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Co-culture of an oleaginous yeast Rhodotorula glutinis and a microalga Chlorella vulgaris for biomass and lipid production using pure and crude glycerol as a sole carbon source
Abstract
This study has shown that a co-culture of an oleaginous yeast Rhodotorula glutinis TISTR 5159 and a microalga Chlorella vulgaris var. vulgaris TISTR 8261 enhanced biomass and lipid production from glycerol. It is possible that the microalga may function as an oxygen
producer in the co-culture and enhance the growth of yeast. The use of 3% pure glycerol as a carbon source and urea as a nitrogen
source with a molar carbon-to-nitrogen (C/N) ratio of 32 gave the highest biomass and lipid production. These produced a 5.7-fold
and 3.8-fold of biomass and lipid, respectively, compared to the initial unoptimized condition. The co-culture system was
also applied to convert crude glycerol, a by-product from a biodiesel plant, to biomass and lipid. The lipid produced from
the crude glycerol by the co-culture was mainly composed of palmitic acid (C16:0) 40.52% and oleic acid (C18:1) 21.30%, which
was a plant oil-like fatty acid composition. This suggests that it has a high potential to be used as a biodiesel feedstock.
... Global demand cannot be met by vegetable oils, animal fats, and waste oils alone [6]. As a result, there is growing interest in biodiesel production from non-edible oils including those derived from oleaginous microorganisms [7][8][9]. ...
... The BCG must be purified before use to prepare the cultivation medium. Among the glycerol purification processes, the acidification method provided higher glycerol yield at minimum relative cost, suitable for industrial operations [9,11]. Therefore, the acidification method was used to purify the BCG in this study. ...
... The ion exchange resin and simple distillation provide very low glycerol yield [8]. The nano-cavitation technology is an exciting process, but it is not practical in large-scale operations [9]. The membrane separation method produces ultra-high-purity glycerol if the crude glycerol is previously filtered to remove salts and organic non-glycerol matter [10]. ...
Biodiesel-derived crude glycerol (BCG) was investigated as a cost-effective carbon source for microbial oil (MO) production using Pseudozyma parantarctica CHC28. Optimal conditions were determined for repeated batch fermentation (RBF) strategies to enhance MO production as glycerol concentration 56.20 g/L, KH2PO4 3.98 g/L, and fermentation temperature 27.2 °C. Under these conditions, maximum biomass and MO yields of 5.80 g/L and 2.46 g/L were achieved, respectively with an oil content of 42.41% (w/w). The RBF strategy, employing a 90% (v/v) repeated batch ratio, significantly increased MO production by 9.4% compared to batch fermentation (BF), resulting in an oil content of 44.64% (w/w). RBF eliminated the need for repeated starter preparation, a requirement in BF. Fatty acid (FA) analysis revealed that the MO primarily comprised long-chain C16 and C18 FAs. The estimated biodiesel properties derived from these FA compositions met the ASTM and EU biodiesel standards. Results demonstrated the potential of BCG as a valuable carbon source for MO synthesis and highlighted the efficacy of the RBF strategy in boosting MO production rates as an alternative feedstock.
Graphical Abstract
... The production of biomass and functional metabolites could be increased by a mixed culture of a given microalga with a productive microorganism such as yeast and biologically converted carbon substrates to valuable bioactive fatty acids (FA). As previously reported, the coculture of yeast and microalga could improve lipid production owing to generation of oxygen by microalgae used by the yeast and, provide CO 2 via yeast growth to the microalgae (9,10). Rhodotorula glutinis is a strict aerobic yeast with the ability to produce carotenoids, proteins, lipids and enzymes utilized in food and pharmaceutical industries (11). ...
... This interaction of metabolites can cause to balance the intrinsic O 2 /CO 2 and pH in the culture leading to promote the growth rate of both microorganisms (20). It is documented that in co-culture, the increase in biomass mainly results from the increased number of yeast cells, since the microalgae produce oxygen for enhancing yeast growth requirement (9,40). ...
... In previous studies, various parameters were assessed to enhance the lipid accumulation in microalgae, such as the effect of temperature, light, carbon source concentration, and dissolved oxygen levels (10,49). In addition, it was demonstrated that the synergistic effects of oleaginous microorganisms might influence lipid contents and improve the production of bio-functional FAs (9,10,49). Various researches have investigated the effects of different parameters on the FA profile in the co-culture of yeast and microalgae, but according to the author's knowledge, the impact of the mixed culture on NA (C24:1) and BA (C22:0) has not been addressed up to now. In this regard, two valuable and useful FAs in biomedical applications were selected, i.e., NA (C24:1) and BA (C22:0), and their levels were compared in different conditions (Fig. 6). ...
In recent years, some studies have reported that co-culturing green algae and yeast improve lipid and biomass concentration. In this study, a co-culture of the oleaginous yeast Rhodotorula glutinis and the microalgae Chlorella vulgaris was consequently conducted with inoculation of microalga and yeast in growth and stationary phases, respectively. For the first time, the expression of two pivotal enzymes in fatty acids synthetic pathway, acetyl-CoA carboxylase and Glycerol-3-phosphate acyltransferase, was evaluated. To evaluate the synergistic impacts of the mixed culture on the enzymes expression, several co-culture models were designed, including the use of different ratio of microalgae to yeast or the use of residual cell-free medium of yeast; a positive impact on enzymes overexpression was shown in the case of the co-culture of the two microorganisms, and when the remaining cell-free medium of yeast was added to the microalgal culture. The results of in vitro co-culture demonstrated increased 6- and 5-fold of nervonic acid (C24:1) and behenic acid (C22:0) concentrations, respectively, in 2:1 microalgae to yeast co-culture as compared to the monoculture batches. Addition of yeast residual cell-free medium in the 2:1 ratio to the microalgal culture enhanced 9 and 6 times nervonic acid (C24:1) and behenic acid (C22:0) amounts, respectively.
... The increase in lipid yield, till 20 C/N ratio, would be an effect of deficit nitrogen. It creates catabolic repression in the RmCvcc, where the decreased activity of the Krebs cycle causes the suppression of AMP deaminase activity with increased citric acid production (a precursor for lipid biosynthesis) (Cheirsilp et al., 2012). A further increase in the C/N ratio significantly increases the citric acid content and induces cell apoptosis by endocytosis, which is evident with the increase in significant Biomass on the ratio of 20 C/N. ...
... The free fatty acids produced by the microbial cells are stored as lipid bodies as triglycerides in the endoplasmic reticulum of the cell after the reaction with glycerol-3-phosphate. The maximum yield of biodiesel through this bioremediation process is due to enhanced lipid accumulation (Cheirsilp et al., 2012). The maximum bio-oil yield in RmCvcc (69 %) is due to the increased and simultaneous uptake of organics and inorganics. ...
... In the coculture of oleaginous microalgae and yeast, various carbon substrates such as crude glycerol [171,175], starch processing efuent [176], and brewery wastewater [177], amongst others, have been used in the production of SCOs. Kitcha and Cheirsilp [171] showed enhancement of biomass and lipid productivity by using glycerol as a carbon source in the cocultivation of oleaginous yeast Trichosporonoides spathulata with mixotrophic microalga C. vulgaris var vulgaris TISTR 8261. ...
... Te overall biomass and lipid productivity were 0.155 and 0.063 g/L/h, which was 1.1-fold higher than 0.138 and 0.055 g/L/h in pure yeast culture [171]. Coculturing of R. glutinis and C. vulgaris in 3% pure glycerol and urea medium with a C/N ratio of 32 yielded a 5.7-fold and 3.8-fold increase in biomass and lipid, respectively, over the monoculture [175]. When starch processing efuent was used, the coculturing of R. glutinis and C. vulgaris resulted in a lipid yield of 1.81 g/L, which was higher than the yield obtained in monoculture (i.e., 0.81 g/L), [176]. ...
The use of food-based biomass and arable land for bio-oil and biofuel production could compromise global food security. Therefore, renewable and environmentally friendly oils for biofuels from oleaginous microorganisms such as yeasts and microalgae (heterotrophic and mixotrophic) are gaining interest within the scientific community. These microorganisms have shorter cultivation times and higher lipid productivity when compared to higher plants/food crops/autotrophic microorganisms. Despite many advantages, the high carbon requirements and production cost are limiting factors that hinder their deployment at a commercial scale. Lignocellulosic waste substrates are abundant and inexpensive materials that are rich in organic carbon in the form of cellulose and hemicellulose, which release bioavailable forms of sugars upon hydrolysis. Recent studies have shown the tremendous potential of the hydrolysates of these substrates to be utilized as carbon sources for biomass production and the accumulation of lipids in oleaginous hetero-/mixotrophic microorganisms. Therefore, this review highlights the potential use of lignocellulosic biomass as a low-cost carbon substrate for the cultivation of hetero-/mixotrophic microalgae and yeast for microbial oil production for commercial applications. It also examines the current status, challenges, and future prospects for the utilization of lignocellulose biomass.
... Including O 2 /CO 2 exchange, microalgae acted an O 2 generator for yeast while yeast provided CO 2 to microalgae; the synergistic effect on pH adjustment and substance exchange between microalgae and yeast can contribute to the biomass enhancement [20,21]. However, there was no considerable increase of biomass in mixed culture when compared with the sum of the two mono cultures, T4 with (T1 + T2) and T9 with (T6 + T7), maybe due to the depletion of nutrients, and the attenuation of light intensity caused by the increased yeast cell which contributes to the poor growth of microalgae in the mixed culture [12,22]. ...
... COD in liquid digestate includes volatile fatty acids (VFAs) (mainly in the form of acetic acid) and inorganic carbon sources (mainly in the form of bicarbonate) which can be utilized by microalgae and/or yeast [27,32]. Glycerol as sole carbon source could enhance biomass and lipid production in the mixed culture of Rhodotorula glutinis and C. vulgaris [22]. In this study, to ensure the growth of microbes, and then fulfill the removal of N and P from the YILD, glycerol (initial concentration 40 mM) as a carbon source was introduced into culture system. ...
The symbiosis potential of microalgae and yeast is inherited with distinct advantages, providing an economical venue for their scale-up application. To assess the advantage of the mixed culture of microalgae Chlorella vulgaris and yeast Yarrowia lipolytica for treatment of liquid digestate of yeast industry (YILD) and cogeneration of biofuel feedstock, the cell growth characteristic, the nutrient removal efficiency, the energy storage potential of the mono, and mixed culture were investigated. The results indicated that the biomass concentration of the mixed culture (1.39–1.56 g/L of 5 times dilution group and 1.23–1.53 g/L of 10 times dilution group) was higher than those of mono cultures. The NH3-N and SO42− removal rates of the mixed culture were superior to mono cultures. Besides the higher lipid yield (0.073–0.154 g/L of 5 times dilution group and 0.112–0.183 g/L of 10 times dilution group), the higher yield of higher heating value (20.06–29.76 kJ/L of 5 times dilution group and 21.83–29.85 kJ/L of 10 times dilution group) was also obtained in the mixed culture. This study provides feasibility for remediation of YILD and cogeneration of biofuel feedstock using the mixed culture of microalgae and yeast.
... Both C. zofingiensis and X. dendrorhous have been proposed as promising producers of algal fatty acids and high-value pigments Xueya Jiang and Lu Liu contributed equally to the work and should be regarded as co-first authors * Dong Wei fewd304@scut.edu.cn 1 (Dominguez-Bocanegra et al. 2007;Sun et al. 2008). The correlations between lipid accumulation and the synthesis of fat soluble pigment under stress conditions have been widely reported (Zhekisheva et al. 2002;Cheirsilp et al. 2011). Therefore, the integration of natural astaxanthin production with other high-value products by those two species could provide promising approaches for profitable production of algal biomass. ...
... In this study, the mixed cultivation used glucose and urea in a molar C/N ratio of 180 as the sole carbon and nitrogen sources. It has been reported that a molar C/N ratio of 180 results in a higher astaxanthin content than a ratio of 30, urea is also regarded as better nitrogen for growth and lipid accumulation of microalgae compared with several cheap inorganic nitrogen sources (Cheirsilp et al. 2011;Liu et al. 2013). Due to the rapid growth of yeast in the early cultivation of the mixed culture, the nitrogen was consumed which then resulted in the lipid and astaxanthin accumulation. ...
The alga Chromochloris zofingiensis and the yeast Xanthophyllomyces dendrorhous are typical microorganisms which can accumulate high-value astaxanthin and lipid simultaneously. This study investigated the synergistic effects of X. dendrorhous on the cell growth, lipid, and astaxanthin production of C. zofingiensis by a mixed culture approach. Compared to the pure culture of C. zofingiensis, enhanced lipid and astaxanthin production were obtained in the mixed culture. The maximum astaxanthin and lipid yield achieved in the mixed culture with the ratio of 3:1 (algae to yeast) were 5.50 mg L⁻¹ and 2.37 g L⁻¹, respectively, which were 1.10- and 2.72-fold that of C. zofingiensis monoculture. Additionally, lipid obtained from the mixed culture had a plant oil-like fatty acid composition. This study provides a new insight into the integration of natural astaxanthin production with microbial lipid.
... Microalgae/yeast strain Enhance biomass and lipid production from glycerol Improved biomass and lipid production from glycerol were achieved using a co-culture; the lipid production was not as high as in previously reported work. Cheirsilp et al. (2012) Chlorella pyrenoidosa/ Rhodosporidium toruloides 150 mL flasks containing 25 mL mixed wastewater NA Enhance lipid production from distillery and domestic mixed wastewater The approach of using mixed culture is feasible to improve the lipid production from real mixed wastewater under non-sterile conditions. ...
... In order to ensure the growth of microbes, and then fulfill the removal of nitrogen and phosphorus from the LDDW, glycerol (initial concentration 250 mM) was introduced into culture system as a carbon source. It was reported that glycerol as sole carbon source could enhanced biomass and lipid production in the mixed culture of R. glutinis and C. vulgaris (Cheirsilp et al., 2012). In this study, the COD removal profiles is presented in Fig. 2d, and from this figure it can be observed that the mixed culture and the mono Y. lipolytica culture exhibited higher COD removal rate than the mono microalgae culture, which might be due to the fact that glycerol acts very well as the carbon source for Y. lipolytica (Tomaszewska et al., 2012). ...
To determine the feasibility of microalgae-yeast mixed culture using the liquid digestate of dairy wastewater (LDDW) for biofuels and single cell protein (SCP) production, the cell growth, nutrient removal and outputs evaluation of the mono and mixed culture of Chlorella vulgaris and Yarrowia lipolytica in LDDW were investigated by adding glycerol as carbon source. The results showed that the mixed culture could enhance the biological utilization efficiency of nitrogen and phosphorus, and obtain higher yield of biomass (1.62 g/L), lipid (0.31 g/L), protein (0.51 g/L), and higher heating value (34.06 KJ/L). Compared with the mono culture of C. vulgaris, a decline of the transcription level in nitrate reductase and glutamine synthetase II genes in C. vulgaris was observed in the mixed culture when ammonia was sufficient. The results suggest the possibility of using the mixed culture for the efficient treatment of LDDW and resources recycling.
... However, the number of cells in the PH and MP cultures was higher than in other cultures from day 4. The stationary phase of the pure yeast growth curve (Fig. 1b was observed earlier than in the MP yeast growth curve, indicating that the yeast may have dominated the co-culture in terms of the number of cells (Cheirsilp et al. 2012). From days 3 to 6, the difference in the biomass of microorganisms among the four culture methods was not significant (Fig. 1c) possibly due to cell weight accumulation. ...
... Xue et al. (2010) monitored dissolved oxygen in R. glutinis culture after A. platensis was added and found that microalgae could provide additional oxygen for yeast, thereby enhancing aerobic metabolism. CO 2 produced during yeast metabolism can be used by microalgae during photosynthesis (Cheirsilp et al. 2012). ...
The photoautotrophic co-culture of Chlorella vulgaris and the yeast Rhodotorula glutinis (MP culture) from industrial wastewater was investigated. Cell numbers, biomass, lipid production, and fatty acid content were measured. Owing to co-culture interaction with yeast and microalgae, the MP culture resulted in the highest number of cells (27.73 × 10⁵ cells mL⁻¹) and biomass (0.808 g L⁻¹). Lipid production in the MP culture (117.73 mg L⁻¹) was fourfold higher than that in the photoautotrophic pure culture (23.1 mg L⁻¹). The content of palmitic acid (C16:0) was 24.65%, whereas that of oleic acid (C18:1) was 56.34% in the MP culture, which was higher than in other cultures. The results of this study indicate that MP cultures can be used to effectively support the growth of microorganisms and as an approach for biodiesel production.
... For examples, Shaigani et al. showed that Cutaneotrichosporon oleaginosus produced high levels of lipids when grown on brown algae hydrolysate and that coculture of two oleaginous yeasts C. oleaginosus and Rhodosporidium toruloides were able to effectively co-utilize mannitol, glucose, and xylose present in the hydrolysate (43). Coculture of an oleaginous yeast Rhodotorula glutinis and a microalga Chlorella vulgaris enhanced lipid production from glycerol (44). However, it remains unknown whether alginate can be metabolized by any oleaginous yeast in monoculture. ...
The ability of microorganisms to decompose brown algae has attracted attention. This study aims to clarify the characteristics of marine microbial communities in which prokaryotic and eukaryotic microorganisms interact via the metabolism of brown algae carbohydrates. Amplicon-based microbiome analysis revealed the predominance of the genera Marinomonas and Vibrio in seawater and seaweed samples mixed with alginate and mannitol, which are the primary carbohydrates in brown algae. Three Vibrio species and Candida intermedia were isolated via alginate enrichment culture. Although C. intermedia did not utilize alginate as a nutrient source, the yeast grew in the spent alginate medium in which Vibrio algivorus had been cultured. Coculture with C. intermedia and the Vibrio isolates, especially V. algivorus, also enhanced the growth of the yeast on alginate. These results suggested that C. intermedia grew because of the supply of nutrients via alginate metabolism by Vibrio species. In the coculture medium, the amount of phosphatidylserine increased in the early phase but decreased with the growth of C. intermedia, indicating that phosphatidylserine secreted by Vibrio is involved in the putative mutualistic interaction. We examined whether such interaction is applicable to the production of useful substances and succeeded in lipid production by oleaginous marine yeast Yarrowia lipolytica through coculture with V. algivorus. Our study suggested the potential of mutualistic interaction via degradation of alginate by marine Vibrio for production of industrially useful substances in yeast cells.
IMPORTANCE
In this study, we analyzed the microbiome of seawater and seaweed in the presence of brown algae carbohydrates and reconstructed the putative mutualistic relationship of marine Vibrio and Candida intermedia mediated by metabolism of brown algae in the ocean.
... Thus, H. lacustris has shown more than two-fold increase in its major secondary carotenoid astaxanthin yield in co-culture with the bacteria Sphingomonas hankookensis or Paenarthrobacter ureafaciens, or the fungus Simplicillium lanosoniveum . Co-culturing of microalgae with certain yeast species results in beneficial cross-feeding that either increases the rate of carbon dioxide assimilation or enables the utilization of organic carbon sources for higher biomass accumulation (Cheirsilp et al. 2012;Wang et al. 2016;Gao et al. 2023b). Such co-cultures are designed by high-throughput screening of suitable auxotrophs among microalga, bacteria, and fungi to arrange the most efficient trophic interactions (Saleski et al. 2019). ...
Although established biotechnological applications of microalgae e.g., the production of high-value metabolites is based on axenic cultures, exploitation of the mutualistic consortia of microalgae and bacteria quickly comes to foreground, especially in bioremediation and wastewater treatment. This trend shifts the focus from genomic research of certain microalgal species to metagenomic studies of interactions between microalgae and bacteria in natural communities and in artificial consortia. Dissection of the genetic determinants of the robustness and productivity of the consortia become a hot research direction, too. Admirable contribution to this topic had been made by high-throughput sequencing (HTS), while recent breakthrough in this field was entailed by the advent and rapid development of the 3rd generation nanopore sequencing which becomes increasingly accurate while providing unprecedented sequencing performance. Recent progress of the Oxford Nanopore Technologies (ONT) enabled both classical metagenomic analysis of microalgal-bacterial communities based on whole metagenome sequencing as well as taxonomic and genetic profiling based on the amplicon sequencing. The parallel emergence of novel bioinformatic algorithms for processing the metagenomic datasets opened new opportunities for the analysis of structure and physiology of microalgal-bacterial communities. From the practical perspective, the new HTS techniques became a time- and labor-savers in discovery of new microalgae with a high potential for the accumulation of valuable metabolites, biodegradation of hazardous micropollutants, and biosequestration of nutrients from waste streams. Search for prokaryotic species boosting the biotechnological potential of eukaryotic microalgae via mutualistic interactions with them is another important goal. The insights from the both short-read and long-read metagenomics will form a solid foundation for the rational design of microalgal-bacterial consortia for biotechnology. In this review, we briefly outline the benefits of the long-read sequencing for structural and functional investigation of algal-bacterial consortia and summarize recent reports on using this approach for achieving the biotechnology-related goals.
... Further, they noticed generated biomass at an average concentration of 1.62 g/L with a lipid content of 18.98%. Cheirsilp et al. (2012) explored the possibility of improving lipid synthesis from pure glycerol with urea using a symbiotic mixed culture of Rhodotorula glutinis and Chlorella vulgaris. The results showed a substantial improvement in total biomass (3.20 g/L) and lipid content (34.40%) when these two species were co-cultivated. ...
Liquid biofuels like biodiesel and bioethanol are crucial in the transition to low-carbon and high-energy alternatives to fossil fuels. One significant by-product of biodiesel production is glycerol, which accounts for about 10% of the total conversion output. While waste glycerol poses challenges due to its impurities and contaminants, it also holds potential as a metabolic resource for essential cellular components in microorganisms. Crude glycerol production is reviewed, highlighting relevance in current biodiesel technologies and its biochemical composition. To efficiently utilize waste glycerol, co-valorization with low-cost substrates through biocircular platforms using various microorganisms or insects for second and third-generation oxy-biofuels has been explored. Among these, the black soldier fly larvae have demonstrated higher competitiveness for lipid contents (35–43%), making them a promising organism for recycling waste glycerol into biodiesel production, alongside microalgae and oleaginous yeast. The microbial biodiesel productivity from oleaginous yeast is notably higher (3546 kg ha−1 y−1) than soybean biodiesel (562 kg ha−1 y−1), while microalgal biodiesel productivity surpasses palm biodiesel by more than 25 times. Remarkably, black soldier fly larvae biodiesel productivity was reported to be ~ 1.7 times higher than microalgae and an impressive ~ 43 times higher than palm biodiesel. Despite their potential for biodiesel production, waste glycerol from biodiesel industry still represents a challenge because of high impurities, high viscosity, and limited direct applications in existing processes. To further enhance energy sustainability and address the challenge of waste glycerol, biocircular platforms are discussed for waste glycerol utilization with domestic wastewater sludge, lignocellulosic biomass, and protein-rich wastes. These platforms offer opportunities to create other sustainable agricultural products while minimizing their environmental footprint.
... A control sample was placed in a vacuum chamber without radiation. After mutating the samples with different nitrogen concentrations, the mutated samples were rinsed using a sterile saline solution containing 8.5 g of NaCl in 1 L of double distilled water [23]. ...
The overconsumption of energy results in the depletion of fossil fuels. Generally, biodiesels are produced from wastes of animal fats and vegetable oils. In this study, we have tried to produce biodiesel from both the wild strain and ion beam mutated strain and compared the concentration of lipids produced from both the strains and their properties. Lipids were extracted from microbes using the Bligh and Dyer method and analyzed using gas chromatography and mass spectrophotometry (GCMS) and Fourier-transform infrared (FTIR) spectroscopy. Extracted lipids (free fatty acids) were converted into biodiesel (fatty acid methyl esters) using a base catalyst. The end product biodiesel was characterized and analyzed based on ASTM standards.
... composition, pH, temperature, and incubation time reported 50:50, 6.50, 32.5 °C, and 90 h, respectively 41 . Many other species have been reported to have high growth coupled with other microbial species, such R. glutinis and Ambrosiozyma cicatricose [42][43][44] and Monoraphidium sp FXY10 45,46 . The composition of the coculture inoculum can have a fundamental effect on the performance of microbial growth and lipid production in a coculture system. ...
This study aimed to improve lipid and gamma-linolenic acid (GLA) production of an oleaginous fungus, Mucor plumbeus, through coculturing with Bacillus subtilis bacteria, optimising the environmental and nutritional culture conditions, and scaling them for batch fermentation. The maximum levels of biomass, lipid, fatty acid, and GLA in a 5 L bioreactor containing cellobiose and ammonium sulfate as the optimal carbon and nitrogen sources, respectively, achieved during the coculturing processes were 14.5 ± 0.4 g/L, 41.5 ± 1.3, 24 ± 0.8, and 20 ± 0.5%, respectively. This strategy uses cellobiose in place of glucose, decreasing production costs. The nutritional and abiotic factor results suggest that the highest production efficiency is achieved at 6.5 pH, 30 °C temperature, 10% (v/v) inoculum composition, 200 rpm agitation speed, and a 5-day incubation period. Interestingly, the GLA concentration of cocultures (20.0 ± 0.5%) was twofold higher than that of monocultures (8.27 ± 0.11%). More importantly, the GC chromatograms of cocultures indicated the presence of one additional peak corresponding to decanoic acid (5.32 ± 0.20%) that is absent in monocultures, indicating activation of silent gene clusters via cocultivation with bacteria. This study is the first to show that coculturing of Mucor plumbeus with Bacillus subtilis is a promising strategy with industrialisation potential for the production of GLA-rich microbial lipids and prospective biosynthesis of new products.
... After centrifugation, the supernatant was discarded and the pellet was collected and dried in a hot air oven at 65 • C for 1 day. The dried pellet was then weighed in a vacuum weighing machine [29,30]. ...
The future of petroleum-based fuel is biodiesel. Biodiesel is an eco-friendly fuel that can be used in any diesel engine without any alterations. Researchers have focused on biodiesel that can be produced from microbial lipids extracted from high lipid-yielding microbes. In this study, microbial cultures were screened for high lipid-yielding capabilities and mutated using UV radiation at three different time intervals of 30, 75, and 90 min. The Nile red fluorescence method was used to analyze high lipid-yielding microbes. An outstanding increase in biomass and lipid productivity was noted when the microbes were exposed to UV for 30 min. For example, an M30-8 UV-mutated strain produced a lipid yield of 68.5%. The lipids produced from the wild and mutated strains were analyzed using GCMS and FTIR spectrophotometric analysis. Then, the lipids extracted from both wild VS3 and UV-mutated M30-8 strains were transesterified using a base catalyst and the produced biodiesel was analyzed using ASTM standards. The aim and objective of the research was to mutate high lipid-yielding microbes by using UV radiation and produce biodiesel from the lipids extracted from both wild and UV-mutated strains.
... In general, research on this method has been described for algae species e.g., the co-culture of C. sorokiniana and Chlorella vulgaris with Azospirillum brasilense increased not only lipid, but also variety of fatty acids, (244). Many other species have been reported to have high growth coupled with other microbial species, such Rhodotorula glutinis and Ambrosiozyma cicatricose (245)(246)(247) and Monoraphidium sp FXY10 (248,249), Dostalek found that the co-culture of R. toruloides and Saccharomycopsis fibuligera in starch media resulted in a greater lipid and biomass content (250). ...
Microbes have gained a lot of attention for their potential in producing polyunsaturated fatty acids (PUFAs). PUFAs are gaining scientific interest due to their important health-promoting effects on higher organisms including humans. The current sources of PUFAs (animal and plant) have associated limitations that have led to increased interest in microbial PUFAs as most reliable alternative source. The focus is on increasing the product value of existing oleaginous microbes or discovering new microbes by implementing new biotechnological strategies in order to compete with other sources. The multidisciplinary approaches, including metabolic engineering, high-throughput screening, tapping new microbial sources, genome-mining as well as co-culturing and elicitation for the production of PUFAs, have been considered and discussed in this review. The usage of agro-industrial wastes as alternative low-cost substrates in fermentation for high-value single-cell oil production has also been discussed. Multidisciplinary approaches combined with new technologies may help to uncover new microbial PUFA sources that may have nutraceutical and biotechnological importance.
... Furthermore, other advantages such as metabolite exchanges and medium pH auto-adjustment are observed in mixed cultures [1]. Therefore, yeast and algae when cultivated in mixed cultures produce intracellular compounds more efficiently with potential commercial interest, such as lipids and carotenoids [3,4]. ...
In this work, primary brewery wastewater (PBWW) and secondary brewery wastewater (SBWW) separately, or mixed at the ratios of 1:1 (PBWW:SBWW) and 1:7 (PBWW:SBWW), with or without supplementation with sugarcane molasses (SCM), were used as culture media for lipid production by a mixed culture of the oleaginous yeast Rhodosporidium toruloides NCYC 921 and the microalgae Tetradesmus obliquus (ACOI 204/07). Flow cytometry was used to understand the dynamics of the two micro-organisms during the mixed cultures evolution, as well as to evaluate the physiological states of each micro-organism, in order to assess the impact of the different brewery effluent media composition on the microbial consortium performance. Both brewery wastewaters (primary and secondary) without supplementation did not allow R. toruloides heterotrophic growth. Nevertheless, all brewery wastewater media, with and without SCM supplementation, allowed the microalgae growth, although the yeast was the dominant population. The maximum total biomass concentration of 2.17 g L⁻¹ was achieved in the PBWW mixed cultivation with 10 g L⁻¹ of SCM. The maximum lipid content (14.86% (w/w DCW)) was obtained for the mixed culture developed on SBWW supplemented with 10 g L⁻¹ of SCM. This work demonstrated the potential of using brewery wastewater supplemented with SCM as a low-cost culture medium to grow R. toruloides and T. obliquus in a mixed culture for brewery wastewater treatment with concomitant lipid production.
... A higher lipid yield ( 4.6 g/ L) was obtained as compared to the monocultures of yeast and algae. Co-cultivation of C. vulgaris and R. glutinis on crude glycerol revealed an enhanced production of lipid ( Cheirsilp et al., 2012). Co-cultivation of R. glutinis and C. vulgaris on seafood processing plant effluent showed a greater production of lipid than each strain culture individually ( Cheirsilp et al., 2011). ...
... A higher lipid yield ( 4.6 g/ L) was obtained as compared to the monocultures of yeast and algae. Co-cultivation of C. vulgaris and R. glutinis on crude glycerol revealed an enhanced production of lipid ( Cheirsilp et al., 2012). Co-cultivation of R. glutinis and C. vulgaris on seafood processing plant effluent showed a greater production of lipid than each strain culture individually ( Cheirsilp et al., 2011). ...
Depending upon the source of carbon, biofuels have been categorized into first, second, and third generations. Second-generation biofuels offer the most promising industrial target as they neither compete with edible crops nor lack advanced scientific development in the field, which plagues both first and third generations, respectively. To efficiently metabolize second-generation lignocellulosic carbon, microbial strains should be tolerant against the inhibitors generated during the treatment of biomass. The potent common inhibitors are furfural, 5-hydroxymethylfurfural and acetic acid which stress microbial metabolism and lower the productivity of synthesis of biofuel compounds. This chapter covers the genetic modifications pursued on Escherichia coli and Saccharomyces cerevisiae to increase the productivity of biofuel of interest under stressful conditions. High-energy advanced biofuels such as bioalcohols and hydrocarbons have sparked widespread interest due to their high energy densities and compatibility with the existing infrastructure. This chapter will also discuss the development on the production pathways and metabolic engineering of various species used in the processing of these advanced biofuels.
... Apart from these, marine microalgae are also referred as potential source of biofuels or in cosmeceuticals (Quinn et al. 2008, Bhatnagar & Kim 2010, Mayer et al. 2010, Penesyan et al. 2010, Gomma et al. 2015, Mourelle et al. 2017, Maeda et al. 2018. Especially in recent times, different species of Chlorella has become an important source for biodiesel production (Cheirsilp et al. 2012, Kirrolia et al. 2014, Mathimani et al. 2017, Mathimani et al. 2018, Chi et al. 2019, wastewater treatment and heavy metal removal (Kumar & Goyal 2010, Das et al. 2018, Amin & Chetpattananondh 2019. Our present study involves disc diffusion method for identification of the antibacterial compounds from Southern Ocean (Indian sector) origin marine microalgae Chlorella sp. ...
Marine microalgae has been attracting the researcher’s attention for centuries. Development on microalgal research is majorly favoured by its medicinal, pharmaceutical or cosmeceutical properties. The advancement in the investigation related to microalgal products have been concentrated in the coastal zones because of the greater supply of raw material. The Southern Ocean, highly productive and relatively poorly studied ecosystem, constitutes approximately 10% of the global volume of the oceans. In this study, marine microalgae Chlorella sp. PR-1 was isolated from the Southern Ocean (Indian Sector) for the identification of bioactive antibacterial compounds. The antimicrobial activity of this microalgal extracts was evaluated against three gram positive (Staphylococcus aureus, Bacillus licheniformis, and Bacillus subtilis) and three gram negative bacteria (Pseudomonas aeruginosa, Salmonella typhimurium, and Escherichia coli). The extract showing antibacterial activity was further purified by thin layer and column chromatography. The antimicrobial activity was again evaluated and confirmed with the purified fraction against the same bacteria. The identification of the functional groups in the purified fraction was performed by infrared spectroscopic analysis. Based on gas chromatographic and mass spectroscopic analysis, the principle bioactive compound was proved to be 2,4-bis (1,1-dimethylethyl)- phenol. Thus, the bioactive compound isolated from marine microalga of Southern Ocean origin may be a novel alternative source of antibacterials in the future.
... and C18:1 (21.14-24.21%) FAs were obtained as major compounds in the study, which suggests that co-culture system could be a reliable approach for biodiesel production (Cheirsilp et al. 2012). ...
In recent years, research initiatives on renewable bioenergy or biofuels have been gaining momentum, not only due to fast depletion of finite reserves of fossil fuels but also because of the associated concerns for the environment and future energy security. In the last few decades, interest is growing concerning microalgae as the third-generation biofuel feedstock. The CO2 fixation ability and conversion of it into value-added compounds, devoid of challenging food and feed crops, make these photosynthetic microorganisms an optimistic producer of biofuel from an environmental point of view. Microalgal-derived fuels are currently being considered as clean, renewable, and promising sustainable biofuel. Therefore, most research targets to obtain strains with the highest lipid productivity and a high growth rate at the lowest cultivation costs. Different methods and strategies to attain higher biomass and lipid accumulation in microalgae have been extensively reported in the previous research, but there are fewer inclusive reports that summarize the conventional methods with the modern techniques for lipid enhancement and biodiesel production from microalgae. Therefore, the current review focuses on the latest techniques and advances in different cultivation conditions, the effect of different abiotic and heavy metal stress, and the role of nanoparticles (NPs) in the stimulation of lipid accumulation in microalgae. Techniques such as genetic engineering, where particular genes associated with lipid metabolism, are modified to boost lipid synthesis within the microalgae, the contribution of “Omics” in metabolic pathway studies. Further, the contribution of CRISPR/Cas9 system technique to the production of microalgae biofuel is also briefly described.
... Lipids (mainly as free fatty acids) produced by R. glutinis have a profile similar to some vegetable oils Vasconcelos et al. 2018), being thus promising alternatives as food additives, diet supplements, substituents of unhealthy fats, or as raw material in oleochemistry industries (Saxena et al. 1998;Papanikolaou and Aggelis 2011;Lopes et al. 2018). In turn, R. glutinis carotenoids are natural pigments (Kirti et al. 2014 reported Saini and Keum 2018;, the most applied for the recovery of intracellular lipids are Soxhlet (Dai et al. 2007;Chuck et al. 2014;Karamerou et al. 2016), Folch (Pan et al. 1986;Cheirsilp et al. 2012;Vasconcelos et al. 2018), and Bligh and Dyer Kuan et al. 2018;Santos Ribeiro et al. 2019) methods, which require the use of significant amounts of volatile organic compounds (VOCs) such as hexane, chloroform, and methanol (Kot et al. 2017;Tkáčová et al. 2017). Likewise, the recovery of carotenoids from R. glutinis is not environmentally friendly, since conventional techniques using petroleum ether, dimethyl sulfoxide ( Tisochrysis lutea (Gallego et al. 2020). ...
This thesis consists of the investigation and optimization of sustainable strategies to enhance carotenoids production from yeast Rhodotorula glutinis CCT- 2186 (R. glutinis) and their extraction by means of ionic liquids (ILs) and bio-based solvents. The interest on carotenoids such as β-carotene, torulene and torularhodin relies on the plethora of relevant properties of these products and their commercial value for food, feed, cosmetic and pharmaceutical industries. However, to make carotenoids more accessible in a sustainable way, their production from microbial sources is of paramount importance. Concerning the production of carotenoids from R. glutinis yeast there are still processual challenges, such as the improvement of production yields and development of efficient and sustainable extraction platforms. Initially, different statistical experimental designs were applied to improve carotenoids production and the best bioprocess was scaled-up to a 5 L stirred-tank bioreactor. Since the carotenoids are produced intracellularly, requiring appropriate cell-disrupting and extraction methodologies for their recovery, subsequently, the development of more benign and effective extraction/purification platforms was evaluated. A comprehensive study using aqueous solutions of ionic liquids (ILs) for solid-liquid extraction (SLE) processes was carried out, following the carotenoids purification using a three-phase partitioning system composed of aqueous solutions of ILs and inorganic salts. To gather additional information on the phase separation mechanisms, aqueous biphasic systems (ABS) composed of ILs and inorganic salts were determined and characterized. Afterward, the potential of bio-based solvents was evaluated, with the purpose of designing a more efficient and ecofriendly extraction process for recovery of the intracellular carotenoids from R. glutinis. In this study it was designed and optimized an integrated downstream platform using a ternary mixture of bio-based solvents (ethyl acetate/ethanol/water) with isolation and polishing of carotenoids as well as the recycling of the solvents. The overall sustainability of the proposed technology was assessed in terms of solvents recyclability and carotenoids polishing, and the environmental impact of the platform through a life cycle assessment (carbon footprint). This thesis demonstrates the importance of combined organic and inorganic nitrogen sources to supplement the nutritional media for cultivation of R. glutinis and production of carotenoids, and that ILs and mixed bio-based solvents can be used to design simple, efficient and sustainable platforms for the recovery of intracellular carotenoids from microbial biomass.
... Even though the nitrogen source used in the present work was known to promote satisfactory cell growth, its high concentrations in the culture medium might have suppressed the biosynthesis of carotenoids and other secondary metabolites, such as lipids (Saenge et al., 2011). Some researchers have stated that the C:N ratio influences the production of colorants by several microorganisms (Cheirsilp et al., 2012;Braunwald et al., 2013;Machado and Burkert, 2015;Tkáčová et al., 2017). In the present work, it was observed that R. toruloides required only minimum quantities of nitrogen to maintain cell development, and utilised the excess of carbon in the culture medium to synthesise carotene. ...
Several artificial colouring agents are used as food additives to improve the foods' visual appearance. A recent increase in the use of natural colouring with bioactive properties (antioxidants) as a substitute for additives used in the food industry has led to the search for novel sources to produce such substances with functional colouring. The present work was aimed to isolate and select yeasts from the Cerrado (Savannah) biome (Central region, Brazil) to produce carotenoids. Sixty-nine of the 470 colonies, selected after screening, presented colours from yellow to red. These yeasts were grouped into three colours: yellow, pink and orange. Yeast belonging to the pink group, identified as Rhodotorula toruloides, was chosen for improvement of the factors (physical and nutritional) involved in submerged cultivation. Carotenoid bioproduction was improved by using an experimental design which evaluated the characteristics of the physical processes (agitation and temperature), followed by 2 5-1 factorial experimental design to select the relevant factors for the culture medium. Following statistical analysis, a complete second-order experimental design was employed to optimise the composition of the culture medium. The maximum carotenoid production obtained was 1,333.11 µg.L-1 (106.92 µg.g-1) after 144 h at 25°C and 130 rpm in yeast malt (YM) medium containing 45.95 g.L-1 glucose, 1 g.L-1 malt extract, 0.7 g.L-1 yeast extract, and 0.4 g.L-1 peptone, with an initial pH value of 6. This result showed the potential of this yeast as a viable source of biopigments.
... It can utilize different sugars and simple compounds as a carbon source [15]. In light of these microalgae lacunas, oleaginous yeasts may be a potential alternative option for biodiesel production owing to their fast growth rate, short life cycle, easy scale-up, capability to utilize cost-effective fermentation media, and rapid lipid accumulation ability in distinct lipid bodies [16,17]. ...
The cost of biodiesel production and the requirement of raw ingredients are the primary constraints that need to be addressed while searching for viable alternative fuels to petrol and diesel. Oleaginous yeasts are gaining wider acceptance as biofuel candidates among oil-rich crops/microbes. The present investigation aimed to integrate the agro-industrial wastewater stream as a nutrient source for the cultivation of oleaginous yeast and to use the resultant biomass and lipid as a feedstock for biofuel synthesis. The yeast biomass grown in sago processing wastewater contained 7.21% moisture content, 69.01% volatile matter, 12.61% fixed carbon, and 11.16% ash content. The ultimate analysis determined the contents of carbon (40.43%), nitrogen (5.14%), hydrogen (4.62%), sulfur (0.54%), and oxygen (49.27%). The heating value of yeast biomass was 16.54 MJ kg⁻¹. The thermal behavior of yeast biomass also suggests its potential use as an energy source. The FTIR spectrum of biomass had major lipid (3030–2800 cm⁻¹ and 1500–1350 cm⁻¹) and carbohydrate (1250 cm⁻¹ and 1000 cm⁻¹) functional peaks. Further FAME profiling revealed that the yeast biomass is primarily composed of stearic, oleic, linoleic, and linolenic acids, similar to the vegetable oils. The fuel characteristics of yeast biodiesel (SV, 168.87 mg KOH g⁻¹; IV, 120 mg I2 100 g⁻¹; CN, 61.79; and KV, 3.16 mm² s⁻¹) are also within the ASTM standard limits, suggesting that yeast biomass could be a sustainable and economically viable feedstock for both solid and liquid biofuel production.
... Yeast and microalgae mixed cultures take advantage of the complementary heterotrophic/ autotrophic nutritional modes: the microalgae consume carbon dioxide and produce oxygen, through photosynthesis, which is consumed by the yeast, which in turn supply carbon dioxide through respiration, to be consumed by the microalgae. It has been reported that yeast and microalgae co-cultures allow both microorganisms to produce higher biomass yields (Cheirsilp et al. 2011;Santos et al. 2013). Likewise, there are also metabolites exchanges and pH medium adjustment that are benefic for both microorganisms (Dias et al. 2019). ...
Recently, yeast and microalgae mixed cultures have been widely used in biological effluent treatments and biofuel production because such cultures show many advantages over pure cultures. However, industrial effluents often contain toxic compounds; therefore, it is important to evaluate the cell stress response when growing in such conditions during the mixed culture development. In this work, flow cytometry (FC) was used to differentiate Rhodosporidium toruloides cells from Tetradesmus obliquus cells, based on their size, internal complexity, and chlorophyll content. FC coupled with SYTOX Green and CFDA fluorochromes was also used to characterize the cell stress response of R. toruloides and T. obliquus individual cells in a mixed culture. This work describes, for the first time, a simple and easy method to monitor individual stress response of R. toruloides and T. obliquus cells growing in mixed cultures on brewery effluents, using FC coupled with fluorescent dyes.
... Clearly, the ability of OYC-Y.BC.SH to utilize different types of carbon sources reflects upon its ability to valorize wastes derived from lignocellulosic biomass (Ali et al., 2020a) as well as other industries such as dairy industry, sugar mill effluents and those obtained from FAME transesterification wastes (Mahari et al., 2020). Table 1 depicts lipid production parameters obtained in this study compared with those of oleaginous yeast species reported in literature (Vyas and Molitoris, 1995;Cheirsilp et al., 2011;Ling et al., 2014;Cheirsilp et al., 2012;Nascimento et al., 2013;Zhao et al., 2014;Ayadi et al., 2016). On the other hand, OYC-Y.BC.SH showed a significant difference in lipase activity when grown on various carbon sources (Fig. 4). ...
Construction of a multipurpose yeast consortium suitable for lipid production, textile dye/effluent removal and lignin valorization is critical for both biorefinery and bioremediation. Therefore, a novel oleaginous consortium, designated as OYC-Y.BC.SH has been developed using three yeast cultures viz. Yarrowia sp. SSA1642, Barnettozyma californica SSA1518 and Sterigmatomyces halophilus SSA1511. The OYC-Y.BC.SH was able to grow on different carbon sources and accumulate lipids, with its highest lipid productivity (1.56 g/L/day) and lipase activity (170.3 U/mL) exhibited in xylose. The total saturated fatty acid content was 36.09 %, while the mono-unsaturated and poly-unsaturated fatty acids were 45.44 and 18.30 %, respectively, making OYC-Y.BC.SH valuable for biodiesel production. The OYC-Y.BC.SH showed its highest decolorization efficiency of Red HE3B dye (above 82 %) in presence of sorghum husk as agricultural co-substrate, suggesting its feasibility for simultaneous lignin valorization. The significant higher performance of OYC-Y.BC.SH on decolorizing the real dyeing effluent sample at pH 8.0 suggests its potential and suitability for degrading most of the wastewater textile effluents. Clearly, toxicological studies underline the additional advantage of using OYC-Y.BC.SH for bioremediation of industrial dyeing effluents in terms of decolorization and detoxification. A possible mechanism of Red HE3B biodegradation and ATP synthesis was also proposed.
... In this study, C. vulgaris and 13218 bacteria could cooperate with each other in the utilization of limited resources under nutrition-deprived conditions, benefitting growth and biomass accumulation in the co-culture system. In addition, the synergistic effects were more likely to have an O 2 /CO 2 balance effect in co-cultures (Cheirsilp et al. 2012). Bacteria release CO 2 as a byproduct of respiration (Moroney and Somanchi 1999), and algal cells can then capture the CO 2 by photosynthesis and convert this into algal biomass (Sayre 2010). ...
Microalgal lipids have been considered as one of the most promising feedstocks for biodiesel production. In order to solve the contradiction of algae growth and lipid accumulation, Chlorella vulgaris was co-cultured with Mesorhizobium sangaii under nitrogen deficiency conditions. The biomass and lipid production of C. vulgaris–bacteria co-culture with initial ratio of algae/bacteria = 40:1 were significantly improved compared with the pure algae culture. The maximum biomass, lipid content and productivity of algae in the co-cultures at 40:1 ratio were 1.89 mg L⁻¹, 51.2%, and 96.77 mg L⁻¹ day⁻¹, respectively, which were 1.5, 2.2, and 3.3 times higher than those of the pure algae culture. Furthermore, the proportion of unsaturated fatty acids and C18:1 fatty acid of the consortium system was also significantly increased. Our study clearly suggests that co-cultivation of algae–bacteria can effectively contribute to the quality and quantity of microalgal bio-oil and shows promising applications for production of algal biomass and biodiesel.
Graphical abstract
... There are a few examples of yeasts and microalgae coculture for lipid production from sugar or glycerol using yeast species such as Rhodotorula glutinis, T. spathulata, and microalgae C. vulgaris, Chlorella pyrenoidosa and Scenedesmus obliquus (Cheirsilp, Kitcha, & Torpee, 2012;Kitcha & Cheirsilp, 2014;L. Liu, Chen, Lim, & Wei, 2018;Wang, Wu, & Wang, 2016;Yen, Chen, & Chen, 2015). ...
Microbial oils are proposed as a suitable alternative to petroleum‐based chemistry in terms of environmental preservation. These oils have traditionally been studied using sugar‐based feedstock, which implies high costs, substrate limitation, and high contamination risks. In this sense, low‐cost carbon sources such as volatile fatty acids (VFAs) are envisaged as promising building blocks for lipid biosynthesis to produce oil‐based bioproducts. VFAs can be generated from a wide variety of organic wastes through anaerobic digestion and further converted into lipids by oleaginous yeasts (OYs) in a fermentation process. These microorganisms can accumulate in the form of lipid bodies, lipids of up to 60% wt/wt of their biomass. In this context, OY is a promising biotechnological tool for biofuel and bioproduct generation using low‐cost VFA media as substrates. This review covers recent advances in microbial oil production from VFAs. Production of VFAs via anaerobic digestion processes and the involved metabolic pathways are reviewed. The main challenges as well as recent approaches for lipid overproduction are also discussed.
... Recent concerns in the microbial lipid production field for biofuel production have been focused towards the mixed culture of microalgae and yeast. A number of microalgae and yeast species in various combinations have been studied and the enhanced lipid production in the mixed culture was confirmed [7][8][9][10]. For example, 40-50% Electronic supplementary material The online version of this article (https ://doi.org/10.1007/s0044 ...
Microbial biomass which mostly generated from the microbial processes of bacteria, yeasts, and microalgae is an important resource. Recent concerns in microbial biomass production field, especially microbial lipid production for biofuel, have been focused towards the mixed culture of microalgae and yeast. To more comprehensive understanding of the mixed culture for microbial biomass, mono Chlorella pyrenoidosa, mono Yarrowia lipolytica and the mixed culture were investigated in the present work. Results showed that the mixed culture achieved significantly faster cell propagation of microalga and yeast, smaller individual cell size of yeast and higher relative chlorophyll content of microalga. The mixed culture facilitated the assimilation of carbon and nitrogen and drove the carbon flow to carbohydrate. Besides higher lipid yield (0.77 g/L), higher yields of carbohydrates (1.82 g/L), protein (1.99 g/L) and heating value (114.64 kJ/L) indicated the microbial biomass harvested from the mixed culture have more potential utilization in renewable energy, feedstuff, and chemical industry.
... In order to reduce the aeration requirements, different aeration rates can be used promoting a lower aeration in the lipid accumulation stage, and another option is to select yeasts that require lower aeration rates. Contributions to reduce the aeration requirements were mentioned in co-culture studies of oleaginous yeasts with microalgae due to the release of oxygen to the medium by the microalgae (Xue et al. 2010;Cheirsilp et al. 2012). Xue, when assessing the lipid production of mix cultivation of Spirulina platensis and Rhodotorula glutinis, registered a rapid increase in dissolved oxygen from 7.45 to 120.5% in 5 h when Spirulina platensis was added to the culture (Xue et al. 2010). ...
A growing world population and a growing number of applications for vegetable oils are generating an increasing demand for these oils, causing serious environmental problems. A sustainable lipid production is then fundamental to address these problems. Oleaginous yeasts are a promising solution for sustainable lipid production, but, with the current knowledge and technology, they are still not a serious alternative in the market. In this review, the potential of these yeasts is highlighted and a discussion is made mainly focused on the economics of the oleaginous yeast oil production and identification of the key points to be improved to achieve lower production costs and higher income. Three main stages of the production process, where costs are higher, were identified. To render economically feasible the production of oils using oleaginous yeasts, a reduction in production costs must occur in all stages, lipid yields and productivities must be improved, and production must be targeted to high-value product applications.
... Oleaginous yeasts are considered as a promising alternative lipid source for biodiesel fuel production [34]. Yeasts offer credible candidature for biodiesel production owing to their unicellular high growth rate, short life cycle, easy scale-up and rapid lipid-accumulating ability in discrete lipid bodies [35,36]. Furthermore, they are also capable of utilizing inexpensive fermentation media such as nutritional residues from agriculture and industry [37][38][39]. ...
The depletion of exhaustible underground petroleum resources has put the present civilization at stake, thereby warranting intense research on non-exhaustible fuel. With this energy crisis hitting the block, microorganisms such as yeasts are gaining wider importance as potential biofuel candidates. An indigenous yeast strain Saccharomyces cerevisiae isolated from laboratory-scale brewing was investigated for biodiesel production. Biodiesel was produced by in situ transesterification approach using 1,1,3,3-tetramethylguanidine as the catalyst. The fuel properties such as viscosity, density, calorific value and cetane number (CN) were determined to assess the fuel quality of S. cerevisiae biodiesel. Additionally, the investigation also focuses on theoretical studies considering the yeast de-oiled cake (low-value biomass refuse). Fatty acid methyl ester analysis revealed that biodiesel was primarily composed of tricosylic acid (C23:0, 28.71%), palmitoleic acid (C16:1, 28.96%) and oleic acid (C18:1, 18.13%). Eicosapentaenoic acid (C20:5, 2.01%), one of the most commonly known polyunsaturated fatty acid, was present in the yeast strain. The CN of yeast biodiesel was 71.58, which was much higher than petro-diesel. The theoretical findings suggest the competitiveness of yeast biomass conversion technologies with petroleum refining process economics. The overall study warrants the feasibility of co-production of biodiesel from S. cerevisiae and cracked biofuel products (from S. cerevisiae de-oiled cake) under the aegis of biorefining applications.
... High-cell-density continuous culture can provide much higher volumetric productivity than other suspension systems, such as batch and fedbatch cultivation [3]. To significantly improve the lipid productivity, it is necessary to boost enough cell density and control the concentration of nitrogen source to achieve a nutrient-limited condition [6]. ...
As a potential feedstock for biofuel production, a high-cell-density continuous culture for the lipid production by Cryptococcus albidus was investigated in this study. The influences of dilution rates in the single-stage continuous cultures were explored first. To reach a high-cell-density culture, a single-stage continuous culture coupled with a membrane cell recycling system was carried out at a constant dilution rate of 0.36/h with varied bleeding ratios. The maximum lipid productivity of 0.69 g/L/h was achieved with the highest bleeding ratio of 0.4. To reach a better lipid yield and content, a two-stage continuous cultivation was performed by adjusting the C/N ratio in two different stages. Finally, a lipid yield of 0.32 g/g and lipid content of 56.4% were obtained. This two-stage continuous cultivation, which provided a higher lipid production performance, shows a great potential for an industrial-scale biotechnological production of microbial lipids and biofuel production.
... Wastewater characteristics and levels of pollutants vary significantly from industry to industry based on the process involved, raw material used, by-products obtained, etc. Biodiesel production using industrial wastes is a novel technique since it resulted in the fuel generation on par with waste valorisation. Few microorganisms metabolise wastes as a feedstock and store intracellular lipids which can be converted to biodiesel (Cheirsilp et al., 2012). Biodiesel cost is the main hurdle for commercialisation and it can also be minimised by utilising waste as cheap raw material, adoption of continuous transesterification process and recovery of high-quality glycerol as biodiesel by-product. ...
... Literatürde R. glutinis'ten yararlı kimyasalların üretimi; gliserol, tek ya da ek karbon kaynağı olarak kullanılarak, araştırılmıştır. Bunlar arasında R. glutinis' ten lipid üretimi [1, 4,5] karoten üretimi [6] ve önemli antioksidan enzimler olan süperoksit dismutaz ve katalaz enzimleri [7] [8], sensör üretiminde [9], bira, meyve suyu, şarap, kurutulmuş gıda ve mayonezden oksijenin uzaklaştırtılmasında [10], tekstil endüstrisinde ve kozmetik endüstrisinde (lens bakımı) ürünlerden peroksit uzaklaştırılmasında [11] kullanılmaktadır. Ayrıca, antioksidan, yaşlanma karşıtı, hücre yenileyici, yağ eritici ve ömür uzatıcı özellikleriyle kapsüller halinde çeşitli markalar bünyesinde ticari olarak satılmaktadır. ...
... However, microalgal cell numbers in the monoculture were higher than that in the mixed culture, especially when CBH with high sugar concentration was used. This phenomenon might be caused by the suppressed photoautotrophic growth of algae cells due to the scarcely penetrated light by the high concentration of yeast cells (Cheirsilp et al., 2011a). The cell distributions of the two species in the mixed culture with different concentrations of CBH were dynamic. ...
The single cell oil (SCO) production by the mono and mixed culture of microalgae Chlorella pyrenoidosa and red yeast Rhodotorula glutinis was investigated using non-detoxified cassava bagasse hydrolysate (CBH) as carbon source. The results suggested that the two strains were able to tolerate and even degrade some byproducts presented in the CBH, and the mixed culture approach enhanced the degradation of certain byproducts. Biomass (20.37 ± 0.38 g/L) and lipid yield (10.42 ± 1.21 g/L) of the mixed culture achieved in the batch culture were significantly higher than that of the mono-cultures (p < 0.05). The fed-batch culture further raised the biomass and lipid yield to 31.45 ± 4.93 g/L and 18.47 ± 3.25 g/L, respectively. The lipids mainly composed of oleic acid and palmitic acid, suggesting the potential applications such as biofuel feedstock, cosmetics, food additives and lubricant. This study provided new insights for the integration of the economical SCO production with agro-industrial waste disposal.
The production process of microbial lipids through oleaginous microbial fermentation requires suitable strains for the rational design, operation, control, optimization, and enhancement of the entire fermentation process to maximize the efficiency of the fermentation process. Regarding the selection of suitable strains, the previous chapters detailed the screening of high-yield strains for oleaginous microorganisms and the use of metabolic engineering methods to improve oleaginous microorganisms. This chapter will introduce in detail how to use process engineering strategies to optimize the fermentation process of oleaginous microorganisms and strengthen the process. In the process of oleaginous microbial fermentation to produce lipids, “oleaginous microbial strains” and “reasonable process engineering technologies” are indispensable for oleaginous microbial fermentation technology.
Oleaginous fungi have attracted a great deal of interest for their potency to accumulate high amounts of lipids (more than 20% of biomass dry weight) and polyunsaturated fatty acids (PUFAs), which have a variety of industrial and biological applications. Lipids of plant and animal origin are related to some restrictions and thus lead to attention towards oleaginous microorganisms as reliable substitute resources. Lipids are traditionally biosynthesized intra-cellularly and involved in the building structure of a variety of cellular compartments. In oleaginous fungi, under certain conditions of elevated carbon ratio and decreased nitrogen in the growth medium, a change in metabolic pathway occurred by switching the whole central carbon metabolism to fatty acid anabolism, which subsequently resulted in high lipid accumulation. The present review illustrates the bio-lipid structure, fatty acid classes and biosynthesis within oleaginous fungi with certain key enzymes, and the advantages of oleaginous fungi over other lipid bio-sources. Qualitative and quantitative techniques for detecting the lipid accumulation capability of oleaginous microbes including visual, and analytical (convenient and non-convenient) were debated. Factors affecting lipid production, and different approaches followed to enhance the lipid content in oleaginous yeasts and fungi, including optimization, utilization of cost-effective wastes, co-culturing, as well as metabolic and genetic engineering, were discussed. A better understanding of the oleaginous fungi regarding screening, detection, and maximization of lipid content using different strategies could help to discover new potent oleaginous isolates, exploit and recycle low-cost wastes, and improve the efficiency of bio-lipids cumulation with biotechnological significance.
Microorganisms have a high potential as biofuel sources. Co-culture of microalgae and yeasts can result in high lipid production as a modification treatment. The goal of this study was to see how the co-culture of the Glagah consortium (diversity of associated microalgae and bacteria from Glagah Lagoon, Yogyakarta) and Lipomyces starkeyi affected the production of biomass, lipids, proteins, and carbohydrates. The culture was performed under airtight conditions on a shaker at 127 rpm, with a light intensity of 27.75 mol/m2/s and a temperature of 30°C. The culture was subjected to a dark: light (6:18) treatment. Biomass was measured by dry weight, lipids by the Bligh and Dyer method, proteins by the Bradford method and carbohydrates by the phenol-sulfuric acid method. On day 3, L. starkey culture produced the most biomass, yielding 2.21 g/L with a productivity of 0.49 g/L/day. On day 4, the highest lipids produced from co-culture treatment yielded 1.03 g/g with a productivity of 0.21 g/L/day. The highest protein yield was obtained from L. starkeyi culture treatment on day 4, yielding 0.60 g/g with a productivity of 0.12 g/L/day. On day 6, co-culture produced the total carbohydrates, yielding 4.78 g/g with a productivity of 0.68 g/L/day. The co-culture treatment produced the highest lipids and carbohydrates production (1.03 g/g and 4.78 g/g) and productivity (0.21 g/L/day and 0.68 g/L/day), while L. starkeyi culture produced the highest total biomass and protein production (2.21 g/L and 0.6 g/g) and productivity (0.49 g\L\day and 0.12 g/L/day). In microalgae culture, CO2 generally given directly through the aeration process. In this study, the source of CO2 was yeast, whereas yeast also obtained O2 from microalgae in the consortium for their metabolic process. This mutualism symbiosis will help in providing benefits in reducing the costs for the cultivation process, especially in optimizing the production of biomass an lipids.
Microbial biomass and lipid production with mixed-culture of Rhodotorula glutinis and Chlorella vulgaris using acetate as sole carbon source was investigated. Synergistic effect of mixed-culture using 20 g/L acetate significantly promoted cell growth and acetate utilization efficiency. Increasing the proportion of algae in co-culture was beneficial for biomass and lipid accumulation and the optimal ratio of yeast/algae was 1:2. Light exposure further enhanced biomass and lipid titer with 6.9 g/L biomass and 2.6 g/L lipid (38.3% lipid content) obtained in a 5L bioreactor. The results of lipid classes and fatty acid profiles moreover indicated that more neutral lipids and linolenic acid were synthesized in mixed-culture under light exposure condition, suggesting the great potential in applications of biofuels production. This study provided new insight and strategy for economical microbial biomass and lipid production by light-exposed mixed-culture using inexpensive acetate as carbon source.
Microalgae is considered an alternative source for biodiesel production producing renewable, sustainable and carbon-neutral energy. Microalgae property changes among species, which determines the efficiency of biodiesel production. Besides the lipid content evaluation, multi-principles (including high lipid productivity, high biomass yield, pollution resistance and desired fatty acid, etc.) for superior oil-producing species screening was proposed in this review and three microalgae species (Chlorella vulgaris, Scenedesmus obliquus and Mychonastes afer) with high bio-lipid producing prospect were screened out based on big data digging and analysis. The multilateral strategies for algal-lipid stimulating were also compared, among which, nutrient restriction, temperature control, heterotrophy and chemicals addition showed high potential in enhancing lipid accumulation; while electromagnetic field showed little effect. Interestingly, it was found that the lipid accumulation was more sensitive to nitrogen (N)-limitation other than phosphorus (P). Nutrient restriction, salinity stress etc. enhanced lipid accumulation by creating a stressed environment. Hence, optimum conditions (e.g. N:15–35 mg/L and P:4–16 mg/L) should be set to balance the lipid accumulation and biomass growth, and further guarantee the algal-lipid productivity. Otherwise, two-step cultivation could be applied during all the stressed stimulation. Different from lab study, effectiveness, operability and economy should be all considered for stimulation strategy selection. Nutrient restriction, temperature control and heterotrophy were highly feasible after the multidimensional evaluation.
Aims: The synergistic bio-activity between oleaginous yeast and microalga has been recognized, which would enhance lipid production as biodiesel feedstock. Nevertheless, yeast and microalga require different conditions for optimal growth. In this study, the locally isolated oleaginous yeast Rhodotorula toruloides and microalga Chaetoceros muelleri were co-cultivated to enhance biomass and lipid production. Methodology and results: The growth characteristics of both yeast and microalga monocultures were initially determined prior to optimizing the co-cultivation conditions. The biomass and lipid productivity of the co-culture were investigated and compared to their monocultures. The results showed that R. toruloides grew actively within 3 days while C. muelleri exhibited more prolonged cultivation, up to 21 days. The co-cultivation could be carried out optimally using growth media at pH 6, light intensity of 15,000 lux and yeast/microalga ratio of 1:2, yielding the highest biomass productivity determined at 0.18 g/l/day and lipid production of 17%. The lipid productivity of the co-culture increased by 42% and 75% as compared to monocultures of yeast and microalga, respectively. Furthermore, the biomass productivity was also higher than the monoculture, about 1.2-fold for the yeast and 13-fold for the microalga. Conclusion, significance and impact of study: The findings revealed that co-cultivation of yeast and microalga is a viable technique for long-term microbial oil production. © 2022, Malaysian Journal of Microbiology. All Rights Reserved.
Oleaginous microorganisms have the potential to convert an array of carbon sources into intracellular lipid molecules resulting in high lipid production. These lipids are also called single cell oils (SCOs) that are produced during nitrogen limitation and excess of carbon source conditions in the stationary phase of microbial growth. Depending upon different types of oleaginous species such as yeast, microalgae, bacteria, and filamentous fungi, microbial lipids differ in their fatty acid profile composition and are thus, suitably utilized in different industrial applications. Among different oleaginous species, oleaginous yeasts are considered as a prime candidate for microbial lipid or oil production owing to their high lipid content that, in turn, can be valorized into different products of commercial interest. Oleaginous yeasts have received attention because of their easy acclimatization to the surroundings and ease of genetic modifications that produce high cell density in terms of lipid or oils utilizing low-cost raw materials. In this chapter, we focus on the oleaginous yeasts and advantages of yeasts lipid, the biochemical mechanism of yeasts lipid accumulation, lipid content and fatty acid compositional analysis, innovation and state of the art in oleaginous yeast lipid production, applications of derived lipids along with major issues in lipid production technology.
The adverse impacts of fossil fuels on the environment, specifically climate change, have intensified the need for finding a sustainable alternative source of energy. Numerous studies have postulated that biotechnology development, focusing on biofuel production processes, could be a suitable solution for sustainable energy production. The utilization of microorganisms, such as microalgae, is one of the basic strategies to produce biodiesel, pharmaceuticals, and nutraceuticals. Co-cultivation has overtaken mono-cultivation to improve the production of microalgae due to its endurance, foreseeability, and stability. However, further development of the co-cultivation process requires elaborate efforts to make it safe, practical, and optimal. Some dominant factors affecting the co-culture system control are the diversity of the cell groups, mass transfer, scale-up, population ratio, and time. In this review article, we will discuss some critical topics related to the co-cultivation process, such as data collection, modelling, cultivation methods, and interaction varieties. An overview of the quantification techniques for biomass concentration and lipid content is also provided. Moreover, the utilization of microalgal co-cultures will be analyzed, depicting the difficulties associated with their efficient control. As knowledge of the reactions and their entailing kinetics is elemental for analyzing the microalgal systems, which are used to synthesize intermediate products and chemicals, some studies focusing on the kinetics of microalgal biomass conversion into biofuels are presented. Since the microbial fuel cells (MFCs), as a new bioelectrochemical process, are utilized in the mixed-culture systems to treat wastewater and produce biofuels and other valuable by-products, a summary of the microalgal MFCs is also provided. Finally, arguments about the challenges and advantages of the co-cultivation systems are presented.
Microalgae, especially oleaginous species, have gained much attention as bioenergy feedstocks in response to uprising energy crisis, lessening natural resources, and climate change. Microalgae are also used to extract high value co-products such as pigments, vitamins, proteins, long-chain polyunsaturated fatty acids, and carbohydrates, those are beneficial in various sectors. These benefits authorize microalgae as a promising source for biorefinery. The focus on zero waste approach would help promote sustainability of microalgal biorefinery. This review offers depiction of evolving zero waste and biorefinery concept for the cost/energy-effective and maintainable processes. Sequential fractionation of microalgal biomass into promising raw materials for biorefinery access and recent conversion technologies applied on microalgal biomass, are discussed. The applications for high-to-low value added-products including nutraceuticals/pharmaceuticals, food and feed, bioenergy and biofuels, and fertilizer are proposed. Key challenges for zero waste microalgae biorefinery are summarized. These strategies may greatly contribute to sustainability of microalgae-based bioproducts and biofuels.
Natural bioactive compounds have been attracting growing interest from the industries as a "greener" alternative to synthetic raw materials/products. Rhodotorula glutinis yeast naturally synthesizes added value compounds such as lipids and carotenoids, commonly used for cosmetic, pharmaceutical, and food applications. R. glutinis constitutes a rigid cell-wall structure, requiring energy-saving and efficient cell disruption methods for a sustainable recovery of the intracellular compounds. A simple and eco-friendly technology using mixed bio-based solvents (biosolvents) was evaluated here as an alternative platform to permeabilize yeast cells and to improve the selective recovery of β-carotene, torularhodin, torulene and lipids. The extraction ability of pure and solvent mixtures (methanol, ethanol, ethyl acetate, isopropanol, cyclohexane and 2-methyl tetrahydrofuran) was initially screened, demonstrating the clear impact of using mixtures to improve the extraction yields (up to threefold increase). After identifying ethyl acetate/ethanol/water as the solvent mixture with a greater capacity to extract carotenoids and lipids, the selective recovery of carotenoids and lipids was enhanced by optimizing the solvent mixture composition ratio. Envisioning the industrial application, an integrated biosolvent-based downstream platform was designed. Two different strategies were investigated to further isolate carotenoids and lipids from R. glutinis biomass and to recycle the ethyl acetate/ethanol/water mixture: (i) precipitation using cold acetone; (ii) sequential liquid-liquid extraction. The integrated process for each strategy was compared with a conventional extraction procedure in terms of recovery efficiencies and its environmental impact. Regardless of the strategy, it is shown that the mixture of ethyl acetate, ethanol and water (15/27/ 58% w/w) can be reused up to three consecutive extractive cycles, ensuring high extraction efficiency yields, while decreasing the process carbon footprint by about 75% compared to the conventional method.
Some yeast and microalgae species are able to accumulate >20% of lipids (w/w) and have complementary nutritional requirements. The use of symbiotic (mixed or sequential) cultures of yeasts and microalgae show several advantages over pure cultures, and can be a strategy to enhance microbial lipid and carotenoid production, in comparison to pure cultures. In addition, if low cost substrates are used, such as industrial residues or effluents, the process costs may be reduced. This review presents an overview of different strategies of oleaginous yeast and microalgae symbiotic cultures for lipid production using low cost substrates, highlighting the major benefits and disadvantages of such strategies in comparison with pure cultures performance.
Yeast and microalgae are microorganisms widely studied for the production of high-value compounds used in food and energy area. This work proposes a process of mixed culture of Saccharomyces cerevisiae and Chlorella vulgaris for both growth and CO2 mitigation. The process relies on mutual symbiosis between the two organisms through gas exchange, which is possible by engineering the co-dominance of populations. The two populations must be balanced in such a way so that microalgae can cope with the rate of CO2 production by the yeast activity. The process is performed in non-aerated 5l-photo-bioreactor fitted with a fermentation lock to prevent gas exchange with the outside atmosphere. With this set-up, the CO2 is produced in dissolved form and is available to the microalgae avoiding degassing and dissolution phenomena. The two organism populations are balanced at approximately 20 millions cells per ml, 12% CO2 produced by yeast was reutilized by microalgae within 168 hours of culture. A yeast and microalgae growth model in mixed culture is developed by combining each individual growth model. The predictive yeast model considers the possible metabolic pathways involved in fermentation and respiration and imposes limitation factors on these pathways, in this manner, the model can predict the partition of these pathways. The microalgae individual model is based on the photosynthetic activity. The results of this work show the feasibility of such process and could provide a basis for the development of a green process of low environmental impact.
Microalgae have been regarded as a sustainable feedstock to produce biofuel to meet the serious problems arising from the depletion of fossil fuels. However, the pure cultivation of microalgae for biofuel production is commercially infeasible due to the high costs associated with culturing and harvesting microalgae in this way. The coupling of microalgae biofuel production with wastewater treatment provides a method of circumventing this deadlock and highlights the necessity of research on the interactions between microalgae and wastewater-borne microorganisms. This paper reviews the interactions between microalgae and other microorganisms, such as microzooplankton, bacteria, fungi, algae, and viruses, and the mechanisms involved in these interactions that have been identified in recent years. Several factors affecting the outcomes of co-culture and wastewater-culture are involved. At present, the interactions between microalgae and wastewater-borne microorganisms are still unclear and merit further study.
The catabolic glycerol pathways have long been elucidated, and the regulatory properties of the enzymes involved in the major pathways have been studied in some detail. The advent of molecular biology allowed for the identification and characterization of the genes coding for the enzymes of the major catabolic pathways. Characterization of the glycerol genes, still in its infancy, produced rather confusing results and thus many of these findings are subject to revision.
Continuous energy crises and increasing demand for conventional fuels has resulted in the need for biofuels on a commercial scale. Transesterification of oils to yield biodiesel – one of the principal biofuels currently produced in large-scale operations – is coupled with significant production of a glycerol-rich water (so-called “crude” or “raw” glycerol), as an important side-product of the process. The increasing demand for biodiesel leads to abundant quantities of this glycerol-rich material on the market. Therefore, glycerol valorization has much to offer in the cost reduction of biodiesel production. To this end, various chemical or biotechnological strategies have been developed to obtain added-value products using crude glycerol as substrate. This review combines an account of our attempts to achieve a biotechnological valorization of raw glycerol with a review of appropriate literature.
Raw glycerol, byproduct from bio-diesel production process, is used as carbon substrate in several biotechnological applications. Using Clostridium butyricum F2b, 47.1 g L−1 of 1,3-propanediol was produced in batch anaerobic cultures while substrate uptake rate (rS, expressed in g L−1 h−1) increased with increase in glycerol concentration in the medium. In continuous cultures, microbial behaviour was studied in transitory states after addition of 1,3-propanediol in the chemostat vessel. Microbial growth was not affected by the high 1,3-propanediol (which was added in the chemostat vessel) concentration, while butyric and acetic acids concentrations were increased. In a two-stage continuous culture, 43.5 g L−1 of 1,3-propanediol was produced with a total volumetric productivity of 1.33 g L−1 h−1.Yarrowia lipolytica ACA-DC 50109 was grown in nitrogen-limited aerobic cultures on raw glycerol and it exhibited remarkable biomass production even at high glycerol concentration media, while rS decreased with increase in glycerol concentration. Citric acid was produced after nitrogen depletion in the medium, with the highest quantity of 62.5 g L−1, and yield on glycerol consumed was 0.56 g g−1. Fatty acid analysis of total cellular lipids showed that glycerol concentration increase in the growth medium somehow increased the cellular unsaturated fatty acids content of lipids.Mortierella isabellina ATHUM 2935 exhibited satisfactory growth in nitrogen-limited aerobic cultures with raw glycerol used as sole substrate. When high initial glycerol quantities were employed (e.g. 100 g L−1), 4.4 g L−1 of lipid were accumulated corresponding to around 51% (wt/wt) of lipid in dry weight. rS constantly decreased with increase in glycerol concentration in the medium, and in all cases notable glycerol quantities remained unconsumed in the medium.
A possible source of biological material for the production of biodiesel is represented by microalgae, in particular by their lipid content. The aim of the present work was to study of the effects of temperature and nitrogen concentration on the lipid content of Nannochloropsis oculata and Chlorella vulgaris in view of their possible utilization as novel raw materials for biodiesel production. In addition, various lipid extraction methods were investigated. The extracted lipids were quantitatively and qualitatively analyzed by gravimetric and gas chromatographic methods, respectively, in order to check their suitability according to the European standards for biodiesel. The lipid content of microalgae was strongly influenced by the variation of tested parameters; indeed, an increase in temperature from 20 to 25 °C practically doubled the lipid content of N. oculata (from 7.90 to 14.92%), while an increase from 25 to 30 °C brought about a decrease of the lipid content of C. vulgaris from 14.71 to 5.90%. On the other hand, a 75% decrease of the nitrogen concentration in the medium, with respect to the optimal values for growth, increased the lipid fractions of N. oculata from 7.90 to 15.31% and of C. vulgaris from 5.90 to 16.41%, respectively.
The growth of Yarrowia lipolytica on glycerol was studied in bioreactor repeated batch cultures and three distinct phases, namely biomass production phase, lipogenic phase and citric acid production phase were identified during growth cycle. In each phase, yeast cells were characterised by specific morphological and biochemical features. Though high activity of NAD(+) dependent iso-citric dehydrogenase (NAD(+)-ICDH) was detected during biomass production phase, this activity was significantly decreased afterwards inducing lipogenesis. A further drop in NAD(+)-ICDH activity to minimal levels and a decrease in glycerol kinase activity were observed during the citric acid production phase. Surprisingly, citric acid production was accompanied by storage (neutral) lipid turnover, along with remarkable biosynthesis of glycolipids, sphingolipids and phospholipids. Oleic acid was the major fatty acid in all lipid fractions and phosphatidylcholine was the main phospholipid. This study allows concluding that Y. lipolytica successfully converts glycerol via phosphorylation pathway into valuable biotechnological products, such as single cell oil and citric acid.
Biomass and lipid productivities of Chlorella vulgaris under different growth conditions were investigated. While autotrophic growth did provide higher cellular lipid content (38%), the lipid productivity was much lower compared with those from heterotrophic growth with acetate, glucose, or glycerol. Optimal cell growth (2 g l(-1)) and lipid productivity (54 mg l(-1) day(-1)) were attained using glucose at 1% (w/v) whereas higher concentrations were inhibitory. Growth of C. vulgaris on glycerol had a similar dose effects as those from glucose. Overall, C. vulgaris is mixotrophic.
Yarrowia lipolytica LGAM S(7)1 presented remarkable growth on industrial glycerol used as sole carbon substrate. Nitrogen-limited flask cultures were accompanied by restricted synthesis of reserve lipid, whilst amounts of citric acid were produced extracellularly. On the contrary, high amounts of reserve lipid (up to 3.5 g/l, 43% w/w of lipids in dry biomass) were produced in highly aerated continuous cultures. Lipid production was favoured at low specific dilution rates whilst fat-free material yield increased over the whole range of D (h(-1)). The maximum volumetric productivity obtained was 0.12 g lipid/1 h. Storage lipid composition did not present remarkable changes in the specific dilution rates tested. Oleate and linoleate were the dominant cellular fatty acids.
The oleaginous fungus Cunninghamella echinulata when cultivated on a tomato waste hydrolysate medium accumulated 7.8gl−1 of reserve lipid, while, after the exhaustion of the carbon source in the growth environment, 44% of this lipid was consumed and 3.2gl−1 of lipid-free biomass were synthesized. It was demonstrated that lipid fractions and individual lipid classes varied in amount, relative proportions and fatty acid profile during the turnover phase. Triacylglycerols (TAG) were preferentially consumed as their percentage proportion decreased from 26.6 to 6.9% (w/w) of lipid-free biomass, while TAG structures containing more unsaturated fatty acids were partially discriminated. Consequently, the relative proportion of γ-linolenic acid (GLA) increased in TAG from 9.2% (end of the lipogenic phase) to 15.3% (w/w), whereas C16:0 decreased from 22.7 to 15.6% (w/w). Concomitantly membrane polar lipid fractions were synthesized during lipid turnover. During the transition, glycolipids plus sphingolipids fraction was enriched in polyunsaturated fatty acids, especially in GLA, while phospholipids fraction was enriched in GLA but not in C18:2.
Crude glycerol, a by-product of biodiesel plants, was used as the sole carbon source for concomitant production of lipids and carotenoids by oleaginous red yeast Rhodotorula glutinis TISTR 5159. The addition of ammonium sulfate as a nitrogen source and Tween 20 as a surfactant increased the accumulation of lipids and carotenoids. Among the factors investigated using response surface methodology, the C/N ratio contributed a significant effect on biomass, lipid content and production of carotenoids. The synergic effects of the C/N ratio with glycerol concentration and Tween 20 concentration were observed in the accumulation of lipids. The optimum condition for biomass was glycerol concentration of 8.5% and C/N ratio of 60, while that for lipid content and carotenoids production was glycerol concentration of 9.5% and C/N ratio of 85. The production of lipids and carotenoids were further improved in a stirred tank bioreactor with pH controlled at 6.0 and aeration rate at 2 vvm. In fed-batch fermentation, the highest lipids production of 6.05 g/L with a cellular lipid content of 60.7% and carotenoids production of 135.25 mg/L were obtained. The yeast lipids obtained have shown the favorable properties for being used as feedstock in the production of biodiesel.
Raman spectrometry and electron microscopy show that, in the hydrocarbon-rich alga Botryococcus braunii, hydrocarbons accumulate in two distinct sites; internally in cytoplasmic inclusions and externally in successive outer walls and derived globules. No other classes of lipid are present in noticeable amounts in the cytoplasmic inclusions and in the external globules. The same hydrocarbons are observed in the internal and external pools but with different relative abundances, the shorter hydrocarbons being more abundant in the internal pool. The bulk of B. braunii hydrocarbons (ca 95%) is located in the external pool. Such an extracellular location allows this species to exhibit both an unusually high hydrocarbon content (15% of dry wt) and a normal level (0.75%) within the cells. The hydrocarbon pattern and location of B. braunii were compared with that of other organisms; a close relation appears between higher plant epidermal cells and this green alga. The trilaminar outer walls of B. braunii, at whose contact external hydrocarbon globules accumulate, contain a sporopollenin-like compound.
The green colonial alga Botryococcus braunii has unusually high levels of hydrocarbons. Two distinct sites of hydrocarbon accumulation are present in the species: an internal pool present in cytoplasmic inclusions and an external pool in the trilaminar outer walls and associated globules. It is generally assumed that the hydrocarbons are produced within the cells and then excreted into the external pool to maintain the intracellular content at a normal value. Various feeding experiments showed, however, that the radioactivity of the external pool is much higher than the internal one. On the other hand, there was no decrease in the labelling of internal hydrocarbons in chase experiments. Therefore, an excretory process apparently does not take place in B. braunii. The outer wall, therefore, is the main site of hydrocarbon accumulation and also the place where the bulk of B. braunii hydrocarbons are produced. The outer wall also is involved in the matrix of colony formation and the above findings account for the sharp decrease of hydrocarbon production which is associated with the loss of colonial habit. The cultures were also shown to be unable, under usual growth conditions, to catabolize their own hydrocarbons. Such a feature, along with the extracellular location of the main site of production, may account for the abnormally high content of hydrocarbons typical of B. braunii.
Fatty acid composition of three species of Chlorella were studied under conditions of photoautotrophic and heterotrophic cultivation, nitrogen starvation, and outdoor in a photobioreactor. The composition 14:0, 16:0, 16:1, 16:2, 16:3, 18:0, 18:1, 18:2, α-18:3 is confirmed for Chlorella. Fatty acids with 20 carbon atoms and four or five double bonds are considered not originating from Chlorella. Other exceptions of this composition are interpreted as mixed algal culture, bacterial contamination or impurities.
Free fatty acid (FFA), monoacylglycerol (MG) and diacylglycerol (DG) in high-FFA rice bran oil were continuously converted with glycerol (G) to form triacylglycerol (TG), using lipase fromRhizomucor miehei immobilized on anion-exchange resin. The reaction was continued for more than 1 month by a reactor with two circulation loops, each being connected to a fixed-bed reactor and a dehydrator. The reaction of 2 FFA + G DG + 2H2O appeared to occur until the glycerol was exhausted; the reaction of FFA + DG TG + H2O then followed. The consecutive esterificaion continued in the presence of 2–8 ppm water and the TG content reached 74%–88%. The industrial feasibility of this process was assessed from the standpoints of enzyme cost and value added by esterification.
Rhodotorula glutinis TISTR 5159 is oleaginous red yeast that accumulates both lipids and carotenoids. It was cultured in palm oil mill effluent
(POME) with only the addition of ammonium sulfate and Tween 20 as a suitable nitrogen source and surfactant, respectively.
Response surface methodology (RSM) was applied to optimize initial chemical oxygen demand (COD) in POME, C/N ratio, and Tween
20 concentration for concomitant production of lipids and carotenoids. Among three investigated factors, C/N ratio contributed
a significant effect upon lipid and carotenoids production. Analysis of response surface plots revealed that the optimum C/N
ratio for the biomass was 140, while that for lipid content and carotenoids were higher at 180 and 170, respectively. The
high level of the nitrogen source (with a low C/N ratio) enhanced the biomass, making the accumulation of lipids and carotenoids
less preferable. Hence, the two-stage process was attempted as an optimal way for cell growth in the first stage and product
accumulation in the second stage. The lipid yield and carotenoid production obtained in the two-stage process were higher
than those in the one-stage process. In the semi-continuous fermentation, R. glutinis TISTR 5159 accumulated high lipid content and produced a considerably high concentration of carotenoids during long-term
cultivation. Additionally, efficient COD removal by R. glutinis TISTR 5159 was observed. The biodiesel produced from yeast lipids was composed mainly of oleic and palmitic acids, similar
to those from plant oil.
Keywordscarotenoids–lipid–palm oil mill effluent–response surface methodology–
Rhodotorula glutinis
A quick, reliable and very inexpensive method is described for the analysis of fatty acids derived from soybean lipids. The
method involves extraction of soybean lipids with petroleum ether, followed by hydrolysis of lipids with KOH/MeOH (0.5 M)
for 5 min at 100 C followed by esterification with aq. HC1 (36%)/MeOH (4:1, v/v) for 15 min at 100 C. No problems were encountered
with the esterifica-tion procedure in the presence of water and the procedure gave results comparable to die more conventional
BF3/MeOH reagent. The aq. HCI/MeOH reagent is several hundred times cheaper than BF3/MeOH, and does not compromise the efficiency of the reagent.
Cryptococcus curvatus is a yeast with industrial potential because it can grow and accumulate lipid on a very broad range of substrates. In this
study we describe growth and lipid accumulation on glycerol in a fed-batch fermentation mode. We performed a fermentation
consisting of two phases. The first phase is the biomass production phase in which there is no nutrient limitation except
for very short periods of glycerol exhaustion. The substrate feed was controlled by the dissolved oxygen tension. In the second
phase nitrogen limitation was introduced, which causes lipid accumulation. This way very high cell densities of 118 g/l in
a 50-h fermentation could be reached. With a lipid production rate of 0.59 g lipid l-1h-1, a cellular lipid content of 25% was obtained. The growth and lipid accumulation phase are characterized by different cellular
fatty acid compositions. In the growth phase, a relatively high amount of C18:2 (linoleic acid) is present, which is a major
component of membrane lipids. C18:0 (stearic acid) and C18:1 (oleic acid) are major constituents of the accumulated triglycerides
and therefore the relative amount of C18:2 decreases during the lipid accumulation phase.
Rice bran oil containing 30–50% free fatty acid was continually converted to an oil containing more than 75% of triacylglycerol
(TG) by means of immobilized lipase. The reaction was carried out at 60°C for 24 h with dehydration and reactant mixing by
dry nitrogen flow under a positive nitrogen atmosphere. Enzymatic TG synthesis with evaporation by heating was not suitable
because of the increasing peroxide value of the oil.
In this study, we found that Rhodotorula mucilaginosa TJY15a could accumulate 48.8% (w/w) oil from hydrolysate of inulin and its cell dry weight reached 14.8 g/l during the batch cultivation while it could accumulate 48.6% (w/w) oil and 52.2% (w/w) oil from hydrolysate of extract of Jerusalem artichoke tubers and its cell dry weight reached 14.4 g/l and 19.5 g/l during the batch and fed-batch cultivations, respectively. At the end of the fed-batch cultivation, only 0.04% of reducing sugar and 0.08% of total sugar were left in the fermented medium. Over 87.6% of the fatty acids from the yeast strain TJY15a cultivated in the hydrolysate of extract of Jerusalem artichoke tubers was C16:0, C18:1 and C18:2, especially C18:1 (54.7%). Therefore, the results show that hydrolysates of inulin and extract of Jerusalem artichoke tubers were also the good materials for single cell oil production.
The effects of nitrate, ammonium, and urea as nitrogen sources on the heterotrophic growth of Chlorella protothecoides were investigated using flask cultures. No appreciable inhibitory effect on the algal growth was observed over a nitrogen concentration range of 0.85–1.7 g l−1. In contrast, differences in specific growth rate and biomass production were found among the cultures with the various nitrogen compounds. The influence of different nitrogen sources at a concentration equivalent to 1.7 g l−1 nitrogen on the heterotrophic production of biomass and lutein by C. protothecoides was investigated using the culture medium containing 40 g l−1 glucose as the sole carbon and energy source in fermentors. The maximum biomass concentrations in the three cultures with nitrate, ammonium, and urea were 18.4, 18.9, and 19.6 g l−1 dry cells, respectively. The maximum lutein yields in these cultures were between 68.42 and 83.81 mg l−1. The highest yields of both biomass and lutein were achieved in the culture with urea. It was therefore concluded that urea was the best nitrogen source for the production of biomass and lutein. Based on the experimental results, a group of kinetic models describing cell growth, lutein production, and glucose and nitrogen consumption were proposed and a satisfactory fit was found between the experimental results and predicted values. Dynamic analysis of models demonstrated that enhancing initial nitrogen concentration in fermentor cultures, which correspondingly enhances cell growth and lutein formation, may shorten the fermentation cycle by 25–46%.
Hydrogenolysis of glycerol to propylene glycol was performed using nickel, palladium, platinum, copper, and copper-chromite catalysts. The effects of temperature, hydrogen pressure, initial water content, choice of catalyst, catalyst reduction temperature, and the amount of catalyst were evaluated. At temperatures above 200 °C and hydrogen pressure of 200 psi, the selectivity to propylene glycol decreased due to excessive hydrogenolysis of the propylene glycol. At 200 psi and 200 °C the pressures and temperaures were significantly lower than those reported in the literature while maintaining high selectivities and good conversions. The yield of propylene glycol increased with decreasing water content. A new reaction pathway for converting glycerol to propylene glycol via an intermediate was validated by isolating the acetol intermediate.
Microbial lipid production by the oleaginous yeast Rhorosporidium toruloides Y4 was studied using glucose as carbon source, in order to realize high-density cell culture. Batch cultures demonstrated that there was little inhibitory effect with a substrate concentration of up to 150 g l−1. Flask fed-batch cultures were run for 25 days and reached a dry biomass and cellular lipid content of 151.5 g l−1 and 48.0% (w/w), respectively. Using pilot-scale fed-batch cultures in a 15-l stirred-tank fermenter for 134 h resulted in dry biomass, lipid content and lipid productivity of 106.5 g l−1, 67.5% (w/w) and 0.54 g l−1 h−1, respectively. The fed-batch culture model used here featured initial nutrient-rich media and a pure carbon source with discontinuous feeding. Gas chromatography analysis revealed that lipids from R. toruloides Y4 contained mainly long-chain fatty acids with 16 and 18 carbon atoms. The four major constituent fatty acids were oleic acid, palmitic acid, stearic acid and linoleic acid. A slight increase in stearic acid production was found during the culture process. Based on these compositional data, microbial lipids from R. toruloides Y4 are a potential alternative oil resource for biodiesel production.
Rhodotorula mucilaginosa TJY15a which was isolated from surface of marine fish could accumulate a large amount of lipid from hydrolysate of cassava starch. The cells contained 47.9% (w/w) oil during batch cultivation, whereas 52.9% (w/w) of lipid was obtained during the fed-batch cultivation. At the end of the fed-batch cultivation, all the starch were converted into reducing sugar and only 0.34 g dm−3 of reducing sugar was left in the fermented medium. Therefore, the marine-derived R. mucilaginosa TJY15a was another candidate for single cell oil production. The fatty acids from R. mucilaginosa TJY15a were mainly composed of palmitic acid (C16:0), palmitoleic acid (C16:1), stearic acid (C18:0), oleic acid (C18:1) and linolenic acid (C18:2), suggesting that the fatty acids could be used as feedstock for biodiesel production.
A mixed culture of oleaginous yeast Rhodotorula glutinis and microalga Chlorella vulgaris was performed to enhance lipid production from industrial wastes. These included effluent from seafood processing plant and molasses from sugar cane plant. In the mixed culture, the yeast grew faster and the lipid production was higher than that in the pure cultures. This could be because microalga acted as an oxygen generator for yeast, while yeast provided CO(2) to microalga and both carried out the production of lipids. The optimal conditions for lipid production by the mixed culture were as follows: ratio of yeast to microalga at 1:1; initial pH at 5.0; molasses concentration at 1%; shaking speed at 200 rpm; and light intensity at 5.0 klux under 16:8 hours light and dark cycles. Under these conditions, the highest biomass of 4.63±0.15 g/L and lipid production of 2.88±0.16 g/L were obtained after five days of cultivation. In addition, the plant oil-like fatty acid composition of yeast and microalgal lipids suggested their high potential for use as biodiesel feedstock.
Cryptococcus curvatus, an oleaginous yeast was observed to grow on crude glycerol derived from yellow grease. When cultured in a one-stage fed-batch process wherein crude glycerol and nitrogen source were fed intermittently for 12 days, the final biomass density and lipid content were 31.2 g/l and 44.2%, respectively. When cultured in a two-stage fed-batch operation wherein crude glycerol was supplemented at different time points while nitrogen source addition was discontinued at the middle of the experiment, the biomass density was 32.9 g/l and the lipid content was 52% at the end of 12 days. Compared with other oil feedstocks for biodiesel production, lipid accumulated by C. curvatus grown on glycerol has high concentration of monounsaturated fatty acid, which makes it an excellent source for biodiesel use.
Mix cultivation of microalgae (Spirulina platensis) and yeast (Rhodotorula glutinis) for lipid production was studied. Mixing cultivation of the two microorganisms significantly increased the accumulation of total biomass and total lipid yield. Dissolved oxygen and medium components in the mixed fermentation medium were analyzed. Mix cultivation in monosodium glutamate wastewater was further studied. Result indicated 1,600 mg/L of biomass was obtained and 73% of COD were removed.
The calorific value of five strains of Chlorella grown in Watanabe and low-nitrogen medium was determined. The algae were grown in small (2L) stirred tank bioreactors and the best growth was obtained with Chlorella vulgaris with a growth rate of 0.99 d(-1) and the highest calorific value (29 KJ/g) was obtained with C. emersonii. The cellular components were assayed at the end of the growth period and the calorific value appears to be linked to the lipid content rather than any other component.
To study patterns of reserve lipid biosynthesis and turnover (degradation) in two oleaginous Zygomycetes, namely Cunninghamella echinulata and Mortierella isabellina under various growth conditions. Fatty acid composition of the reserve lipid of both strains was also studied in all growth steps.
Cunninghamella echinulata and Mortierella isabellina were grown in carbon-excess batch cultures. In the investigated strains, accumulation of reserve lipid occurred only when the activity of both NAD(+)-isocitrate dehydrogenase (ICDH) and NADP(+)-ICDH were not detectable in the cell-free extract. Specifically, in C. echinulata, NAD(+)-ICDH activity was detected even after depletion of ammonium nitrogen in the medium, resulting in a delay of the initiation of lipid accumulation period. On the contrary, in M. isabellina, lipid accumulation occurred simultaneously with ammonium nitrogen exhaustion in the growth medium, as the activity of both NAD(+)- and NADP(+)-ICDH were not detectable after nitrogen depletion. In C. echinulata reserve lipid was not degraded after glucose had been exhausted. Supplementations of the medium with Fe(3+), yeast extract or Mg(2+) induced, however, reserve lipid breakdown and formation of lipid-free material. In M. isabellina after glucose exhaustion, notable lipid degradation occurred, accompanied by a significant lipid-free material biosynthesis. Nevertheless, in multiple-limited media, in which Mg(2+) or yeast extract, besides carbon and nitrogen, were limiting nutrients, reserve lipid breakdown was repressed. In both strains, the quantity of gamma-linolenic acid (GLA) in the reserve lipids [varying between 9 and 16% (w/w) in C. echinulata and 1.5-4.5% (w/w) in M. isabellina] was proportional to lipid-free biomass.
Lipid accumulation period in Zygomycetes is initiated by the attenuation of ICDH activity in the mycelium while the regulation of ICDH from ammonium nitrogen is strain specific. While a single nitrogen limitation was enough to induce lipid accumulation, however, multiple limitations were needed in order to repress lipid turnover in oleaginous Zygomycetes. As for GLA, its biosynthesis in the mycelium seemed proportional to lipid-free biomass synthesis.
Several nutrients are indispensable for functioning the mechanisms involved in the mobilization of reserve lipid in oleaginous moulds. Therefore, reserve lipid turnover in oleaginous moulds could be repressed in multiple-limited media.
The potential of accumulation of lipids by Lipomyces starkeyi when grown on sewage sludge was assessed. On a synthetic medium, accumulation of lipids strongly depended on the C/N ratio. The highest content of lipids was measured at a C/N-ratio of 150 with 68% lipids of the dry matter while at a C/N-ratio of 60 only 40% were accumulated. Within a pH range from 5.0 to 7.5 the highest lipid accumulation was found at pH 5.0 while the highest yield per litre was pH 6.5. Although sewage sludge had no inhibitory effects on growth or accumulation on L. starkeyi when added to synthetic medium, there was no significant growth on untreated sewage sludge. However, pretreatment of sludge by alkaline or acid hydrolysis, thermal or ultrasonic treatment lead to accumulation of lipids by L. starkeyi with highest values of 1 g L(-1) obtained with ultrasound pre-treatment. Based on the content of free fatty acids and phosphorus, lipids accumulated from sewage sludge could serve as a substrate for the production of biodiesel.
The alga Isochrysis galbana 8701 and the yeast Ambrosiozyma cicatricosa were mix-cultivated in the same medium for 7 d to compare their growth performance and biochemical composition with those of the same organisms cultured under monoculture conditions. The specific growth rates of both species were significantly higher (p<0.05) in the mixed culture than in the monocultures during the corresponding experimental phases. At the end of experiment, the biomass concentration obtained in the mixed culture reached 1.32+/-0.04 g/l of dry weight, which was significantly higher (p<0.05) than those obtained in monocultures, and the alga I. galbana in the mixed culture dominated the cell numbers making up 96.64% of the cells. The biochemical profile of the mixed culture is similar to that of the I. galbana monoculture with some variations; The percentages of both the fatty acids 14:00 and 18:00 detected in the mixed culture were significantly higher (p<0.05) than those detected in the I. galbana monoculture, while the content of the fatty acid 18:2(n-6) detected in the mixed culture was significantly lower (p<0.05) than that detected in the I. galbana monoculture. This study indicates the improved growth performance in mixed culture compared with monocultures and the similarities between the biochemical compositions of the mixed culture and the I. galbana monoculture.
Conversion of sewage sludge into lipids by Lipomyces starkeyi for biodiesel production Comparisons of growth and biochemical composition between mixed culture of alga and yeast and monocultures
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Increase in Chlorella strains calorific values when grown in low nitrogen medium Use of aqueous HCl/MeOH as esterification reagent for analysis of fatty acids derived from soybean lipids
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Metabolic activities of biotechnological interest in Yarrowia lipolytica grown on glycerol in repeated batch cultures High-cell-density cultivation of the lipid accumulating yeast Cryptococcus curvatus using glycerol as a carbon source
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A, Fakas S, Aggelis G (2010) Metabolic activities of biotechnological interest in Yarrowia lipolytica grown on glycerol in repeated batch cultures. Bioresour Technol 101:2351–2358 Meesters PAEP, Huijberts GNM, Eggink G (1996) High-cell-density cultivation of the lipid accumulating yeast Cryptococcus curvatus using glycerol as a carbon source. Appl Microbiol Biotechnol 45:575–579