Third generation biobutanol production by Clostridium beijerinckii in a medium containing mixotrophically cultivated Dunaliella salina biomass.

This study aims the third generation biobutanol production in P2 medium supplemented D. salina biomass mixotrophically cultivated with marble waste (MW). The wastes derived from the marble industry contain approximately 90% of carbon-rich compounds. Microalgal growth in mixotrophic conditions was optimized in the 0.4-2 g/L of MW concentration range. The highest microalgal concentration was obtained as 0.481 g/L in the presence of 1 g/L MW. Furthermore, some important parameters for the production of biobutanol, such as microalgal cultivation conditions, initial mixotrophic microalgal biomass loading (50-300 g/L), and fermentation time (24-96 h) were optimized. The highest biobutanol, total ABE, biobutanol yield and productivity were determined as 11.88 g/L, 13.89 g/L, 0.331 g/g and 0.165 g/L/h at the end of 72 h in P2 medium including 60 g/L glucose and 200 g/L microalgal biomass cultivated in 1 g/L MW, respectively. The results show that D. salina is a suitable raw material for supporting Clostridium beijerinckii DSMZ 6422 cells on biobutanol production. To the best of our knowledge, this is the first study on the use of MW which is a promising feedstock on the mixotrophic cultivation of D. salina for biobutanol production.

Mixotrophic Cultivation of Microalgae Using Biogas as the Substrate.

Biogas utilization through biotechnology represents a potential and novel technology. We propose the microalgal mixotrophic cultivation to convert biogas to microalgae-based biodiesel, in which methanotroph was co-cultured to convert CH4 to organic intermediate (and CO2) for microalgal mixotrophic growth. This study constructed a co-culture of Methylocystis bryophila (methanotroph) and Scenedesmus obliquus (microalgae) with biogas feeding. Compared with the single culture of S. obliquus, higher microalgal biomass but a lower chlorophyll concentration was observed. The organic metabolism-related genes were upregulated, verifying microalgal mixotrophic growth. The stoichiometric calculation of M. bryophila culture shows that M. bryophila tends to release organic matter rather than grow under a low O2 content. M. bryophila rarely grew under five different light intensities, indicating that M. bryophila acts as a biocatalyst in the co-culture. The organic intermediate released by methanotroph increased the maximum biomass of microalgal culture, accelerated nitrogen absorption, accumulated more monounsaturated fatty acids, and improved the adaptation to light. The co-culture of microalgae and methanotroph may provide new opportunities for microalgae-based biodiesel production using biogas as a substrate.

Mixotrophic Cultivation Optimization of Microalga Euglena pisciformis AEW501 for Paramylon Production.

Euglena, a flagellated unicellular protist, has recently received widespread attention for various high-value metabolites, especially paramylon, which was only found in Euglenophyta. The limited species and low biomass of Euglena has impeded paramylon exploitation and utilization. This study established an optimal cultivation method of Euglena pisciformis AEW501 for paramylon production under mixotrophic cultivation. The results showed that the optimum mixotrophic conditions were 20 °C, pH 7.0, and 63 µmol photons m-2∙s-1, and the concentrations of sodium acetate and diammonium hydrogen phosphate were 0.98 g L-1 and 0.79 g L-1, respectively. The maximal biomass and paramylon content were 0.72 g L-1 and 71.39% of dry weight. The algal powder contained more than 16 amino acids, 6 vitamins, and 10 unsaturated fatty acids under the optimal cultivation. E. pisciformis paramylon was pure ß-1,3-glucan-type polysaccharide (the purity was up to 99.13 ± 0.61%) composed of linear glucose chains linked together by ß-1,3-glycosidic bonds. These findings present a valuable basis for the industrial exploitation of paramylon with E. pisciformis AEW501.

Mariculture wastewater treatment with Bacterial-Algal Coupling System (BACS): Effect of light intensity on microalgal biomass production and nutrient removal.

Mariculture wastewater generated from the mariculture industry has increased public concern due to its impact on the sustainability of aquatic environments and aquaculture practices. Herein, the Bacterial-Algal Coupling System was applied for mariculture wastewater treatment. Microalgae growth in heterotrophy and mixotrophy (2000-8000 lux) was first compared. The best microalgal growth and nutrient removal were obtained at 5000 lux, where biomass productivity of microalgae was 0.465 g L-1 d-1, and 98.1% of chemical oxygen demand, 70.7% of ammonia-nitrogen, and 90.0% of total phosphorus were removed. To further understand the nutrient removal through microalgae cultivation, the enzyme activities involved in the Calvin cycle and the Tricarboxylic Acid cycle at different light intensities were determined. Under mixotrophic cultivation, there was a coordination between photosynthesis and heterotrophic metabolism in the agal cell, which resulted in a high algal biomass production and removal efficiency of nutrients. This study provided a novel insight into the bioremediation of mariculture wastewater and microalgae cultivation.

Enhancing growth and oil accumulation of a palmitoleic acid-rich Scenedesmus obliquus in mixotrophic cultivation with acetate and its potential for ammonium-containing wastewater purification and biodiesel production.

A palmitoleic acid-rich Scenedesmus obliquus strain SXND-02 was isolated from ammonium-containing wastewater. Biomass and lipid production were examined for this microalgal strain in photoautotrophic, heterotrophic, and mixotrophic cultivations, respectively, in order to extend its application in wastewater purification coupled with production of valued bio-products. Among the tested conditions, the microalga had better growth and higher lipid accumulation in mixotrophy. NH4Cl inhibited the microalgal growth in photoautotrophic cultivation. However, NaAc alleviated this inhibition in both heterotrophy and mixotrophy. Using 7 g L-1 NaAc and 0.5 g L-1 NH4Cl as carbon and nitrogen sources significantly increased the algal biomass and lipid yields under mixotrophic cultivation, with the highest levels up to 1.0 g L-1 and 59.88%, respectively. Fatty acid profiling indicated that palmitoleic acid was 23% in the S. obliquus SXND-02 under mixotrophic condition, which was about 21-fold higher than that in the control S. obliquus. Furthermore, this microalgal strain was tested in the chicken farm wastewater (CFW) containing high ammonium. Compared with other treatments, the S. obliquus SXND-02 cultivated in the 1/2 CFW + NaAc medium produced larger amounts of biomass (2.18 g L-1) and lipids (50.22%), and simultaneously higher removal rates of total nitrogen (TN) (80%), total ammonium nitrogen (TAN) (68%), total phosphate (TP) (82%), biological oxygen demand (BOD) (86%) and chemical oxygen demand (COD) (89%) from wastewater. The present data indicate that this excellent microalga can be used in mixotrophic cultivation for wastewater purification coupled with commercial production of valued biomass and high-quality algal oils.

Growth behavior, glucose consumption and phenol removal efficiency of Chlorella vulgaris under the synergistic effects of glucose and phenol.

The use of algae is an effective approach to remove phenol and its derivatives from polluted water. The growth behavior, glucose consumption and phenol removal efficiency of Chlorella vulgaris under the synergistic effects of glucose and phenol were investigated. The evolutions of tolerance and removal efficiency of C. vulgaris to phenol under different trophic modes and glucose contents were observed. The results revealed that growth of C. vulgaris were inhibited with the increase of phenol from 0 to 400 mg L-1 in culture media; the tolerance to phenol enhanced with the addition of glucose from 2 to 10 g L-1, while glucose consumption was inhibited with the increase of phenol content; phenol removal efficiency varied with glucose concentrations in mixotrophic media. The finding suggested that phenol inhibited the growth of C. vulgaris and glucose assimilation under mixotrophic cultivation, while appropriate glucose addition could enhance the tolerance of C. vulgaris to phenol and affect the phenol removal efficiency.

Outdoor microalgae cultivation in airlift photobioreactor at high irradiance and temperature conditions: effect of batch and fed-batch strategies, photoinhibition, and temperature stress.

The microalgae Scenedesmus abundans cultivated in five identical airlift photobioreactors (PBRs) in batch and fed-batch modes at the outdoor tropical condition. The microalgae strain S. abundans was found to tolerate high temperature (35-45 °C) and high light intensity (770-1690 µmol m- 2 s- 1). The highest biomass productivities were 152.5-162.5 mg L- 1 day- 1 for fed-batch strategy. The biomass productivity was drastically reduced due to photoinhibition effect at a culture temperature of > 45 °C. The lipid compositions showed fatty acids mainly in the form of saturated and monounsaturated fatty acids (> 80%) in all PBRs with Cetane number more than 51. The fed-batch strategies efficiently produced higher biomass and lipid productivities at harsh outdoor conditions. Furthermore, the microalgae also accumulated omega-3 fatty acid (C18:3) up to 14% (w/w) of total fatty acid at given outdoor condition.

New insight into Clostridium butyricum-ferroferric oxide hybrid system in exogenous carbon dioxide-assisted anaerobic fermentation for acetate and butyrate production.

Mixotrophic cultivation, utilizing both gas and organic substances, is commonly employed to minimize the carbon loss during anaerobic fermentation of bulk chemicals. Herein, a novel Clostridium butyricum-ferroferric oxide (Fe3O4) hybrid system, enhanced by exogenous carbon dioxide (CO2), was proposed to improve carbon recovery and optimize metabolite production. The results demonstrated that exogenous CO2 improved metabolite selectivity towards acetate/butyrate, while also accelerating CO2 fixation. Compared to pure Clostridium butyricum, the hybrid system significantly increased carbon conversion to primary metabolites, boosting butyrate and acetate production by 18.7 % and 18.4 %, respectively. Enzyme activity assays revealed that Fe3O4 and exogenous CO2 acted synergistically, enhancing the activities of key enzymes involved in CO2 assimilation. Additionally, Fe3O4 facilitated intra- and extracellular electron transfer, further improving the fermentation process. This study offers new insights into the combined effects of exogenous CO2 and Fe3O4 on anaerobic fermentation, providing an efficient strategy for carbon recovery and selective acetate/butyrate production.

A new microalgal negative carbon technology for landfill leachate treatment: Simultaneous removal of nitrogen and phosphorus.

Replete with ammonia nitrogen and organic pollutants, landfill leachate typically undergoes treatment employing expensive and carbon-intensive integrated techniques. We propose a novel microalgae technology for efficient, low-carbon simultaneous treatment of carbon, nitrogen, and phosphorus in landfill leachate (LL). The microbial composition comprises a mixed microalgae culture with Chlorella accounting for 82.58%. After seven days, the process with an N/P ratio of approximately 14:1 removed 98.81% of NH4+-N, 88.62 % of TN, and 99.55% of TP. Notably, the concentrations of NH4+-N and TP met the discharge standards, while the removal rate of NH4+-N was nearly three times higher than previously reported in relevant studies. The microalgae achieved a removal efficiency of 64.27% for Total Organic Carbon (TOC) and 99.26% for Inorganic Carbon (IC) under mixotrophic cultivation, yielding a biomass of 1.18 g/L. The treatment process employed in this study results in a carbon emissions equivalent of -8.25 kgCO2/kgNremoved, representing a reduction of 33.56 kgCO2 compared to the 2AO + MBR process. In addition, shake flask experiments were conducted to evaluate the biodegradability of leachate after microalgae treatment. After microalgae treatment, the TOCB (Biodegradable Total Organic Carbon)/TOC ratio decreased from 56.54% to 27.71%, with no significant improvement in biodegradability. It establishes a fundamental foundation for further applied research in microalgae treatment of leachate.

A Response Surface Methodology Study for Chlorella vulgaris Mixotrophic Culture Optimization.

Glycerol is a carbon source that produces good biomass under mixotrophic conditions. Enhancing the composition of culture media in algae biomass production improves growth rates, biomass yield, nutrient utilization efficiency, and overall cost-effectiveness. Among the key nutrients in the medium, nitrogen plays a pivotal role. Urea can be effectively used as a nitrogen source and is considered a low-cost form of nitrogen compared to other sources. Urea metabolism releases some CO2 in photosynthesis, and magnesium plays a major role in urea uptake. Magnesium is another key nutrient that is key in photosynthesis and other metabolic reactions. To maximize glycerol consumption in the mixotrophic system and to obtain high biomass and lipid productions, the variations in MgSO4·7H2O and urea concentrations were evaluated in the growth medium of the microalgae. A response surface methodology (RSM) using a central composite design (CCD) was designed to maximize glycerol consumption at the initial cellular growth rates (up to four days). The magnesium and urea supply varied from 0.3 to 1.7 g L-1. Response surface methodology was utilized to analyze the results, and the highest glycerol consumption rate, 770.2 mg L-1 d-1, was observed when C. vulgaris was grown at 1.7 g L-1 urea, 1.0 g L-1 MgSO4·7H2O. Using the optimal urea and magnesium concentrations with acetate, glucose, and glycerol as carbon sources, the same lipid content (10% average) was achieved on day 4 of mixotrophic C. vulgaris culture. Overall, the results show that mixotrophic growth of C. vulgaris using urea with an optimum magnesium concentration yields large amounts of fatty acids and that the carbon source greatly influences the profile of the fatty acids.

Insights into carbon utilization under mixotrophic conditions in Chlamydomonas.

Mixotrophic microalgae cultivation with various carbon resources is considered as a strategy that could increase biomass. However, the mechanism of carbon utilization between inorganic carbon (IC) and organic carbon (OC) remains unknown. In this study, IC and OC consumption, chlorophyll fluorescence parameters, intracellular Nicotinamide adenine dinucleotide phosphate content and transcriptional changes in related genes were characterized. The results showed that IC was utilized preferentially, whereas 76% IC was consumed at 8 h. Subsequently, OC was the dominant carbon resource for fermentation. The cell density in the IC group was 100% higher than that in the group without IC at 24 h. Bicarbonate addition enhanced photosynthesis by dissipating less energy and generating more electrons and energy, which benefited OC assimilation. This finding was verified by qRT-PCR analysis. These results elucidate the carbon utilization mechanism under mixotrophic conditions, which provide clues for promoting microalgae growth by regulating carbon utilization.

Enhancement of microalgal biomass productivity through mixotrophic culture process utilizing waste soy sauce and industrial flue gas.

Wastewater treatment plants are indispensable facilities, which emit a massive amount of greenhouse gases. To boost CO2 mitigation and wastewater treatment performance, mixotrophic microalgae cultivation using wastewater has recently been proposed. In this study, food industry wastewater (waste soy sauce) was applied to Chlorella sorokiniana UTEX 2714 cultivation. By using a medium with 20% (v/v) of 10-fold diluted soy sauce, the biomass and fatty acid methyl ester (FAME) productivity enhanced by 1.93 and 1.76 times, respectively. Biomass productivity increased up to 5.2 times when using medium with high soy sauce content under high-intensity light that inhibits cell growth in photoautotrophic environments. Furthermore, industrial flue gas treatment with wastewater was demonstrated by outdoor semi-continuous cultivation with 42% improved biomass production. Consequently, these results suggest that mixotrophic microalgal cultivation has great potential to address both climate change and water pollution while producing valuable products and can contribute to building a sustainable society.

Astaxanthin production from the microalga Haematococcus lacustris with a dual substrate mixotrophy strategy.

This study investigates the development of dual-substrate mixotrophy strategy to cultivate the microalga Haematococcus lacustris for astaxanthin production. The influence of different concentrations of acetate and pyruvate on biomass productivity was first assessed individually, and then both substrates were used together to improve biomass growth in the green phase and astaxanthin accumulation in red the phase. The results showed that dual-substrates mixotrophy significantly increased the biomass productivity during green growth phase up to 2-fold compared to phototrophic controls. Furthermore, supplementation of dual-substrate to the red phase increased astaxanthin accumulation by 10% in the dual-substrate group compared to single-substrate acetate and no substrate. This dual-substrate mixotrophy approach shows promise for cultivating Haematococcus for commercial production of biological astaxanthin in indoor closed systems.

New insights into the production of fucoxanthin by mixotrophic cultivation of Ochromonas and Microcystis aeruginosa.

Fucoxanthin (Fx) has attracted great interest due to its remarkable biological activities such as antioxidant and anti-obesity, and its increasing demands in biopharmaceutical and cosmetic fields. However, its commercial production is limited by low yield and high cost. In this study, we isolated and identified a species of golden algae (Ochromonas sp.) capable of engulfing Microcystis aeruginosa (M. aeruginosa) and accumulating Fx. After 72 h mixotrophic cultivation of Ochromonas sp. and M. aeruginosa, the algal culture changed from green to yellow-brown, and the content of Fx and the daily production rate were up to 11.58 mg g-1, and 1.315 mg L-1 d-1, respectively. The utilization rate of M. aeruginosa was 527.27 fg cell-1. This study will not only provide a new thought to produce Fx in an efficient, low-cost, and sustainable way but an innovative method for the control and treatment of harmful cyanobacterial blooms from eutrophic freshwaters as well.

Mixotrophic microalgal-biofilm reactor augmenting biomass and biofuel productivity.

The present work aimed to evaluate the mixotrophic growth of Chlorella pyrenoidosa in a microalgal-biofilm reactor (MBR) using waste glycerol as an organic carbon source. The biomass productivity of C. pyrenoidosa (10.14 g m-2 d-1) under the mixotrophic mode was remarkably higher than that observed during the phototrophic mode (4.16 g m-2 d-1), under similar incubation conditions. The hydraulic retention time (HRT) of 6 d was found optimal for the higher productivity of microalgae in the MBR. Notably, based on biofuel quality, mixotrophically grown microalgal biomass was noted to have better suitability for biomethane production compared to biodiesel. Besides, up to 98.09, 75.74, and 55.86% removal of phosphate, nitrate, and COD, respectively, was recorded within 6 d under mixotrophic growth. Overall, the present findings magnificently demonstrate the efficient recycling of waste glycerol for higher biomass production coupled with phycoremediation using mixotrophic MBR.

Paradigm shift in algal biomass refinery and its challenges.

Microalgae have been studied and tested for over 70 years. However, biodiesel, the prime target of the algal industry, has suffered from low competitiveness and current steps toward banning the internal combustion engine all over the world. Meanwhile, interest in reducing CO2 emissions has grown as the world has witnessed disasters caused by global warming. In this situation, in order to maximize the benefits of the microalgal industry and surmount current limitations, new breakthroughs are being sought. First, drop-in fuel, mandatory for the aviation and maritime industries, has been discussed as a new product. Second, methods to secure stable and feasible outdoor cultivation focusing on CO2 sequestration were investigated. Lastly, the need for an integrated refinery process to simultaneously produce multiple products has been discussed. While the merits of microalgae industry remain valid, further investigations into these new frontiers would put algal industry at the core of future bio-based economy.

Insight into nitrogen and phosphorus coupling effects on mixotrophic Chlorella vulgaris growth under stably controlled nutrient conditions.

In water environment, nitrogen (N) and phosphorus (P) are biochemically dependent nutrients following the co-limitation concept for algae growth under mixotrophic mode. From a practical viewpoint, algae growth may not bring about significant change of the background nutrient concentration of an actual waterbody in contrast to a conventional batch system. In order to better understand the growth pattern of microalgae in aquatic environments, a series of experiments were conducted under stably controlled N-P levels for studying the N-P coupling effect on mixotrophic Chlorella vulgaris growth process, with attention paid to the physiological and biochemical characteristics. It was found that within the concentration range of N = 1-8 mg·L-1 and P = 0.1-1.0 mg·L-1, the variation of the N-P level slightly affected the specific growth rate, but significantly influenced nutrients uptake, biomass dry weight, chlorophyll contents of the grown C. vulgaris. The biochemical and elemental composition of the microalgae tended to be more sensitive to the N-P concentrations and ratios in the lower nutrient range (1-2 mg N·L-1, 0.1-0.4 mg P·L-1) in which the highest N and P conversion rates were gained as 90.18 ± 1.23% and 60.47 ± 1.59%, respectively. The P assimilation and conversion efficiencies were much affected by both N and P supplies, while the P supply showed little influence on N assimilation and conversion efficiencies. It was also noticed that the N level greatly affected the metabolic pathway involving nutrient assimilation, carbohydrate fixation and monosaccharide profile, resulting in conversion of the dominant fraction of protein at N ≤ 2 mg·L-1 into other biochemical compositions including lipids at N ≥ 3 mg·L-1. The fatty acid methyl esters (FAMEs) composition tended to differ with varied nutrient levels. These findings may deepen our understanding of algal growth in aquatic environment and provide perspective for eutrophication control.

Enhancement of Biomass Production in Colony-Forming Green Algae, Botryosphaerella sudetica, Under Mixotrophic Cultivation.

In this study, we characterized the potential of colony-forming green algae, Botryosphaerella sudetica KNUA107, isolated from Ulleung Island, South Korea, as a bioresource and analyzed the effects of mixotrophic cultivation on its bioresource production efficiency. Internal transcribed spacer (ITS) (ITS1, 5.8S, and ITS2), ribulose bisphosphate carboxylase large subunit (rbcL), and elongation factor Tu (tufa) regions were used for molecular identification and phylogenetic analysis. B. sudetica KNUA107 had a strong relationship with the green algae of Botryococcus and Botryosphaerella genera, which are colony-forming species, and was also associated with members of the Neochloris genus. To improve biomass productivity, we tested mixotrophic cultivation conditions using several organic carbon sources. Glucose supplementation stimulated B. sudetica KNUA107 growth and reduced the time needed to reach the stationary phase. In addition, the colony size was 1.5-2.0 times larger with glucose than in photoautotrophic cultures, and settleability improved in proportion to colony size. The total lipid content and biomass productivity were also higher in cultures supplemented with glucose. Among the lipid components, saturated fatty acids and monounsaturated fatty acids had the highest proportion. Our study suggests that B. sudetica KNUA107, which has enhanced efficiency in biomass production and lipid components under mixotrophic cultivation, has high potential as a bioresource.

Mixotrophic Growth of Chlorella sorokiniana on Acetate and Butyrate: Interplay Between Substrate, C:N Ratio and pH.

Microalgae can be cultivated on waste dark fermentation effluents containing volatile fatty acids (VFA) such as acetate or butyrate. These VFA can however inhibit microalgae growth at concentrations above 0.5-1 gC.L-1. This study used the model strain Chlorella sorokiniana to investigate the effects of acetate or butyrate concentration on biomass growth rates and yields alongside C:N:P ratios and pH control. Decreasing undissociated acid levels by raising the initial pH to 8.0 allowed growth without inhibition up to 5 gC.L-1 VFAs. However, VFA concentration strongly affected biomass yields irrespective of pH control or C:N:P ratios. Biomass yields on 1.0 gC.L-1 acetate were around 1.3-1.5 gC.gC -1 but decreased by 26-48% when increasing initial acetate to 2.0 gC.L-1. This was also observed for butyrate with yields decreasing up to 25%. This decrease in yield in suggested to be due to the prevalence of heterotrophic metabolism at high organic acid concentration, which reduced the amount of carbon fixed by autotrophy. Finally, the effects of C:N:P on biomass, lipids and carbohydrates production dynamics were assessed using a mixture of both substrates. In nutrient replete conditions, C. sorokiniana accumulated up to 20.5% carbohydrates and 16.4% lipids while nutrient limitation triggered carbohydrates accumulation up to 45.3%.

Role of C/N ratio on microalgae growth in mixotrophy and incorporation of titanium nanoparticles for cell flocculation and lipid enhancement in economical biodiesel application.

Present study aimed to evaluate the influence of carbon/nitrogen ratio (C/N) on mixotrophic growth of microalgae and role of nanomaterial in cell recovery and lipid improvement. In this study, three microalgae species were isolated, screened from local freshwater body for lipid assimilation. The microalgae were identified as Chlorococcum sp., Scenedesmus sp., and Euglena sp. Mixotrophic cultivation of each microalgae strain using various organic carbon sources was preferred in contrast with photoautotrophic mode. Sucrose represented as the preeminent source for enhancing the microalgae biomass of 3.5 g/L and lipid content of 58.35%, which was a significant improvement as compared to control. Later, response surface methodology-central composite design (RSM-CCD), tool was employed to optimize the C/N ratio and demonstrated the maximum biomass production of 5.02 g/L along with the increased lipid content of 60.34%. Ti nanoparticles (Ti nps) were added to the culture for lipid enhancement in the stationary phase and biomass removal was performed by nanoparticle (np)-mediated flocculation technique. Optimized concentration of 15 ppm Ti nps determined the cell harvesting efficacy of 82.46% during 45 min of sedimentation time and 1.23-fold lipid enhancement was reported. Extracted lipid was converted to fatty acid methyl esters (FAME) by the process of transesterification and analyzed by gas chromatography-mass spectrometry (GC-MS). Characterization of FAME revealed the presence of 56.31% of saturated fatty acid (SFA) and 29.06% unsaturated fatty acids (UFA) that could be processed towards sustainable biodiesel production. Hence, our results suggested that integration of mixotrophic cultivation and Ti nps emerged as a new cost-effective approach for biomass and lipid enhancement in microalgae Chlorococcum sp.