixotrophic cultivation of Chlorella vulgaris in a mixture of primary and secondary municipal wastewater

Microalgae's biomass productivity and oil content depend heavily on the method of its cultivation. In this study, nutrient removal from municipal wastewater by Chlorella vulgaris in batch culture was investigated. Carbon dioxide was supplied from sodium hydrogen carbonate. Effect of parameters including light intensity, sodium hydrogen carbonate concentration, and daily illumination time on the productivity of biomass and lipid was investigated. Lipid and biomass production of C. vulgaris increased at higher concentration of sodium hydrogen carbonate concentration and higher light intensity until a certain value and then decreased, but longer daily illumination time, increased both biomass and lipid productivity. Cultivation of C. vulgairs in mixotrophic mode was also studied in a mixture of primary and secondary wastewater with different ratios (25, 50 and 75 volume percent of the primary wastewater). It was observed that using 25% of the primary wastewater results in 100% COD removal, 100% ammonium removal and 82% nitrate elimination. Biomass productivity and lipid productivity of C. vulgaris in a mixture of primary (25%) and secondary wastewater were 138.76 mg/L/d and 45.49 mg/L/d, respectively.     

Effect of carbon supply mode on biomass and lipid in CSMCRI's Chlorella variabilis (ATCC 12198)

CSIR-CSMCRI's Chlorella variabilis (ATCC 12198) was evaluated through autotrophic, mixotrophic and heterotrophic growth for lipid production. Autotrophic growth was assessed by providing sodium bicarbonate/sodium carbonate/CO₂ (air in a medium). Higher lipid productivity (115.94 mg L⁻¹ d⁻¹) with higher biomass productivity (724.98 mg L⁻¹ d⁻¹) of this strain was attained through bicarbonate and CO₂ sequestration in a photobioreactor. Ability to regulate the pH in favorable bicarbonate/carbonate ratio showed its potential in alkaline effluent based carbon sequestration system for biofuel generation. The simultaneous study was also conducted to understand the effect of elevated CO₂ (0.4, 1 and 1.2 g L⁻¹) in air on the culture to assess adaptation, growth and lipid in the closed chamber conditions. It was observed that CO₂ sequestration by the microalgae from the CO₂ enriched environment was optimum at 1 g L⁻¹ C. variabilis adapted to comparatively higher CO₂ (1 g L⁻¹) but grew better in low CO₂ (0.4 g L⁻¹). It was also observed that the growth, lipid content and fatty acid composition was significantly affected by CO₂ supply strategies. The effect of intermittently added sodium bicarbonate at different pH on microalgal lipid content and composition of fatty acids was observed which could affect the quality of biodiesel. The effect on fatty acid composition was observed in response to carbon supply mode during the microalgal growth at different pH dictating the properties of biodiesel.     

Biohydrogen production by a novel integration of dark fermentation and mixotrophic microalgae cultivation

Biohydrogen is usually produced via dark fermentation, which generates CO₂ emissions and produces soluble metabolites (e.g., volatile fatty acids) with high chemical oxygen demand (COD) as the by-products, which require further treatments. In this study, mixotrophic culture of an isolated microalga (Chlorella vulgaris ESP6) was utilized to simultaneously consume CO₂ and COD by-products from dark fermentation, converting them to valuable microalgae biomass. Light intensity and food to microorganism (F/M) ratio were adjusted to 150 μmol m⁻² s⁻¹ and F/M ratio, 4.5, respectively, to improve the efficiency of assimilating the soluble metabolites. The mixotrophic microalgae culture could reduce the CO₂ content of dark fermentation effluent from 34% to 5% with nearly 100% consumption of soluble metabolites (mainly butyrate and acetate) in 9 days. The obtained microalgal biomass was hydrolyzed with 1.5% HCl and subsequently used as the substrate for bioH₂ production with Clostridium butyricum CGS5, giving a cumulative H₂ production of 1276 ml/L, a H₂ production rate of 240 ml/L/h, and a H₂ yield of 0.94 mol/mol sugar.     

Biomass and lipid production of marine microalgae using municipal wastewater and high concentration of CO2

In order to reduce the cost of the production of microalgae for biodiesel, the feasibility of using the mixture of seawater and municipal wastewater as culture medium and CO₂ from flue gas for the cultivation of marine microalgae was investigated in this study. Effects of different ratios of municipal wastewater and 15% CO₂ aeration on the growth of Nannochloropsis sp. were examined, and lipid accumulation of microalgae was also studied under nitrogen starvation and high light. It was found that optimal growth of microalgae occurred in 50% municipal wastewater, and the growth was further significantly enhanced by aeration with 15% CO₂. When Nannochloropsis sp. cells were transferred from the first growth phase to the second lipid accumulation phase under the combination of nitrogen deprivation and high light, both biomass and lipid production of Nannochloropsis sp. were significantly increased. After 12 days of the second-phase cultivation, the biomass concentration and total lipid content increased from 0.71 to 2.23 g L⁻¹ and 33.8–59.9%, respectively. This study suggests that it is possible to utilize municipal wastewater to replace nutrients in seawater medium and use flue gas to provide CO₂ in the cultivation of oil-bearing marine microalgae for biodiesel.     

Fermentative hydrogen production by Clostridium butyricum CGS5 using carbohydrate-rich microalgal biomass as feedstock

In this work, a carbohydrate-rich microalga, Chlorella vulgaris ESP6, was grown photoautotrophically to fix the CO₂. The resulting microalgal biomass was hydrolyzed by acid or alkaline/enzymatic treatment and was then used for biohydrogen production with Clostridium butyricum CGS5. The C. vulgaris biomass could be effectively hydrolyzed by acid pretreatment while similar hydrolysis efficiency was achieved by combination of alkaline pretreatment and enzymatic hydrolysis. The biomass of C. vulgaris ESP6 containing a carbohydrate content of 57% (dry weight basis) was efficiently hydrolyzed by acid treatment with 1.5% HCl, giving a reducing sugars (RS) yield of nearly 100%. C. butyricum CGS5 could utilize RS from C. vulgaris ESP6 biomass to produce hydrogen without any additional organic carbon sources. The optimal conditions for hydrogen production were 37 °C and a microalgal hydrolysate loading of 9 g RS/L with pH-controlled at 5.5. Under the optimal conditions, the cumulative H₂ production, H₂ production rate, and H₂ yield were 1476 ml/L, 246 ml/L/h, and 1.15 mol/mol RS, respectively. The results demonstrate that the C. vulgaris biomass has the potential to serve as effective feedstock for dark fermentative H₂ production.     

Waste-free technology of wastewater treatment to obtain microalgal biomass for biodiesel production

Five axenic cultures of microalgae were isolated from the wastewater of Almaty city and identified as Chlorella vulgaris strain № 1, Chlorella sp. strain № 3, Scenedesmus obliquus, Phormidium foveolarum and Lyngbya limnetica. Among these strains, C. vulgaris strain № 1 was characterized by the maximum growth rate and the highest productivity. Mass cultivation of this strain in wastewater resulted in accumulation of 5 × 10⁷ cells per ml in 16 days, and in the removal of ～95% of pollutants from water. Cells of C. vulgaris consisted of ～35% proteins, 29% carbohydrates, 30% lipids, and 6% ash, as calculated on a dry weight basis. The major fatty-acids of C. vulgaris were represented by palmitic, cis-7,10-hexadecenoic acid, linoleic, and α-linolenic acids. Culturing in wastewater decreased the unsaturation index of FAs. Thus, C. vulgaris cells are suitable for both waste water purification and accumulation of biomass for further biodiesel production.     

Renewable biomass production by mixotrophic algae in the presence of various carbon sources and wastewaters

This study evaluated mixotrophic growth potential of native microalgae in media supplemented with different organic carbon substrates and wastewaters. Three robust mixotrophic microalgae viz. Chlamydomonas globosa, Chlorella minutissima and Scenedesmus bijuga were isolated after long-term enrichments from industrial wastewater. The mixotrophic growth of these microalgae resulted in 3–10 times more biomass production relative to phototrophy. Glucose, sucrose and acetate supported significant mixotrophic growth. Poultry litter extract (PLE) as growth medium recorded up to 180% more biomass growth compared to standard growth medium BG11, while treated and untreated carpet industry wastewaters also supported higher biomass, compared to BG11 growth with no significant effect of additional nitrogen supplementation. Supplementing treated wastewater and PLE with glucose and nitrogen resulted in 2–7 times increase in biomass relative to the unamended wastewaters or PLE. The consortia of Chlamydomonas–Chlorella and Scenedesmus–Chlorella were the best for PLE and untreated wastewater respectively, while a combination all three strains was suitable for both PLE and wastewater. These algae can be good candidates for biofuel feedstock generation as they would not require freshwater or fertilizers. Such mixotrophic algal consortia offer great promise for production of renewable biomass for bioenergy applications using wastewaters.     

Growth and lipid accumulation properties of a freshwater microalga, Chlorella ellipsoidea YJ1, in domestic secondary effluents

The combination of microalga-based biodiesel production and wastewater treatment is a promising approach to solve problems related to the energy crisis as well as eutrophication in bodies of water. A freshwater microalga, Chlorella ellipsoidea YJ1, with a high capacity for biomass production and lipid accumulation in secondary effluent was isolated. C. ellipsoidea YJ1 could achieve a biomass of 425 mg L⁻¹ (dry weight) in domestic secondary effluent treated with activated sludge technology; and the lipid content per unit of algal biomass was as high as 43% (w/w) in this condition. The lipid growth rate of C. ellipsoidea YJ1 in domestic secondary effluents could attain 11.4 mg/L. Furthermore, after the cultivation of C. ellipsoidea YJ1, the removal efficiencies of nitrogen and phosphorus from the secondary effluent studied in this paper were more than 99% and 90%, respectively. Logistic and Monod models were used successfully to simulate the growth of C. ellipsoidea YJ1, and its maximum biomass and maximum population growth rate under different initial concentrations of nitrogen and phosphorus could be simulated and predicted using the models. .     

Evaluating renewable carbon sources as substrates for single cell oil production by Cunninghamella echinulata and Mortierella isabellina

The biochemical behavior (biomass production, accumulation of total lipid, substrate uptake, fatty acid composition of fungal oil) of two oleaginous Mucorales strains, namely Mortierella isabellina ATHUM 2935 and Cunninghamella echinulata ATHUM 4411, was studied when the aforementioned microorganisms were cultivated on xylose, raw glycerol and glucose under nitrogen-limited conditions. Significant differences in the process of lipid accumulation as related to the carbon sources used were observed for both microorganisms. These differences were attributed to the different metabolic pathways involved in the assimilation of the above substrates. Therefore, the various carbon sources were channeled, at different extent, to storage lipid or to lipid-free biomass formation. Although glucose containing media favored the production of mycelial mass (15 g L^?1 of total biomass in the case of C. echinulata and 27 g L^?1 in the case of M. isabellina), the accumulated lipid in dry matter was 46.0% for C. echinulata and 44.6% for M. isabellina. Lipid accumulation was induced on xylose containing media (M. isabellina accumulated 65.5% and C. echinulata 57.7% of lipid, wt wt^?1, in dry mycelial mass). In these conditions, lipids of C. echinulata contained significant quantities of γ-linolenic acid (GLA). This fungus, when cultivated on xylose, produced 6.7 g L^?1 of single cell oil and 1119 mg L^?1 of GLA. Finally, the growth of both C. echinulata and M. isabellina on raw glycerol resulted in lower yields in terms of both biomass and oil produced than the growth on xylose.     

Influence of inoculum cell density and carbon dioxide concentration on fed-batch cultivation of Nannochloropsis oculata

The marine microalgae Nannochloropsis oculata is a promising source of biofuel because of its high lipid content. For achieving high productivity of oil from microalgae, a high cell concentration before harvesting is beneficial. The present study investigated fed-batch cultures of N. oculata fed with vitamins and nutrient solutions and found that the biomass yield of N. oculata in the fed-batch culture was 1.25 times higher than that in batch culture. Fed-batch cultivation, especially at high illumination, decreased the inhibitory effect of high carbon dioxide (CO₂) concentration on the microalgal growth. The specific growth rate was directly proportional to the light intensity in the CO₂ environment. A light intensity of 40,000 Lux was able to achieve high specific growth rates in fed-batch cultivation at a CO₂ volume fraction of 2%–15%. The tolerance of N. oculata to CO₂ was enhanced by the daily feeding of nutrients in the fed-batch cultivation. At 2% CO₂, a final cell density of about OD₆₈₂ = 11.4 was achieved in the fed-batch culture in 30 days. Furthermore, a cell density of 14.4 g L⁻¹ was obtained by outdoor fed-batch cultivation in 27 days.     

Culture conditions affecting H2 production by phototrophic bacterium Rhodobacter sphaeroides KD131

The effect of culture conditions on photo-H₂ production was investigated using the photosynthetic bacterium Rhodobacter sphaeroides KD131. When the initial cell concentrations were either below or above a threshold of 0.56 g-dcw/L, the H₂ production decreased due to an imbalance between the biomass and the substrate. Malate- and succinate-fed cultures exhibited the highest substrate conversion to H₂ production, whereas more than 85% of the substrate was utilized for cell growth in acetate- and butyrate-fed cultures. Compared with (NH₄)₂SO₄, glutamate as a nitrogen source was more appropriate for the initial H₂ production, but inhibited H₂ evolution during extended cultivation due to released NH₄⁺ ion. Even though the KD131 strain grew well under slightly acidic conditions, the pH value should be maintained in a neutral range in order to enhance H₂ production. The highest H₂ yield of 3.65 mol-H₂/mol-succinate was achieved when the KD131 strain grew in the succinate–glutamate medium with an initial cell concentration of 0.56 g-dcw/L and the pH level controlled to 7.5.     

Neochloris oleoabundans grown on anaerobically digested dairy manure for concomitant nutrient removal and biodiesel feedstock production

Microalgae have been investigated as a promising biodiesel feedstock; however, large-scale production is not currently cost-competitive with petroleum diesel, and its environmental impacts have received little attention. Using wastewater to supply nutrients for algal growth obviates synthetic fertilizer use, provides on-site nutrient removal, and reduces greenhouse gas emissions. In this work, anaerobically digested dairy manure was used to grow the oleaginous green alga Neochloris oleoabundans. In batch culture experiments with both synthetic media and anaerobic digester effluent, N. oleoabundans assimilated 90-95% of the initial nitrate and ammonium after 6 d and yielded 10-30% fatty acid methyl esters on a dry weight basis. Cellular lipid content and the N concentration in the growth media were inversely correlated. In addition, the proportion of polyunsaturated fatty acids (i.e. C16:3, C18:2, and C18:3) decreased with N concentration over time while the proportion of C18:1 fatty acid increased. Although N deficiency is likely the primary driver behind lipid accumulation, the influence of culture pH confounded results and requires further study. Other living microorganisms in the digester effluent were not observed to affect algal growth and lipid productivity, though the breakdown of organic nitrogen may have hindered lipid accumulation traditionally achieved through the manipulation of synthetic media. This work highlights the potential for waste-grown mono-algal cultures to produce high quality biodiesel while accomplishing simultaneous wastewater treatment.