Algal Fermentation Research Articles

Life cycle assessment of industrial production of microalgal oil from heterotrophic fermentation

Heterotrophic fermentation has been proposed as an economically promising way to grow microalgae, a feedstock to produce microbial oils, which can substitute for fossil fuels and crop oil products. This study conducted a life cycle assessment (LCA) to evaluate the environmental impacts of microalgal oil produced from two microalgae Chlorella sp. and Schizochytrium sp., which were cultivated in commercial fermenters with a maximal volume of 60 m3. Algal oil produced from Chlorella sp. in autotrophic raceway ponds was also included as a base case this LCA study. The LCA results showed the oil produced by heterotrophic Schizochytrium sp. had generally lower impacts than autotrophic Chlorella sp. whereas oil produced from heterotrophic Chlorella sp. only had lower impacts in acidification, eutrophication, carcinogens, and ecotoxicity when compared to the autotrophic Chlorella sp. An in-depth analysis showed the algal fermentation stage accounted for over 86% of the total impacts in all categories, and nutrients and electricity use in fermentation caused the major impacts. Due to its high biomass density and high oil content properties, the harvest stage with a series of dewatering and lipid extraction steps for heterotrophic algae was processed with fewer impacts than autotrophic algae. This paper also compared four sugar sources used to grow algae in fermenters and found out that the sorghum stem was the best nutrient feedstock for fermentation. Finally, the authors determined the best fermentation time as around 131 h in algae cultivation to obtain a relatively high oil yield while keeping low environmental impacts.

Cofermenting Algal Biomass with Municipal Primary Solids to Enhance Carboxylate Production.

As water resource recovery facilities (WRRFs) implement biological nutrient removal (BNR) processes to remove excess wastewater nutrients, carboxylic acid demands increase; resource recovery processes (e.g., struvite, polyhydroxyalkanoate production) also demand carboxylates. In this regard, interest in algae to achieve tertiary treatment creates a new intraWRRF fermentation substrate. Indeed, fermentation potential tests indicated that algal augmentation could prove beneficial; carboxylate concentrations increased 31 % over primary solids. However, unexpectedly, and disproving a key research hypothesis, algal augmentation in a fed-batch fermenter decreased the production of carboxylic acids (26-34% at SRTs of 5-7 d); preliminary analyses suggest heterotrophic algae consumed carboxylates. Disproving a second research hypothesis, algal biomass did not significantly diversify carboxylate speciation. Finally, and unexpectedly, algal fermentation realized significant ammonia removal (39-96 % at SRTs of 5-7 d). Although decreased carboxylate yield is not desired, reduced ammonia load could potentially decrease WRRF energy demands and decrease carboxylic acid demands to achieve denitrification.

Optimization of lipid production in Chlorella vulgaris for biodiesel production using flux balance analysis

Microalgae, the world’s largest group of photosynthetic organisms, convert atmospheric CO2 to polar and neutral lipids using sunlight, which after esterification can be utilized for biodiesel production. In the present study, a fully compartmentalized metabolic network was developed to describe the metabolism of Chlorella vulgaris based on known enzymatic reactions and typical metabolic pathways of green algae. Flux balance analysis was employed to optimize the specific growth rate and the lipid production rate using measured exchange fluxes of the metabolites. The experimental data for batch and fed batch algal fermentation systems acquired from the literature were used to validate the accuracy of the pseudo- steady state model. The physiological pathways of the microalgae for lipid biosynthesis were identified. The simulation revealed that the microalgae would be able to produce higher levels of lipid content (43.6%) during N-starvation cultivation under 100 μmol m−2 s−1 light intensity, 0.25 vvm aeration with 2% (v/v) CO2, 2 mg L−1 PO4-P, and 5 mg L−1 NO3-N. Sensitivity analysis showed that CO2, light energy, O2, and nitrate were the most important factors affecting the lipid production at N-deficient conditions. The findings consequential for manipulation of the metabolism of the microalgae with optimal activity.

Quantification of oxygen production and respiration rates in mixotrophic cultivation of microalgae in nonstirred photobioreactors.

Online monitoring and controlling of different cellular parameters are key issues in aerobic bioprocesses. Since mixotrophic cultivation, in which we observe a mixture of cellular respiration and oxygen production has gained more popularity, there is a need for an on-process quantification of these parameters. The presented and adapted double gassing-out method applied to a mixotrophic cultivation of Galdieria sulphuraria, will be a tool for monitoring and further optimization of algal fermentation in nonstirred photobioreactors (PBR). We measured the highest net specific oxygen production rate (opr net) as 5.73·10-3molO2g-1h-1 at the lowest oxygen uptake rate (OUR) of 1.00·10-4molO2L-1h-1. Due to higher cell densities, we also demonstrated the increasing shading effect by a decrease of opr net, reaching the lowest value of 1.2510-5molO2g-1h-1. Nevertheless, with this on process measurement, we can predict the relation between the zone in which oxygen is net produced to the area where cell respiration dominates in a PBR, which has a major impact to optimize cell growth along with the formation of different products of interest such as pigments.

A simple and rapid protocol for measuring neutral lipids in algal cells using fluorescence.

Algae are considered excellent candidates for renewable fuel sources due to their natural lipid storage capabilities. Robust monitoring of algal fermentation processes and screening for new oil-rich strains requires a fast and reliable protocol for determination of intracellular lipid content. Current practices rely largely on gravimetric methods to determine oil content, techniques developed decades ago that are time consuming and require large sample volumes. In this paper, Nile Red, a fluorescent dye that has been used to identify the presence of lipid bodies in numerous types of organisms, is incorporated into a simple, fast, and reliable protocol for measuring the neutral lipid content of Auxenochlorella protothecoides, a green alga. The method uses ethanol, a relatively mild solvent, to permeabilize the cell membrane before staining and a 96 well micro-plate to increase sample capacity during fluorescence intensity measurements. It has been designed with the specific application of monitoring bioprocess performance. Previously dried samples or live samples from a growing culture can be used in the assay.

Algal Fermentation^|^mdash;The Seed for a New Fermentation Industry of Foods and Related Products

Many kinds of fermented products are now being consumed as food and dietary items, although those produced from algae have yet to be developed. A recent observation that seaweed could be used as a substrate for lactic acid fermentation opened the possibility of obtaining such products as foods, diets and fertilizers by algal fermentation. This manuscript reviews past studies on the lactic acid fermentation of algae. Both macroalgae (seaweeds) and microalgae can be used as the materials for lactic acid fermentation, as successful fermentation has been observed regarding all the seaweed species tested to date. Saccharification by cellulase treatment is considered a significant element for inducing algal fermentation. The addition of a starter culture of lactic acid bacteria and salt also promotes successful fermentation. A wide range of Lactobacillus species can be used for inducing algal fermentation, with Lactobacillus brevis, Lactobacillus casei and Lactobacillus plantarum in particular showing a superior ability to dominate in seaweed fermentation cultures. A starter culture of halophilic lactic acid bacteria that is now being developed will make it possible to prepare algal fermented products containing a high (>10%) salt content and having long-term preservation. As for application, a prototype of ‘seaweed sauce’ containing a high quantity of amino acids was obtained from Porphyla sp. (Rhodophyta). Some functional effects are also demonstrated when fish and animals were fed algal fermented products. Studies on the ethanol fermentation of seaweeds are also making progress. All these advances in algal fermentation are expected to lead to the creation of a new genre of algal fermentation industry.

DSM Will Acquire Ocean Nutrition

Dutch chemical maker DSM will spend about $530 million to acquire Ocean Nutrition Canada, which calls itself the world’s largest supplier of omega-3 fatty acids to the dietary supplement and food manufacturing markets. DSM says the acquisition is the fifth purchase it has made in the nutrition field since September 2010, when it announced a corporate strategy to expand in the health, nutrition, and industrial materials markets. The largest of these deals was last year’s $1.1 billion purchase of Martek Biosciences, a producer of nutritional fatty acids via algal fermentation. Founded in 1997, Ocean Nutrition derives the omega-3 fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) from oil obtained mostly from anchovies. DSM expects the firm to have sales this year of $185 million. DSM says the purchase will strengthen its newly created nutritional lipids business by adding fish-oil-derived fatty acids to Martek’s fermentation-derived DHA and arachidonic acid. Although ...

The effect of non-marine HUFA supplementation with fish oil removal on growth and survival of the Pacific white shrimp, Litopenaeus vannamei

The use of non-marine arachidonic acid (ArA) and docosahexaenoic acid (DHA) as highly unsaturated fatty acid (HUFA) enrichments was evaluated as complete replacements for marine fish oil in practical diets formulated with solvent-extracted soybean meal (SESM). Litopenaeus vannamei juveniles (0.59 g) were reared over 84 days in an outdoor tank system with no water discharge. Fishmeal was replaced with SESM, while fish oil was replaced with HUFA-rich algal cells, alternative oil and/or fermentation products. Spray-dried Schizochytrium algal cells (Schizomeal-Hi DHA) served as the DHA enrichment source. Oil extracted from Mortierella sp. was used as the ArA enrichment (AquaGrow® ArA). DHA and ArA sources (Advanced BioNutrition Corp., Columbia, MD, USA) were non-marine products obtained from a commercial supplier. Five diets were formulated with ArA inclusion levels of 0, 0.65, 1.3, 2.6 and 5.2 g kg−1. In addition, one diet was formulated to be DHA deficient and another was formulated with menhaden fish oil (control). Different inclusion levels of non-marine ArA had no effect on survival or growth. Shrimp fed the non-marine HUFA-supplemented diets had lower average weight compared to shrimp offered the diet containing fish oil. No differences were detected in average weights of shrimp offered the ArA-deficient and ArA-supplemented diets.

Simultaneous saccharification of cassava starch and fermentation of algae for biodiesel production

A simpler approach to produce biodiesel from cassava starch was established, which successfully integrates the simultaneous saccharification and heterotrophic algal fermentation in an identical system. Batch experiments were investigated to verify the feasibility of raw starchy substrates fermentation for microalgal oil. The highest cell density (49.34 g L−1) and oil content (54.60%) were obtained in 5-L fed-batch cultivation via simultaneous saccharification and fermentation (SSF). It is demonstrated that the previous multistep hydrolysis and fermentation for feedstock oil could be replaced by SSF with higher energy efficiency and lower facility costs.

Use of sweet sorghum juice for lipid production by Schizochytrium limacinum SR21

Stalk juice from sweet sorghum grown in Southern Illinois, USA, was examined for lipid production through microalgal fermentation. Juice concentrations at 100%, 75%, 50%, and 25% led to different biomass, lipid, and docosahexaenoic acid (DHA) production by Schizochytrium limacinum SR21. Biomass dry weight as 9.4 g/l at 50% juice concentration was similar to that from pure glucose (10.9 g/l). But with a 73.4% lipid content, this dose resulted in higher lipid and DHA production than those from pure glucose. Major fatty acids in cells grown on juice were identical to those fed by other substrates. Among the three sugars – glucose, fructose, and sucrose in sorghum juice, only glucose was utilized for growth. Spent medium after algal removal may be further processed for white sugar production in a traditional way since sucrose content remained the same throughout the algal fermentation process. Algal cells or lipids harvested can be utilized as fish meal, human nutrition supplements, or for biodiesel purpose.

Rapid quantitation of lipid in microalgae by time-domain nuclear magnetic resonance

A specific strain of Chlorella protothecoides has been studied in heterotrophic fermentation for increasing cell growth rate and lipid content for biodiesel production. For optimizing the process of fermentation to reduce costs of alga-based biodiesel production, rapid determination of lipid content in microalgal cells is critical. Nile Red (NR) staining and time-domain nuclear magnetic resonance (TD-NMR) have been investigated to quantitate the lipid content in C. protothecoides. Both methods were found feasible and simpler than gravimetric methods that are commonly employed. The TD-NMR method showed better agreement ( R 2 = 0.9973) with the measured values from lipid extraction experiments than the NR staining method ( R 2 = 0.9067). Additionally, the smaller standard deviations of the samples (≤ 0.36) analyzed by TD-NMR revealed that the method is accurate and reproducible. The application of TD-NMR for lipid quantitation in C. protothecoides opens up the possibility of determining lipid content in algal fermentation precisely and quickly.

Producing Docosahexaenoic Acid (DHA)-Rich Algae from Biodiesel-Derived Crude Glycerol: Effects of Impurities on DHA Production and Algal Biomass Composition

Crude glycerol is the primary byproduct of the biodiesel industry. Producing docosahexaenoic acid (DHA, 22:6 n-3) through fermentation of the alga Schizochytrium limacinum on crude glycerol provides a unique opportunity to utilize a large quantity of this byproduct. The objective of this work is to investigate the effects of impurities contained in the crude glycerol on DHA production and algal biomass composition. Crude glycerol streams were obtained from different biodiesel refineries. All of the glycerol samples contained methanol, soaps, and various elements including calcium, phosphorus, potassium, silicon, sodium, and zinc. Both methanol and soap were found to negatively influence algal DHA production; these two impurities can be removed from culture medium by evaporation through autoclaving (for methanol) and by precipitation through pH adjustment (for soap). The glycerol-derived algal biomass contained 45-50% lipid, 14-20% protein, and 25% carbohydrate, with 8-13% ash content. Palmitic acid (C16:0) and DHA were the two major fatty acids in the algal lipid. The algal biomass was rich in lysine and cysteine, relative to many common feedstuffs. Elemental analysis by inductively coupled plasma showed that boron, calcium, copper, iron, magnesium, phosphorus, potassium, silicon, sodium, and sulfur were present in the biomass, whereas no heavy metals (such as mercury) were detected in the algal biomass. Overall, the results show that crude glycerol was a suitable carbon source for algal fermentation. The crude glycerol-derived algal biomass had a high level of DHA and a nutritional profile similar to that of commercial algal biomass, suggesting a great potential for using crude glycerol-derived algae in omega-3-fortified food or feed.

Comparison of Pond Production Efficiency, Fatty Acid Profiles, and Contaminants in Litopenaeus vannamei Fed Organic Plant‐based and Fish‐meal‐based Diets

Abstract Reduction or elimination of fish meal and fish oil from aquaculture diets can help to reduce the potential for contamination and dependence of the industry on pelagic fisheries while improving economic competitiveness. However, fish oil provides important omega‐3 (n‐3) fatty acids (FAs) essential to shrimp health and beneficial to humans. This study evaluated an organic, plant‐based diet formulated to replace fish meal and fish oil with plant proteins and docosahexaenoic acid (DHA) produced by algal fermentation. Shrimp cultured in replicate outdoor ponds at 25/m2 were fed either a diet composed of organically produced plant ingredients or a conventional commercial fish‐meal‐based feed. No significant differences were found in production parameters between the conventional fish‐meal‐based diet and the plant‐based diet (production: 4594 and 4592 kg/ha; harvest size: 18.7 and 19.2 g; survival: 93 and 88%; and feed conversion ratio: 1.4 and 1.3, respectively). At harvest, shrimp were analyzed for 147 chemical contaminants and 71 FAs. Contaminant levels were negligible for shrimp raised on both diets. The fish meal and fish oil diet provided significantly higher quantities of eicosapentaenoic acid and DHA than the plant‐based diet, and the shrimp fed the conventional diet reflected this with higher levels of these beneficial FAs in edible tissues. Differences between feeds and shrimp tissues suggest that essential n‐3 FAs may accumulate in shrimp tissues over time or that natural pond productivity may play a role in providing supplemental nutrition. Shrimp raised on the two diets and wild‐caught shrimp are clearly distinguishable by their FA profiles. Compared to alternative protein sources like beef, pork, or chicken, differences in lipid profiles of shrimp raised on either diet may be insignificant because both offer increased human health benefits.

Thermochemical treatment for algal fermentation

The purpose of this study was to determine the influence of the thermochemical pretreatment process of algal fermentation on the efficiency with which algal energy is converted microbiologically to the energy in methane. The variables studies were pretreatment temperature, duration, concentration, and dosage of sodium hydroxide (NaOH). In order to optimize the thermochemical pretreatment of algae, an independent variables study was selected. The results indicate that pretreatment best efficiency was attained with a temperature of 100°C for 8 h at a concentration 3.7% solids and without NaOH. Compared with untreated algae, pretreatment improved the efficiency of methane fermentation a maximum at 33%. An orthogonal square design was selected to determine the mathematical model to describe the effects of algal thermochemical pretreatment and to determine the relative significance among the pretreatment parameters.

Hydrogen production by photosynthetic microorganisms

Hydrogen is a clean energy alternative to the fossil fuels, the main source of greenhouse gas emissions. We developed a stable system for the conversion of solar energy into hydrogen using photosynthetic microorganisms. Our system consists of the following three stages: We constructed a test plant of this process at Nankoh power plant of Kansai Electric Power Company in Osaka, Japan, and carried out a series of tests using CO2 obtained from a chemical absorption pilot-plant. The photobiological hydrogen production process used a combination of a marine alga,Chlamydomonas sp. MGA 161 and marine photosynthetic bacterium,Rhodopseudomonas sp. W-1S. The dark anaerobic fermentation of algal starch biomass was also investigated. Sustained and stable starch accumulation, starch degradation in the algal cell, and hydrogen production from algal fermentation and photosynthetic bacteria in the light were demonstrated during several experiments.

Hydrogen production by photosynthetic microorganisms

The algal fermentation conditions for the hydrogen production were investigated to improve the photobiological hydrogen production by a combination of a marine green alga Chlamydomonas sp. MGA161 and a marine photosynthetic bacterium Rhodopseudomonas sp. W-1S. The scaling-up of our biophotolysis system with algal-bacterial combination was carried out. The stably sustained starch accumulation and degradation in the algal cells and hydrogen photoproduction from algal fermentation products by photosynthetic bacteria occurred in an alternating light-dark cycle.

Hydrogen Evolution as a Consumption Mode of Reducing Equivalents in Green Algal Fermentation

Dark anaerobic fermentation in the green algae Chlamydomonas MGA 161, Chlamydomonas reinhardtii, Chlorella pyrenoidosa, and Chlorococcum minutum was studied. Our isolate, Chlamydomonas MGA 161, was unusual in having high H(2) but almost no formate. The fermentation pattern in Chlamydomonas MGA 161 was altered by changes in the NaCl or NH(4)Cl concentration. Glycerol formation increased at low (0.1%) and high (7%) NaCl concentrations; starch degradation, and formation of ethanol, H(2), and CO(2) increased with the addition of NH(4)Cl to above 5 millimolar in N-deficient cells. C. reinhardtii and C. pyrenoidosa exhibited a very similar anaerobic metabolism, forming formate, acetate and ethanol in a ratio of about 2:2:1. C. minutum was also unusual in forming acetate, glycerol, and CO(2) as its main products, with H(2), formate, and ethanol being formed in negligible amounts. In the presence of CO, ethanol formation increased twofold in Chlamydomonas MGA 161 and C. reinhardtii, but the fermentation pattern in C. minutum did not change. An experiment with hypophosphite addition showed that dark H(2) evolution of the Escherichia coli type could be ruled out in Chlamydomonas MGA 161 and C. reinhardtii. Among the green algae investigated, three fermentation types were identified by the distribution pattern of the end products, which reflected the consumption mode of reducing equivalents in the cells.

Oscillatory starch degradation and fermentation in the green alga Chlamydomonas reinhardii

After transition to anaerobiosis an oscillatory fermentation of plastidic starch is found in the green alga Chlamydomonas reinhardii. Oscillations are investigated by analysis of fluctuations in starch, fermentation products and metabolites. Period length, amplitudes and phase relationships are determined by a biometrical processing of the biochemical signals, which allows statistical treatment even under limitation of data points. The mean period length is 59 min and oscillations are detectable within 3 h after the onset of anaerobiosis. Metabolized starch carbon is found by 90% in the accumulating fermentation products as formate, acetate and ethanol. From the intermediates only pyruvate increases during fermentative oscillations. A phase shift of 180° between ATP and both, AMP and ADP, indicates activity of adenylate kinase and values for energy charge fluctuates between 0.70 and 0.89. Phase relations of glucose-6- P to both AMP and pyruvate are 190° and 56°, respectively, whereas that of pyruvate to ATP is 134°. Glucose-6- P concentration does not follow the rate of starch degradation. Phase relations of fermentation products to pyruvate indicates enzyme activities of formate pathway. High period length as observed during algal fermentation is thought to be generated by a complex rate control in a different compartmentalized glycolytic pathway.

Starch fermentation via a formate producing pathway in Chlamydomonas reinhardii, Chlorogonium elongatum and Chlorella fusca

Starch degradation was investigated during anaerobic dark incubation in the algae Chlamydomonas reinhardii, Chlorogonium elongatum and Chlorella fusca. The pathway of algal formate fermentation was elucidated by determination of the relationship between substrate consumption and product accumulation. The fate of reducing equivalents was also determined. Investigations were done on dependence of pH, fermentation time, cell cycle, and after addition of H2, hypophosphite and inhibitors of protein synthesis.A mixed acid fermentation that produced formate, acetate and ethanol (2:1:1) with only small amounts of H2 and CO2 was shown for the algal strains used. The failure of inhibition with cycloheximide and chloramphenicol indicated the constitutive presence of all fermenting enzymes. Nevertheless, glycerol, D(–)lactate and stoichiometrical amounts of ethanol and CO2 were found additionally at extreme pH (pH 4.6 and 7.9), and after addition of H2 and hypophosphite (7 mM). During long‐term incubation (28 h) fermentation changed from mixed acid to ethanol production. The pathways of algal fermentation did not depend on cell cycle, and fermentation rate corresponded directly to the actual starch content of algal cells. The results gave evidence for synthesis of formate during anaerobic metabolism in algae by a thioclastic cleavage of pyruvate via the enzyme pyruvate formate lyase. This indicated an algal fermentation pathway thought to be present only in procaryotic organisms.