Single Cell Oil Production Research Articles

Single Cell Oil Production from Waste Biomass: Review of Applicable Industrial By-Products

Abstract Single cell oil (SCO) is an attractive alternative source of oil, which, depending on the fatty acid composition, can be used as a feedstock for biodiesel production, as an ingredient for pharmaceuticals or as a source of essential fatty acids for human and animal consumption. However, the use of SCO is limited due to use of relatively expensive food or feed products in the cultivation of SCO producing microorganisms. In order to reduce SCO production costs, the use of cheaper feedstock such as biodegradable agro-industrial wastes are necessary. At the same time, the microbial treatment of biodegradable wastes ensures the neutralization of environmentally harmful compounds and reduces the negative impact on the environment. Oleaginous microorganisms are capable of fermenting a variety of industrial by-products, waste products and wastewaters, however further discussion on properties of the waste materials is necessary to facilitate the selection of the most appropriate waste materials for SCO production. Thus, this review compares various industrial waste products that can be used as cheap feedstock for the cultivation of SCO producing microorganisms. Industrial waste products, by-products and wastewaters are compared according to their global availability, current use in competing industries, required pre-fermentation treatments, oleaginous microorganism cell concentrations and SCO yields.

Eco-Green Conversion of Watermelon Peels to Single Cell Oils Using a Unique Oleaginous Fungus: Lichtheimia corymbifera AH13

Watermelon peel waste (WPW) is being used for first time in single cell oils (SCOs) production via a promising oleaginous fungus Lichtheimia corymbifera which was isolated from Egyptian ecosystem. Pretreatments of WPW were carried out by mechanical, physical and chemical methods; the most potent pretreatment was selected according to total reducing sugar and total lipid production. Accordingly, the mechanical ptetreatment of WPW was distinctly the best pretreatment method for SCOs production from L. corymbifera. Taguchi design clarified that the most optimal culture conditions were 35 °C and pH 7.0 for 4 days by which the highest potential of SCOs and lipid content was yielded (2.93 gl−1 and 39.56% respectively). Mechanical treatment revealed that the dominant fatty acid was palmitic and oleic acids with 41.98 and 34.65% respectively with appearance of γ linolenic acid (GLA) at low concentration 1.43%. Finally, this study showed that WPW was used as a natural, effective, economic, ecofriendly and integrated substrate without adding any outsource nutrients to produce sustainable SCOs with low cost production.

Production of single cell oil by using cassava peel substrate from oleaginous yeast Rhodotorula glutinis

Abstract Oleaginous yeasts seem to be the promising source for the production of lipid. In this context Rhodotorula glutinis was investigated utilizing an agro-industrial waste cassava peel. Medium optimization was performed by response surface methodology (RSM) with Box-Behnken design under submerged fermentation to improve the lipid yield. The results of RSM revealed that the highest lipid production of 58% lipid/dry biomass may be achieved when the culture was grown for 3 days at 30 °C under 120 rpm shaking, pH 5.45 in a medium containing 12% (w/v) cassava peel and 8.03% (v/v) yeast inoculum. Scanning electron microscopic studies confirmed complete colonization of starch granules by yeast. The process was further up scaled in a 7 L bio-reactor and an yield increment of 9% was observed. Overall, the above results clearly displayed the potential of R. glutinis to utilize cassava peel waste for the single cell oil (SCO) production.

Single cell oil and ethanol production by the oleaginous yeast Trichosporon fermentans utilizing dried sweet sorghum stalks

The ability of the oleaginous yeast Trichosporon fermentans to efficiently produce lipids when cultivated in dried sweet sorghum was evaluated. First, lipid production was evaluated in synthetic media mimicking the composition of sweet sorghum stalks and optimized based on the nitrogen source and C: N ratio. Under optimum conditions, the lipid production reached 3.66 g/L with 21.91% w/w lipid content by using a mixture of sucrose, glucose and fructose and peptone at C: N ratio 160. Cultivation on pre-saccharified sweet sorghum stalks offered 1.97 g/L, while it was found that sweet sorghum stalks can support yeast growth and lipid production without the need for external nitrogen source addition. At an attempt to increase the carbon source concentration for optimizing lipid production, the Crabtree effect was observed in T. fermentans. To this end, the yeast was evaluated for its potential to produce ethanol under anaerobic conditions in synthetic media and sweet sorghum. The ethanol concentration at 100 g/L glucose was 40.31 g/L, while utilizing sweet sorghum by adding a distinct saccharification step and external nitrogen source offered ethanol concentration equal to 23.5 g/L. To the authors’ knowledge, this is the first time that the Crabtree effect is observed in T. fermentans.

Valorization of agro-starchy wastes as substrates for oleaginous microbes

Different types of agro-industrial or household food processing wastes were assessed for their suitability as growth substrates using a set of oleaginous yeasts, for maximizing the production of lipids and useful enzymes under submerged fermentation (SmF). Minimal medium (pH 5.0) containing different wastes (10% w/v) were inoculated with different yeast strains and incubated at 30 °C for 8 days in an incubator shaker (120 rpm). The processing wastes (potato peel, banana peel, cassava peel, corn residue, rice husk, wheat bran, yam peel and barley husk) utilized, without any pre-treatment, were found to support both microbial growth and lipid production. Lipid productivity was observed to be higher in Cryptococcus curvatus as compared to Rhodotorula mucilaginosa, Saccharomyces pastorianus, Trichosporon cutaneum, Lipomyces starkeyi and Rhodotorula glutinis. Among the different substrates, wheat bran proved to be the best substrate for C. curvatus, as it resulted in the production of 16.0 g lipid/g starch, followed by banana peel yielding 14.3 g lipids/g starch. Interestingly, all the cultures (yeast and fungal) were found to exhibit activity of cellulases, as well as amylase and inulinase, which illustrates the novelty of the study and the viability of the technology for lipid production. Positive correlation between lipid productivity and enzyme activities further highlights the promise of these strains. These research findings illustrate the environmental friendly and alternate use of agro-industrial and household food processing wastes, towards their valorization as substrates for commercial single cell oil production.

New Method for the Extraction of Single-Cell Oils from Wet Oleaginous Microbial Biomass: Efficiency, Oil Characterisation and Energy Assessment

Culture medium composition and an efficient lipid extraction process have the largest impact on the cost of industrial production of microbial single-cell oil (SCO). In this study, SCO from the yeast Rhodosporidium thoruloides grown in sugarcane medium was successfully produced in a 1 m3 bioreactor. Biomass was concentrated by sedimentation and the acid-hydrolysed cells were extracted using a recurrent extraction (RE) and a newly-proposed wet extraction (WE) process. RE consisted of Soxhlet extraction of dry hydrolysed biomass with different solvents: hexane, chloroform and chloroform/methanol (2:1). The WE process consisted of the extraction of wet hydrolysed biomass with the same solvents. The highest lipid yields (m/m) were achieved by hexane in RE and chloroform in WE. The chloroform WE extract presented similar density, saponification number, iodine value, moisture and acid value as that from RE with hexane, but much lower peroxide content. The fatty acid methyl ester compositions of WE and RE extracts were also very similar. An energy–cost analysis showed that WE used only 13% of the energy used in RE. Moreover, an energy conservation analysis showed that approx. 65% of the energy from molasses was effectively transformed into microbial lipids. The low energy requirement, the high efficiency and the characteristics of the resulting bio-oil achieved by WE evidence the potential of the proposed methodology for use on an industrial scale.

Sustainability and life cycle assessment (LCA) of macroalgae-derived single cell oils

Marine macroalgae (seaweed) has many advantages over terrestrial crops as a source of renewable biomass but is severely underutilised at present, especially within Europe. In particular, macroalgae has elevated poly- and monosaccharide content, making it an ideal feedstock as a heterotrophic fermentation sugar source for the production of higher value chemicals. Recent reports have detailed the suitability of seaweeds as a feedstock for the production of single-cell oils (SCOs) which have application in food, oleochemicals and fuels. It is proposed that a biorefinery system based on the production of SCOs alongside other secondary metabolites, has the potential to provide a sustainable replacement to terrestrial oils such as palm oil.This work therefore evaluates, for the first time, the environmental and economic sustainability of a production process for SCOs from seaweed Saccharina latissima using the oleaginous yeast Metschnikowia pulcherrima. Two alternative fermentation systems were considered, and uncertainties associated with the seasonal variation in seaweed carbohydrate yield and fermentation performance were integrated into the analysis. From an environmental perspective, the work indicates that seaweed derived SCO lipids and fats can be comparable to a terrestrial oil mix, with a potential climate change impact ranging between 2.5 and 9.9 kg CO2 eq. kg−1 refined SCO. Interestingly and of particular significance, environmental impacts are mainly dominated by energy demand within fermentation and upstream processing steps. From an economic perspective, a break-even selling price for the oil was determined as between €5,300-€31,000 tonne−1 refined SCO, which was highly dependent on cost of the seaweed feedstock.Overall, we demonstrate that key uncertainties relating to seaweed cultivation costs and hydrolysate fermentation at scale result in a large range in values for environmental impact and economic return on investment. Yet even within the constraints and limitations of current knowhow, seaweed already offers a viable proposition for the competitive production of exotic oils similar to cocoa or shea butter in price and nature.

Co-production of single cell oil and gluconic acid using oleaginous Cryptococcus podzolicus DSM 27192

BackgroundThe co-production of single cell oil (SCO) with value-added products could improve the economic viability of industrial SCO production. The newly isolated oleaginous yeast Cryptococcus podzolicus DSM 27192 was able to co-produce SCO intracellularly and gluconic acid (GA) extracellularly. In this study, the metabolic regulation of carbon distribution between SCO and GA through process optimization was comprehensively investigated.ResultsThe carbon flow distribution between SCO and GA was significantly influenced by the cultivation conditions, such as nitrogen sources, glucose concentration and dissolved oxygen concentration. It was found that organic nitrogen sources were beneficial for SCO accumulation, while GA production was decreased. Dissolved oxygen concentration (DOC) was found to enhance SCO accumulation, while high glucose concentration was more favorable for GA accumulation. Hence, a two-stage DOC or glucose concentration-controlled strategy was designed to improve cell growth and direct carbon distribution between SCO and GA. Moreover, C. podzolicus DSM 27192 could degrade its stored lipids to synthesize GA in the late stationary phase, although considerable amounts of glucose remained unconsumed in the culture medium, indicating the importance of fermentation time control in co-production systems. All these observations provide opportunity to favor either the production of SCO or GA or rather their simultaneous production.ConclusionsCo-production of SCO and GA by C. podzolicus DSM 27192 can improve the economical value for microbial lipid-derived biodiesel production. Moreover, the results of the proposed co-production strategy might give guidance for other co-production systems.

Performance enhancement of hydrothermal liquefaction for strategic and sustainable valorization of de-oiled yeast biomass into green bio-crude

While oleaginous yeasts have been extensively investigated for single-cell oil production, less attention has been paid towards valorization of the erstwhile-discarded de-oiled biomass. The current study demonstrates the valorization potential of the de-oiled biomass through production of bio-crude by hydrothermal liquefaction process. The performance of the process was enhanced by using glycerol as co-solvent and also by optimizing the processing conditions. Glycerol addition enhanced the bio-crude yield up to 50 wt% with an improved energy content of 32 MJ/kg. The major organic groups found in the bio-crude are acids, esters and phenolics. Typical energy recovery achieved in bio-crude was in the range of 43–62%. Thus, strategic valorization of yeast into green crude with net energy gain, as evident from the energy balance, implies the process is energetically favourable.

Lipid Production From Waste Materials in Seawater-Based Medium by the Yeast Yarrowia lipolytica.

The global limitation of fossil fuels impels scientists to search for new energy sources. A good alternative is biodiesel produced from crop plants. However, its production requires huge quantities of farmland, fertilizers and fresh water, which is in conflict with the human demand for water for consumption and land for food production. Thus, production of single cell oil (SCO) by oleaginous microorganisms remains the best solution for the coming years. Whereas most microorganisms require fresh water for proper cell metabolism, in this study we demonstrate that the unconventional yeast Yarrowia lipolytica is able to produce huge quantities of fatty acid in seawater-based medium. Here we shown that Y. lipolytica is able to produce fatty acids in medium based on seawater and crude glycerol as the main carbon source, which allows for low-cost production of SCO, is beneficial for industrial application and is ecologically friendly.

Single Cell Oil Production from Undetoxified Arundo donax L. hydrolysate by Cutaneotrichosporon curvatus.

The use of low-cost substrates represents one key issue to make single cell oil production sustainable. Among low-input crops, Arundo donax L. is a perennial herbaceous rhizomatous grass containing both C5 and C6 carbohydrates. The scope of the present work was to investigate and optimize the production of lipids by the oleaginous yeast Cutaneotrichosporon curvatus from undetoxified lignocellulosic hydrolysates of steam-pretreated A. donax. The growth of C. curvatus was first optimized in synthetic media, similar in terms of sugar concentration to hydrolysates, by applying the response surface methodology (RSM) analysis. Then the bioconversion of undetoxified hydrolysates was investigated. A fed-batch process for the fermentation of A. donax hydrolysates was finally implemented in a 2-L bioreactor. Under optimized conditions, the total lipid content was 64% of the dry cell weight and the lipid yield was 63% of the theoretical. The fatty acid profile of C. curvatus triglycerides contained 27% palmitic acid, 33% oleic acid and 32% linoleic acid. These results proved the potential of lipid production from A. donax, which is particularly important for their consideration as substitutes for vegetable oils in many applications such as biodiesel or bioplastics.

Review of inulinase production using solid-state fermentation

The purpose of the present study is to critically analyze the recent literature covering the production of inulinase enzyme from various sources by solid-state fermentation and discuss various approaches to increase its production in solid-state fermentation, purification, and its properties. The review deals with the solid-state fermentative production of inulinase production. Inulinases have many applications in industries, such as for the production of ultra-high fructose syrup, biofuels, lactic acid, citric acid, and single-cell oil. Solid-state fermentation (SSF) is more economic, requires smaller vessels, lowers water intake, reduces wastewater treatments, higher product yield, lesser chance of bacterial contamination, and lowers energy consumption. Furthermore, the crude products obtained from SSF can be directly used as the source of enzyme for biotransformation. Although many reports are available on a wide range of microbes which produces inulinases by SSF, it is important to isolate novel microbes for its production. Also, extensive research is going on to exploit unexplored sources for SSF. Higher yield of inulinases can be achieved by bioreactor modeling and proper monitoring of physical and chemical parameters in SSF.

Nitrogen Starvation of Assessment in the Production of Single Cell Oils and Biodiesel Quality in Heterotrophic Cultures of Cyanobacteria Phormidium autumnale

The cyanobacteria have great potential for aquaculture industries, bioproducts, bioenergy and bioremediation, as in the reduction of ammonia, phosphorus and organic compounds. The manipulation of the medium and the modes of operation of the cultures are necessary to induce the production of biomass and bioproducts, such as lipids. The aim of the study was to evaluate the different nitrogen sources and their depletion in heterotrophic cultures of the cyanobacterium Phormidium autumnale aimed at obtaining high yields lipid. The heterotrophic cultures were carried out in bubble column bioreactor employing cassava starch as organic carbon source. In the first stage, were studied different nitrogen source under the C/N (carbon/nitrogen) ratios in the culture medium of 20, 40 and 60. In the second stage, selected to nitrogen source with the greatest potential for the production of lipids and held its depletion searching for the induction of a cellular stress. The fatty acids profile in the end of cultures was used in the estimation of biodiesel quality. In the first stage, the cultures employing sodium nitrate under CN ratio of 60 resulting in lipid content of 13.22% and lipid productivity of 7.62 mg/L.h. From the depletion of sodium nitrate, second stage, obtained a lipid productivity and lipid content of 10.43 mg/L.h and 25.07%, respectively. The fatty acid profile showed, in the presence of nitrogen, saturated fatty acid fraction and monounsaturated of 76.72% and 23.88%, respectively. The depletion of sodium nitrate induced a change in the lipid profile, directing the concentration of saturated fatty acids of 98.97%. The biodiesel properties obtained meet the standards set by standardization of the quality of biodiesel.

Influence of Agitation and Aeration on Single Cell Oil Production by <i>Rhodotorula glutinis</i> from Glycerol

In the present study, the yeast Rhodotorula glutinis has been assessed with the aim of producing microbial lipids from glycerol under different aeration conditions. For such a purpose, experiments were carried out in shake flasks, under different conditions of agitation (150 to 250 rpm) and aeration (2.5 to 5.0 of flask volume-to-medium volume ratio). Furthermore, their influence on fermentative parameters (lipid and cell concentration, biomass yield; lipid yield; and lipid volumetric productivity) has been investigated using a 22 full factorial design. The statistical analysis has revealed a strong influence of both variables on substrate consumption, lipid accumulation, cell growth and lipid productivity. As a whole, results suggest that higher aeration levels provide greater cell and lipid concentrations, and lipid volumetric productivity. The best results (4.5 g/L of lipids and QP = 0.95 g/L⋅day) were achieved at the highest aeration (5.0 flask volume-to-medium volume ratio) and agitation (250 rpm) levels. Their fatty acid profile showed that oleic acid was produced in greater quantity (53.5%), followed by linoleic acid (18.7%), palmitic acid (6.8%) and stearic acid (9.9%). The microbial oil presented viscosity of 39.3 cP at 50°C and free fatty acid content of 1.93% ± 0.08%. These are significant results and contribute to establishing operational conditions that maximize single-cell oil production from glycerol by Rhodotorula glutinis, i.e. an alternative source as renewable raw material for lipid-based biorefineries.

Production of Fish Feed and Fish Oil from Waste Biomass Using Microorganisms: Overview of Methods Analyzing Resource Availability

Abstract Aquaculture is currently the fastest growing food-producing sector in the world. The growth of this industry has been rapid for the last 25 years, however, aquaculture still relies heavily on feed input from wild capture fisheries. Landings in wild capture fisheries have been stagnant for the last two decades; therefore, new alternatives for conventional fish meal and fish oil need to be found. In this review, various alternatives are described and their advantages and disadvantages are evaluated. Single cell oils (SCO) and single cell proteins (SCP) produced by microorganisms are recognized as the alternative with the most potential for replacing fish meal and fish oil in aquacultures. However, production costs of SCOs and SCPsare still higher than production costs of Omega-3 rich oils from other sources (wild capture, plant derived oils and genetically modified plants); therefore, currently used substrates need to be replaced with cheaper agriculture and industrial biomass residues applicable for microbial fermentation. In order to evaluate various biodegradable residues and find the most suitable ones for SCO and SCP production, methods analysing resource availability are reviewed.

Lipid production from corn stover by a cost-efficient system featuring ammonium carbonate-steam explosion and recirculating enzymatic hydrolysis

A cost-efficient system was developed for the production of single cell oil from lignocellulosic biomass. The pretreatment of corn stover by ammonium carbonate-steam explosion was performed. A maximum lignin removal rate of 47.26% was observed and confirmed by SEM, XRD and FTIR. In the process of enzymatic hydrolysis, higher initial enzyme loadings were used to reduce the reaction time to 24 h. Under these conditions, the sugar conversion, sugar conversion rate and sugar yield of the pretreated corn stover were 74.84%, 2.00 g L-1 h-1 and 479.4 g kg-1 corn stover, respectively. The biomass production, lipid concentration and lipid content of Cryptococcus podzolicus SCTCC300292 were 10.56 g L-1, 5.03 g L-1 and 47.60%, respectively, in the fermentation of enzymatic hydrolysate. An improved cycling process of enzymatic hydrolysis was employed to reduce the enzyme loading by 40%. The sugar conversion increased to 80.17% and the sugar yield to 513.45 g kg-1 corn stover with a higher sugar release rate of 2.14 g L-1 h-1. Furthermore, the lipid yield reached 54.6 g kg-1 corn stover for the whole biorefinery process. These results indicated that the process of ammonium carbonate-steam explosion pretreatment and recirculated enzymatic hydrolysis would be a highly efficient path for converting lignocellulosic feedstocks into lipids.

Enhanced single-cell oil production by cold shock in cyanobacterial cultures

ABSTRACT: The aim of this research was to evaluate the enhanced single-cell oil production by cold shock in Aphanothece microscopica Nägeli using dairy processing wastewater as culture medium. The study focused on (i) temperature optimization for biomass production, (ii) cold shock application to induce lipids biosynthesis and (iii) determination of fatty acids profile under different conditions. Results indicated that temperature of 20°C was the best condition in terms of kinetics parameter, reaching biomass productivities of 160.25mg/L.h. Under these conditions, a lipid content of 12.65% was also observed, resulting in a lipid productivity of 20.27mg/L.h. Additionally, the 0°C cold shock was the most efficient in increasing intracellular lipid content, reaching 28.4% in dry weight. Cold shocks also showed influence on the saturation of fatty acid composition, where the saturated fatty acids decreased, and the monounsaturated and polyunsaturated fatty acids increased by increasing the cold application. Thus, the use of cold shocks indicates to be a key condition for improving the prospects of efficient single-cell oils production.

Dynamic decoupling of biomass and wax ester biosynthesis in Acinetobacter baylyi by an autonomously regulated switch

For improving the microbial production of fuels and chemicals, gene knock-outs and overexpression are routinely applied to intensify the carbon flow from substrate to product. However, their possibilities in dynamic control of the flux between the biomass and product synthesis are limited, whereas dynamic metabolic switches can be used for optimizing the distribution of carbon and resources. The production of single cell oils is especially challenging, as the synthesis is strictly regulated, competes directly with biomass, and requires defined conditions, such as nitrogen limitation. Here, we engineered a metabolic switch for redirecting carbon flow from biomass to wax ester production in Acinetobacter baylyi ADP1 using acetate as a carbon source. Isocitrate lyase, an essential enzyme for growth on acetate, was expressed under an arabinose inducible promoter. The autonomous downregulation of the expression is based on the gradual oxidation of the arabinose inducer by a glucose dehydrogenase gcd. The depletion of the inducer, occurring simultaneously to acetate consumption, switches the cells from a biomass mode to a lipid synthesis mode, enabling the efficient channelling of carbon to wax esters in a simple batch culture. In the engineered strain, the yield and titer of wax esters were improved by 3.8 and 3.1 folds, respectively, over the control strain. In addition, the engineered strain accumulated wax esters 19% of cell dry weight, being the highest reported among microbes. The study provides important insights into the dynamic engineering of the biomass-dependent synthesis pathways for the improved production of biocompounds from low-cost and sustainable substrates.

Lipomyces starkeyi: an emerging cell factory for production of lipids, oleochemicals and biotechnology applications.

Oils and oleochemicals produced by microbial cells offer an attractive alternative to petroleum and food-crop derived oils for the production of transport fuel and oleochemicals. An emerging candidate for industrial single cell oil production is the oleaginous yeast Lipomyces starkeyi. This yeast is capable of accumulating storage lipids to concentrations greater than 60% of the dry cell weight. From the perspective of industrial biotechnology L. starkeyi is an excellent chassis for single-cell oil and oleochemical production as it can use a wide variety of carbon and nitrogen sources as feedstock. The strain has been used to produce lipids from hexose and pentose sugars derived from cellulosic hydrolysates as well as crude glycerol and even sewage sludge. L. starkeyi also produces glucanhydrolases that have a variety of industrial applications and displays potential to be employed for bioremediation. Despite its excellent properties for biotechnology applications, adoption of L. starkeyi as an industrial chassis has been hindered by the difficulty of genetically manipulating the strain. This review will highlight the industrial potential of L. starkeyi as a chassis for the production of lipids, oleochemicals and other biochemicals. Additionally, we consider progress and challenges in engineering this organism for industrial applications.

Banana peel waste management for single-cell oil production

Banana peel waste was explored as a substrate for single-cell oil production using a promising oleaginous yeast Rhodotorula glutinis NRRL Y-1091 and fungus Ganoderma wiiroense under solid-state versus submerged fermentation for 8 days. Higher productivity and yield of lipids of 0.020 g lipid/g starch was recorded in both types of fermentation, with the fungal isolate G. wiiroense as compared to R. glutinis. Amylase, β glucosidase and inulinase activity were also monitored, which followed a similar trend, with highest values on 6th or 8th day of incubation. Lipase activity was not detected in the crude extract of both strains. Inulinase activity was highest at 8th day incubation in submerged fermentation with the fungal isolate G. wiiroense. FAME analyses revealed that the composition of the lipids had promising potential as good-quality biodiesel. The high levels of inulinase activity in the yeast and fungal strains enhanced the production of monomeric sugars from inulin present in banana peel, which, in turn, facilitated greater conversion to lipids and stimulated bio-oil production.