Heterotrophic Cultivation Of Microalgae Research Articles

Integrating microalgae growth in biomethane plants: Process design, modelling, and cost evaluation

The integration of microalgae cultivation in anaerobic digestion (AD) plants can take advantage of relevant nutrients (ammonium and ortho-phosphate) and CO2 loads. The proposed scheme of microalgae integration in existing biogas plants aims at producing approximately 250 t·y−1 of microalgal biomass, targeting the biostimulants market that is currently under rapid expansion. A full-scale biorefinery was designed to treat 50 kt·y−1 of raw liquid digestate from AD and 0.45 kt·y−1 of CO2 from biogas upgrading, and 0.40 kt·y−1 of sugar-rich solid by-products from a local confectionery industry. An innovative three-stage cultivation process was designed, modelled, and verified, including: i) microalgae inoculation in tubular PBRs to select the desired algal strains, ii) microalgae cultivation in raceway ponds under greenhouses, and iii) heterotrophic microalgae cultivation in fermenters. A detailed economic assessment of the proposed biorefinery allowed to compute a biomass production cost of 2.8 ± 0.3 €·kg DW−1, that is compatible with current downstream process costs to produce biostimulants, suggesting that the proposed nutrient recovery route is feasible from the technical and economic perspective. Based on the case study analysis, a discussion of process, bioproducts and policy barriers that currently hinder the development of microalgae-based biorefineries is presented.

Solar-powered algal production on vegetable processing industry wastewater at pilot scale

Τhis paper proposed a sustainable treatment scheme for effluents from fruit and vegetable processing industries with high organic load that combined heterotrophic microalgae cultivation with microalgae spray drying producing end-products of commercial interest (biofertilisers and/or animal feed). A pilot plant was operated with feedstock from a fruit and vegetable processing industry and its final products were assessed. The pilot plant was powered by renewable energy (solar energy supported by biomass), which minimized the carbon footprint and operating costs of the process. Through the ultimate analysis of the produced algae, it was evident that in all cases it could be commercialized as a solid NPK organic fertilizer being in line with the respective EU and Spanish legislation framework. As far as the animal feed perspective is concerned, the end-product of the pilot plant could be efficiently included in the feed of various animals, substituting a significant part of the animal feed required. The results from the implementation were used for the design of a full-scale implementation of the innovative treatment scheme, proving that 1.26 ton/d of microalgae could be produced, which could be later used as fertiliser and/or animal feed from the daily wastewater production of a medium sized fruit and vegetable processing company. From an environmental perspective, the proposed solution provides a gold standard example of the circular economy concept, since 1.15 kg CO2 equivalent per kg of sludge avoided could be saved. Additionally, the use of renewable energy (solar and biomass) will result in a saving of 0.531 kg of CO2 emissions per kWh consumed. Conclusively, the proposed treatment scheme could meet circularity and sustainability since the end-products quality permits their integration into new value chains.

Thermal-tolerant potential of ordinary Chlorella pyrenoidosa and the promotion of cell harvesting by heterotrophic cultivation at high temperature.

During the heterotrophic cultivation of microalgae, a cooled process against temperature rise caused by the metabolism of exogenous organic carbon sources greatly increases cultivation cost. Furthermore, microalgae harvesting is also a cost-consuming process. Cell harvesting efficiency is closely related to the characteristics of the algal cells. It may be possible to change cell characteristics through controlling culture conditions to make harvesting easier. In this study, the mesophilic Chlorella pyrenoidosa was found to be a thermal-tolerant species in the heterotrophic mode. The cells could maintain their maximal specific growth rate at 40°C and reached 1.45day-1, which is equivalent to that of cultures at 35°C but significantly higher than those cultured at lower temperatures. Interestingly, the cells cultured at 40°C were much easier to be harvested than those at lower temperatures. The harvesting efficiency of the cells cultured at 40°C reached 96.83% after sedimentation for 240min, while the cells cultured at lower temperatures were reluctant to settle. Likely, the same circumstance occurred when cells were harvested by centrifugation or flocculation. The promotion of cell harvesting for cells cultured at high temperatures was mainly attributed to increased cell size and decreased cell surface charge. To the best of our knowledge, this is the first report that cells cultured at high temperatures can promote microalgae harvesting. This study explores a new approach to simplify the cultivation and harvesting of microalgae, which effectively reduces the microalgae production cost.

Biosynthesis of microalgal lipids, proteins, lutein, and carbohydrates using fish farming wastewater and forest biomass under photoautotrophic and heterotrophic cultivation

Biorefineries enable the circular, sustainable, and economic use of waste resources if value-added products can be recovered from all the generated fractions at a large-scale. In the present studies the comparison and assessment for the production of value-added compounds (e.g., proteins, lutein, and lipids) by the microalga Chlorella sorokiniana grown under photoautotrophic or heterotrophic conditions was performed. Photoautotrophic cultivation generated little biomass and lipids, but abundant proteins (416.66 mg/gCDW) and lutein (6.40 mg/gCDW). Heterotrophic conditions using spruce hydrolysate as a carbon source favored biomass (8.71 g/L at C/N 20 and 8.28 g/L at C/N 60) and lipid synthesis (2.79 g/L at C/N 20 and 3.61 g/L at C/N 60) after 72 h of cultivation. Therefore, heterotrophic cultivation of microalgae using spruce hydrolysate instead of glucose offers a suitable biorefinery concept at large-scale for biodiesel-grade lipids production, whereas photoautotrophic bioreactors are recommended for sustainable protein and lutein biosynthesis.

Heterotrophic vs autotrophic production of microalgae: Bringing some light into the everlasting cost controversy

Heterotrophic or autotrophic? This is the continuous question the industry faces when microalgae production is the endeavor. Surprisingly, nowadays specialists have not reached a consensus on which is the most economical option. The current work analyses costs for heterotrophic and autotrophic cultivation of microalgae at an industrial scale. Heterotrophic cultivation of microalgae results in a production cost of 4.00 €·kg−1 of dry weight as a centrifuged paste. This is within the range of autotrophic costs, but still above the production cost in some photobioreactors. The study also identifies the current limitations on the technology and studies the effect on the cost of overcoming these. Once achieved, the advances in the process could result in a heterotrophic production cost reduced to 1.08 €·kg−1. Autotrophic cultivation seems competitive with heterotrophic production. It is time to leap forward in the autotrophic production scale to achieve the critical reduction in production cost.

Bioprocessing of volatile fatty acids by oleaginous freshwater microalgae and their potential for biofuel and protein production

To address the issue of high organic carbon costs in heterotrophic cultivation of microalgae, we evaluated the hypotheses by employing microalgae as a biorefinery for proteins and advanced biofuels after cultivation on volatile fatty acids (VFAs) instead of pure glucose. To prevent the inhibitory effect of VFAs on lipid synthesis, strains capable of tolerating high levels of VFAs were selected. Growth and lipid synthesis by two freshwater microalgae, Auxenochlorella protothecoides and Chlorella sorokiniana, was optimized at different VFA concentrations. Maximum biomass and lipid content in A. protothecoides (10.66 g/L, 33.93%) and C. sorokiniana (7.98 g/L, 39.80%) were obtained by replacing glucose with 30 g/L acetate at C/N 60. The generated lipids were compliant with existing standards for biodiesel. Moreover, when grown on acetate, both microalgae contained the complete range of essential and non-essential amino acids. Finally, single-source commercial VFAs were replaced with VFAs mixture after acidogenic fermentation of waste lignocellulosic biomass from brewers’ spent grain. The mixture allowed successful mixotrophic and heterotrophic cultivation of both microalgae, demonstrating feasibility of this low-cost carbon source in fuel-grade biodiesel production.

Effect of Fed-batch Culturing on the Growth and Lipid Production of Chlorella vulgaris fo. tertia Applying pH-auxostat Acetic Acid and Predefined Exponential Glucose Feeding

The techniques of heterotrophic microalgae cultivation used to be resulting in higher productivity and better yield than autotrophic culturing. Batch cultivation strategy is commonly used with high glucose concentration, but its potential is limited for biomass production at an industrial scale. Usually, the best productivity can obtain at lower glucose concentration. Moreover, other carbon sources can cause inhibition at higher concentrations. Therefore, the fed-batch cultivation strategy is an obvious choice, as it can maintain the optimal amount of carbon source can be maintained throughout the fermentation by automating the feeding. Such self-regulatory automation is provided by the pH-auxostat addition of acetic acid, which was investigated in this study for Chlorella vulgaris fo. tertia. The pH-auxostat fermentation was upscaled, then the feeding profile was modelled and transformed to another fermentation where glucose was used as a carbon source instead of acetic acid. Thus, the preferred carbon sources were compared under the same circumstances. It was found that the tested strain consumes dissolved oxygen very fast on both carbon substrates. It favored the acetic acid at high nitrogen and phosphorus concentrations. The final biomass concentration was 29.2 g/L under pH-auxostat fed-batch strategy with acetic acid and 18.8 g/L with glucose, respectively. The highest lipid content (393 mg/g) was measured from the biomass in the case of acetic acid. The fermentation settings need further optimization, but the results concluded that pH-auxostat acetic acid feeding has a great potential for scale-up of Chlorella fermentation.

Utilization of the microalga Scenedesmus quadricauda for hexavalent chromium bioremediation and biodiesel production

The purpose of this study was to evaluate the bioremediation potential of the microalga Scenedesmus quadricauda in removing hexavalent chromium (Cr (VI)) from synthetic wastewater, under autotrophic and heterotrophic conditions and different inoculum concentrations. In both cultivation modes, the highest inoculum density of 0.8 g L-1 led to the highest Cr (VI) removal efficiency. In addition, Cr (VI) stress was more severe in 10 ppm compared to 5 ppm, while heavy metal effects were alleviated under heterotrophic conditions. Concurrently, Cr (VI) stress affected biomass quality, resulting in an increase in lipid and carbohydrate production and decreased proteins. Furthermore, under higher Cr (VI) concentration more saturated and monounsaturated fatty acids were produced, while monounsaturated fatty acids content was also greater under heterotrophic conditions. In total, the findings of this study highlight the advantages of heterotrophic cultivation of microalgae for concomitant industrial wastewater treatment and biofuel production.

Mixotrophic and heterotrophic cultivation of microalgae using winery wastewater as organic carbon source

Mixotrophic and heterotrophic cultivation of microalgae using winery wastewater as organic carbon source

Growth enhancement in marine copepod, Pseudodiaptomus annandalei fed with the sodium acetate containing N/P starved medium grown, Tetraselmis suecica

Algal lipids are considered to be good raw materials towards aqua feed formulation and biofuels production, and hence, they have been investigated for decades. Using external carbon source, microalgal cultivation has been getting attention due to high biomass productivity and lipid content with flexible cultivation of algae. In this present study, a marine microalga, Tetraselmis suecica was cultivated by combined method with sodium acetate as carbon source and reciprocal nutrients concentration. The reciprocal concentration of nitrogen and phosphorus with sodium acetate supply towards lipid and fatty acid methyl esters (FAME) synthesis in terms of live feed efficiency was attempted. The highest FAME content (78.44 ± 1.18% of DW) of alga was obtained in nil nitrogen condition with limited phosphate concentration (N0-PL) followed by N0-P (77.31 ± 2.5% of DW) combination. Limitation of phosphate with combination of nil nitrogen (N0-PL) showed slight increment of total FAME accumulation and its higher increments resulted in mono-unsaturated fatty acids (MUFA) accumulation, indicating nitrogen starvation was a real FAME trigger under heterotrophic cultivation. Effect of heterotrophic cultivated T. suecica feeding on marine copepod Pseudodiaptomus annandalei produced higher population density (119 ± 15 ind. L−1) in N0-P combination and high rate (71.98%) of nauplii production was obtained in N0-PL combination. These findings suggested that the heterotrophic microalgae cultivation can be practiced for aqua feed (especially for copepod cultivations) formulation as well as to phosphorous removal from wastewaters.

Heterotrophic Microalgae Cultivation Using Jellyfish Protein Extract to Produce Bioactive Peptides

Jellyfish are often seen as a nuisance, and while attempts have been made to use jellyfish waste as fertilizer and food, they have not proved fruitful. In this study, proteins extracted from jellyfish (Aurelia aurita) are investigated as a novel nutrient source in culture medium and tested on aerial microalgae under heterotrophic condition. Results demonstrated that Vischeria helvetica KGU-Y001 heterotrophically grew in the extract-containing culture medium in the presence of light. Decomposition of the extracted protein in the culture medium containing V. helvetica cells was verified both in the presence and absence of light. The inhibitory activity of angiotensin I-converting enzyme (ACE) was measured in the culture medium when in the presence of light. The hydrolysates in the medium were separated using a gel filtration column and a peptide fraction with approximately 400 Da, and high ACE inhibitory activity (IC50 = 72 µg mL−1) was obtained. The results presented here will help to facilitate the development of culture systems with jellyfish waste as a resource for culture medium.

Nutrient-driven forward osmosis coupled with microalgae cultivation for energy efficient dewatering of microalgae

In this study, a new forward osmosis (FO) coupled with microalgae cultivation process was developed as a means of energy-efficient dewatering of microalgae, adopting a microalgal culture medium as a draw solution. In so doing, the energy-intensive regeneration of draw solution is omitted, and the resulting dilute solution of nutrients is directly reused as a culture medium. Process feasibility was proved by using glucose, a common carbon source for heterotrophic microalgae cultivation, as draw solute in dewatering the microalgal culture. When glucose and microalgal suspension were used as draw solution and feed solution, respectively, membrane orientation and cross-flow velocity (CFV) were found to have substantial impacts on dewatering performance of forward osmosis: compared to active layer-facing-draw solution orientation (AL-DS) and low cross-flow velocity, active layer-facing-feed solution orientation (AL-FS) and high cross-flow velocity were more effective at reducing reversible and irreversible fouling and thereby improving water flux. In comparison with sea salt, which is considered a conventional draw solute that does not require a regeneration step, glucose showed better dewatering performance due to its lower reverse solute flux (RSF), which caused flux decline due to loss of osmotic pressure difference and cake-enhanced concentration polarization (CECP) in fouling layer. The nutrient-driven forward osmosis process coupled with microalgae cultivation led to the production of highly concentrated biomass (roughly 120 g/L), supporting the idea that this novel approach can offer a promising low-energy alternative for microalgal dewatering.

Heterotrophic cultivation of Ettlia sp. based on sequential hydrolysis of Helianthus tuberosus and algal residue

Heterotrophic cultivation of microalgae results in substantial productivity but requires significant nutrient input. Alternative sources of major nutrients such as C and N should be carefully selected to make heterotrophic production of microalgal biomass feasible. In this study, Helianthus tuberosus and lipid-extracted algae (LEA) were hydrolyzed with sulfuric acid to grow Ettlia sp. YC001. Hydrolysis of H. tuberosus with 0.3 N of sulfuric acid resulted in fructose yield of 0.51 g g−1 and glucose yield of 0.14 g g−1 at 80 °C in 1 h. Proteolysis of algal residue with 2 N of sulfuric acid resulted in amino acid yield of 0.38 g g−1 at 110 °C in 24 h. To simplify the production of hydrolysates, the hydrolysis conditions were combined to establish a tailored sequential hydrolysis process. Then, simultaneous neutralization and salt removal of the hydrolysates were done with barium hydroxide. Consequently, cultivation of Ettlia sp. with the diluted sequential hydrolysate of H. tuberosus and LEA successfully achieved 5.90 g L−1 d−1 of biomass productivity and 1.42 g L−1 d−1 of lipid productivity with Ettlia sp.

O-04 オイルパーム樹液を利用した微細藻類の従属的培養法の検討

O-04 オイルパーム樹液を利用した微細藻類の従属的培養法の検討

Optimization of heterotrophic cultivation of Chlorella sp. HS2 using screening, statistical assessment, and validation

The heterotrophic cultivation of microalgae has a number of notable advantages, which include allowing high culture density levels as well as enabling the production of biomass in consistent and predictable quantities. In this study, the full potential of Chlorella sp. HS2 is explored through optimization of the parameters for its heterotrophic cultivation. First, carbon and nitrogen sources were screened in PhotobioBox. Initial screening using the Plackett-Burman design (PBD) was then adopted and the concentrations of the major nutrients (glucose, sodium nitrate, and dipotassium phosphate) were optimized via response surface methodology (RSM) with a central composite design (CCD). Upon validation of the model via flask-scale cultivation, the optimized BG11 medium was found to result in a three-fold improvement in biomass amounts, from 5.85 to 18.13 g/L, in comparison to a non-optimized BG11 medium containing 72 g/L glucose. Scaling up the cultivation to a 5-L fermenter resulted in a greatly improved biomass concentration of 35.3 g/L owing to more efficient oxygenation of the culture. In addition, phosphorus feeding fermentation was employed in an effort to address early depletion of phosphate, and a maximum biomass concentration of 42.95 g/L was achieved, with biomass productivity of 5.37 g/L/D.

Biological Carbon Recovery from Sugar Refinery Washing Water into Microalgal DHA: Medium Optimization and Stress Induction

Sugar refinery washing water (SRWW) contains abundant levels of carbon sources and lower levels of contaminants than other types of wastewater, which makes it ideal for heterotrophic cultivation of microalgae. Here, carbon sources in SRWW were utilized for conversion into the form of value-added docosahexaenoic acid (DHA) using Aurantiochytrium sp. KRS101. Since SRWW is not a defined medium, serial optimizations were performed to maximize the biomass, lipid, and DHA yields by adjusting the nutrient (carbon, nitrogen, and phosphorus) concentrations as well as the application of salt stress. Optimum growth performance was achieved with 30% dilution of SRWW containing a total organic carbon of 95,488 mg L−1. Increasing the nutrient level in the medium by supplementation of 9 g L−1 KH2PO4 and 20 g L−1 yeast extract further improved the biomass yield by an additional 14%, albeit at the expense of a decrease in the lipid content. Maximum biomass, lipid, and DHA yields (22.9, 6.33, and 2.03 g L−1, respectively) were achieved when 35 g L−1 sea salt was applied on a stationary phase for osmotic stress. These results demonstrate the potential of carbon-rich sugar refinery washing water for DHA production using Aurantiochytrium sp. KRS101 and proper cultivation strategy.

Ultrahigh-cell-density heterotrophic cultivation of the unicellular green microalga Scenedesmus acuminatus and application of the cells to photoautotrophic culture enhance biomass and lipid production.

Although production of biodiesels from microalgae is proved to be technically feasible, a commercially viable system has yet to emerge. High‐cell‐density fermentation of microalgae can be coupled with photoautotrophic cultivation to produce oils. In this study, by optimizing culturing conditions and employing a sophisticated substrate feed control strategy, ultrahigh‐cell‐density of 286 and 283.5 g/L was achieved for the unicellular alga Scenedesmus acuminatus grown in 7.5‐L bench‐scale and 1,000‐L pilot‐scale fermenters, respectively. The outdoor scale‐up experiments indicated that heterotrophically grown S. acuminatus cells are more productive in terms of both biomass and lipid accumulation when they are inoculated in photobioreactors for lipid production as compared to the cells originally grown under photoautotrophic conditions. Technoeconomic analysis based on the pilot‐scale data indicated that the cost of heterotrophic cultivation of microalgae for biomass production is comparable with that of the open‐pond system and much lower than that of tubular PBR, if the biomass yield was higher than 200 g/L. This study demonstrated the economic viability of heterotrophic cultivation on large‐scale microalgal inocula production, but ultrahigh‐productivity fermentation is a prerequisite. Moreover, the advantages of the combined heterotrophic and photoautotrophic cultivation of microalgae for biofuels production were also verified in the pilot‐scale.

Estimation of the economy of heterotrophic microalgae- and insect-based food waste utilization processes

An estimation of the economy of Hermetia illucens and Chlorella pyrenoidosa cultivations as food waste treatment with benefits was carried out. For both organisms, a process scale was assumed to treat 56.3 t of wet food waste per day, which is equivalent to the amount of food waste appearing in a catchment area of 141,000 inhabitants. Using hypothetical insect and heterotrophic microalgae cultivation processes, a daily production of 3.64 t and 7.14 t dried biomass, respectively, can be achieved. For the cultivation of H. illucens, equipment and daily operational costs were estimated at 79,358.15 € and 5,281.56 €, respectively. Equipment and operational costs for the C. pyrenoidosa cultivation was 50 and 6 times higher, respectively. The higher costs reflect the more complex and advanced process compared to H. illucens cultivation. The internal return rate for a plant lifetime of 20 times revealed an economic benefit when C. pyrenoidosa biomass is produced. Nevertheless, both processes were found economically feasible when dried biomass is directly commercialized as food without any further downstream processing. However, extraction and purification of special chemicals, such as unsaturated fatty acids and pigments, can significantly increase the revenue.

Heterotrophic cultivation of microalgae in straw lignocellulose hydrolysate for production of high-value biomass rich in polyunsaturated fatty acids (PUFA)

As the current bioconversion of lignocellulose mainly towards on bioenergy, a bioproduct with higher value would be promising to improve the profit output from the process. Straw lignocellulose, hydrothermally pretreated by ammonium sulfite, was used as the substrate to produce high-value microalgal biomass in this study. Medium based on the lignocellulose hydrolysate were prepared to cultivate four locally isolated microalgal strains, Chlorella and Scenedesmus species. All of the microalgal strains well adapted to the lignocellulose hydrolysate. Among the strains, C. ZTY4 had the highest growth rate in hydrolysate-based medium with a generation time of only 10.2 h, which was much shorter than that in glucose medium (16.7 h) and former reports. The polyunsaturated fatty acids (PUFA) content of C. ZTY4 reached to 66.4% in total fatty acids of C. ZTY4, which was well qualified for high-value applications. C. ZTY4 was identified as Chlorella pyrenoidosa by 18S rDNA sequencing. This research proposed a bioconversion approach from straw lignocellulose to high-value microalgal biomass and screened C. ZTY4 as a qualified strain.

Domestic Wastewater Treatment and Lipid Accumulation for Biodiesel Production by an Isolated Heterotrophic Microalgae from an Arid Climate Zone

The isolation of microalgae that can use carbon-rich pollutant from wastewater and accumulate lipids is of great interest in biodiesel production. The heterotrophic cultivation of microalgae could overcome light dependency, and hence increase the yield of microalgae lipid per unit area. After several microbial screening and acclimation procedures, strains of microalgae were proved to be tolerant of wastewater and to grow heterotrophically. One top-performing strain (AE2) was identified through morphological observation as Chlorella sp and was isolated from an open pond of domestic wastewater. This microalgae is able to grow in raw wastewater at 20°C with no illumination and eliminates 100% of its COD (424 mg/L) in 9 days. The biomass produced in wastewater as growth medium comprises 53% of fats of the dry mass and 68% of fats of the dry mass in BG 11 culture medium supplemented with 5 g of glucose/L. The analysis of fatty acid methyl esters FAME composition was 32.5 % of the total biomass. The extracted microalgae oil was converted to good quality biodiesel. This heterotrophic isolated microalgae is a promising strain in terms of wastewater COD removal and cytoplasmic accumulation of high quality and quantity lipids for prospective biodiesel production.