Oleaginous Fungus Research Articles

Purification and characterization of isocitrate dehydrogenase from Mortierella alpina

Isocitrate dehydrogenase (IDH) in oleaginous microorganisms plays an essential role in fatty acid synthesis. We expressed and purified an IDH isozyme from the oleaginous fungus Mortierella alpina (MaIDH4) and identified its enzymatic properties. MaIDH4 is NADP+-dependent and has no detectable activity with NAD+. It exerts its maximum activity when Mn2+ is used as a cofactor, followed by Mg2+. The optimum temperatures was 60 °C and 65 °C, and the optimum pH was 8.5 and 9.5, in the presence of Mn2+ and Mg2+, respectively. The catalytic efficiency of MaIDH4 for NADP+ was 16.33 (Mn2+) and 3.86 (Mg2+) μM−1s−1, while that for isocitrate was 3.06 (Mn2+) and 0.65 (Mg2+) μM−1s−1. Characterization of the enzymatic properties of MaIDH4 provides a basis for further understanding lipid synthesis in M. alpina.

Identification and Functional Characterization of Adenosine Deaminase in Mucor circinelloides: A Novel Potential Regulator of Nitrogen Utilization and Lipid Biosynthesis.

Adenosine deaminase (ADA) is an enzyme distributed in a wide variety of organisms that cleaves adenosine into inosine. Since inosine plays an important role in nitrogen metabolism, ADA may have a critical function in the regulation of fatty acid synthesis. However, the role of ADA in oleaginous fungi has not been reported so far. Therefore, in this study, we identified one ada gene encoding ADA (with ID scaffold0027.9) in the high lipid-producing fungus, Mucor circinelloides WJ11, and investigated its role in cell growth, lipid production, and nitrogen metabolism by overexpressing and knockout of this gene. The results showed that knockout of the ada altered the efficiency of nitrogen consumption, which led to a 20% increment in the lipid content (25% of cell dry weight) of the engineered strain, while overexpression of the ada showed no significant differences compared with the control strain at the final growth stage; however, interestingly, it increased lipid accumulation at the early growth stage. Additionally, transcriptional analysis was conducted by RT-qPCR and our findings indicated that the deletion of ada activated the committed steps of lipid biosynthesis involved in acetyl-CoA carboxylase (acc1 gene), cytosolic malic acid enzyme (cme1 gene), and fatty acid synthases (fas1 gene), while it suppressed the expression of AMP-activated protein kinase (ampk α1 and ampk β genes), which plays a role in lipolysis, whereas the ada-overexpressed strain displayed reverse trends. Conclusively, this work unraveled a novel role of ADA in governing lipid biosynthesis and nitrogen metabolism in the oleaginous fungus, M. circinelloides.

Homologous Overexpression of Diacylglycerol Acyltransferase in Oleaginous Fungus Mucor circinelloides WJ11 Enhances Lipid Accumulation under Static Solid Cultivation.

Diacylglycerol acyltransferase (DGAT) catalyzes the binding of acyl-CoA to diacylglycerol to form triacylglycerol (TAG). Previous studies strongly indicate that DGAT2, rather than DGAT1, is crucial for TAG accumulation in the oleaginous fungus Mucor circinelloides. To increase the lipid content of M. circinelloides WJ11, McDGAT2 was overexpressed by homologous recombination; compared to the control strain Mc2075, transformants McDGAT2d showed a significant increase in biomass for both spores and mycelia (from 87.7 to 101.2 mg/g in spores and from 75.6 to 93.1 mg/g in mycelia). McDGAT2 overexpression under static solid fermentation gave a greater boost to lipid accumulation in mycelia than in spores. Total fatty acid content in mycelia increased by 68.0% (from 13.6 to 22.8%) and in spores by 26.3% (from 10.6 to 13.4%). However, under submerged fermentation, the lipid content of McDGAT2d was the same as the control, while biomass was slightly reduced. Transcriptomics showed that NADPH was derived mainly from the pentose phosphate pathway, acetyl-CoA was from multiple pathways, and leucine metabolism played an important role in substrate supply for fatty acid biosynthesis. Static solid fermentation may be the more suitable fermentation method for microbial oil production by filamentous fungi due to its lower fermentation costs.

Eco-friendly production of nutraceutical and cosmeceutical fatty acids by oleaginous fungus Lichtheimia hyalospora UCP 1266 using renewable substrates

Polyunsaturated fatty acids (PUFAs) are essential for human functions and can be used in nutraceuticals; however, traditional sources are environmentally unsustainable. A promising strategy to reduce the production costs of PUFAs is the use of economical agro-industrial sources as substrates in culture media for oleaginous microorganisms. In this study, we investigated the potential of the fungus Lichtheimia hyalospora UCP 1266 (isolated from the Caatinga soil, Brazil) in the production of PUFAs, through the metabolic conversion of alternative substrates crude glycerol (CG) and corn steep liquor (CSL). The cultivation of L. hyalospora UCP 1266 in medium containing glucose (Synthetic Medium for Mucorales - SMM) yielded 2.1 g/L of biomass and 0.6 g/L of total lipids. However, assay 4 of the 22 full-factorial design, with CG 8% and CSL 8%, provided higher yields of biomass (15.5 g/L) and total lipids (12.8 g/L). The lipids produced in this medium presented a composition of saturated, monounsaturated and polyunsaturated fatty acids (SFAs=35.13%, MUFAs=46.09% and PUFAs=18.14%). The microorganism proved to be promising in the production of omega 6 (linoleic acid, C18:2), γ-linolenic acid (C18:3), omega 3 (α-linolenic acid, C18:3) and omega 9 (oleic acid, C18:1). The results indicated L. hyalospora UCP 1266 as an oleaginous fungus with great potential for use in the development of nutraceutical ingredients for food or applied in cosmeceuticals and/or nutricosmetics.

Production, Biosynthesis, and Commercial Applications of Fatty Acids From Oleaginous Fungi.

Oleaginous fungi (including fungus-like protists) are attractive in lipid production due to their short growth cycle, large biomass and high yield of lipids. Some typical oleaginous fungi including Galactomyces geotrichum, Thraustochytrids, Mortierella isabellina, and Mucor circinelloides, have been well studied for the ability to accumulate fatty acids with commercial application. Here, we review recent progress toward fermentation, extraction, of fungal fatty acids. To reduce cost of the fatty acids, fatty acid productions from raw materials were also summarized. Then, the synthesis mechanism of fatty acids was introduced. We also review recent studies of the metabolic engineering strategies have been developed as efficient tools in oleaginous fungi to overcome the biochemical limit and to improve production efficiency of the special fatty acids. It also can be predictable that metabolic engineering can further enhance biosynthesis of fatty acids and change the storage mode of fatty acids.

Improved γ-Linolenic Acid Production from Cellulose in Mucor circinelloides via Coexpression of Cellobiohydrolase and Delta-6 Desaturase.

The present study was aimed at facilitating the production of γ-linolenic acid (GLA) from the cellulosic substrate with the engineered oleaginous fungus Mucor circinelloides WJ11. Here, the homologous recombination technology was used to overexpress the cellobiohydrolase (CBH2) derived from Trichoderma longibrachiatum and the original delta-6 fatty acid desaturase (D6) in M. circinelloides to construct genetically engineered strains capable of effectively using cellulose to enhance GLA synthesis. When cultivated in modified K&R medium supplemented with microcrystalline cellulose, the CBH2 and D6 coexpressing strains led to increases in the biomass (up to 12.8 g/L) and lipid yield (up to 3.7 g/L) of 87% and 2.4-fold, respectively, compared to that of the control strain. Notably, when CBH2 and D6 were coexpressed in M. circinelloides, the yield of GLA reached 608 mg/L, which was a dramatic increase of 3.9-fold compared to that of the control strain. This is the first report on promoting the GLA production from the cellulosic substrate via coexpression of CBH2 and delta-6 desaturase. This work provides a theoretical basis for efficient transformation from the cellulosic substrate to functional GLA by CBH2 and D6 coexpressing strains, which might play a positive role in promoting the sustainable development of biological industry.

Role of Cytosolic Malic Enzyme in Oleaginicity of High-Lipid-Producing Fungal Strain Mucor circinelloides WJ11.

Mucor circinelloides, an oleaginous filamentous fungus, is gaining popularity due to its ability to synthesize significant amounts of lipids containing γ-linolenic acid (GLA) that have important health benefits. Malic enzyme (ME), which serves as the main source of NADPH in some fungi, has been found to regulate lipid accumulation in oleaginous fungi. In the present study, the role of two cytosolic ME genes, cmalA and cmalB, in the lipid accumulation of the M. circinelloides high-lipid-producing strain WJ11, was evaluated. Strains overexpressing cmalA and cmalB showed a 9.8- and 6.4-fold rise in specific ME activity, respectively, and an elevation of the lipid content by 23.2% and 5.8%, respectively, suggesting that these genes are involved in lipid biosynthesis. Due to increased lipid accumulation, overall GLA content in biomass was observed to be elevated by 11.42% and 16.85% in cmalA and cmalB overexpressing strains, respectively. Our study gives an important insight into different studies exploring the role of the cmalA gene, while we have for the first time investigated the role of the cmalB gene in the M. circinelloides WJ11 strain.

Biodiesel Production from Bioremediation of Palm Oil Mill Effluent via Oleaginous Fungi

AbstractPalm oil mill effluent (POME) is a by‐product of palm oil production. POME can be considered as harmful waste without any treatment. POME, however, has the potential to be a source of low‐cost carbon for lipid production. This study aims to isolate potential oleaginous fungi from POME and to evaluate the ability of lipid production capacity of the purified isolates and commercial oleaginous fungi strains (Aspergillus niger, Aspergillus oryzae and Rhizopus spp.) from POME. POME is converted into lipid via the metabolic pathway of oleaginous fungi, where the lipids can be further trans‐esterified into microbial biodiesel. In this study, two fungal strains are successfully isolated from POME. The combination of Bligh and Dyer with Soxhlet extraction results in better lipid extraction efficiency. A. niger has the highest lipid yield from the cultivation on POME. The projected fuel properties of microbial biodiesel from A. niger are within the limits of Biodiesel Standard. Chemical oxygen demand (COD) removal from POME using A. niger is proven to be effective with 70% COD removal. The production of biodiesel from wastes of the palm oil industry may improve the industry's sustainability through the use of nonfood feedstock coupled with simultaneous bioremediation of POME.

Regulation of the Leucine Metabolism in Mortierella alpina.

The oleaginous fungus Mortierella alpina is a safe source of polyunsaturated fatty acids (PUFA) in industrial food and feed production. Besides PUFA production, pharmaceutically relevant surface-active and antimicrobial oligopeptides were isolated from this basal fungus. Both production of fatty acids and oligopeptides rely on the biosynthesis and high turnover of branched-chain-amino acids (BCAA), especially l-leucine. However, the regulation of BCAA biosynthesis in basal fungi is largely unknown. Here, we report on the regulation of the leucine, isoleucine, and valine metabolism in M. alpina. In contrast to higher fungi, the biosynthetic genes for BCAA are hardly transcriptionally regulated, as shown by qRT-PCR analysis, which suggests a constant production of BCAAs. However, the enzymes of the leucine metabolism are tightly metabolically regulated. Three enzymes of the leucine metabolism were heterologously produced in Escherichia coli, one of which is inhibited by allosteric feedback loops: The key regulator is the α-isopropylmalate synthase LeuA1, which is strongly disabled by l-leucine, α-ketoisocaproate, and propionyl-CoA, the precursor of the odd-chain fatty acid catabolism. Its gene is not related to homologs from higher fungi, but it has been inherited from a phototrophic ancestor by horizontal gene transfer.

Investigation on Cell Disruption Techniques and Supercritical Carbon Dioxide Extraction of Mortierella alpina Lipid.

Mortierella alpina, an oleaginous fungus, has been shown to be a potential source for arachidonic acid (ARA) production. The recovery of intracellular lipids from M. alpina is an important step for the downstream bioprocessing, and green extraction techniques with a focus on being efficient and eco-friendly have drawn much attention. In this study, different cell disruption techniques (mechanical: high-speed homogenization 10,000 rpm, ultrasonication 20 kHz, high-pressure process (HPP) 200–600 MPa; non- mechanical: acid treatment HCl) were investigated for lipid recovery from M. alpina, and process parameters (A. temperature, B. pressure, C. cosolvent ratio) of supercritical carbon dioxide (SC-CO2) lipid extraction were studied by applying response surface methodology (RSM). Compared with Soxhlet extraction as a control group (100%), high-speed homogenization has the highest lipid recovery (115.40%) among mechanical disruption techniques. Besides, there was no significant difference between high-speed homogenization and 1 M HCl treatment (115.55%) in lipid recovery. However, lipid recovery decreased to 107.36% as the concentration of acid was increased to 3 M, and acid treatment showed a negative effect on the ARA ratio. In HPP treatment, the highest lipid recovery (104.81%) was obtained at 400 MPa, 1 time of treatment and water medium. In the response surface model of SC-CO2 extraction, results showed the major influence of the process parameters to lipid recovery was pressure, and there are interaction effects of AC (temperature and cosolvent ratio) and BC (pressure and cosolvent ratio). Lipid recovery of SC-CO2 extraction reached 92.86% at 201 bar, 58.9 °C and cosolvent ratio 1:15. The microbial lipid recovery process of this study could be used as a reference and an eco-friendly alternative for the future downstream bioprocessing of ARA production by M. alpina.

Isolation of oleaginous fungi from natural sources, and fatty acid profile of Cunninghamella echinulata

Oleaginous microorganisms accumulate 20% or more of their dry weight as lipid. In the present study, 247 fungal isolates were obtained from different types of soil, marine water, and food, and screened for their ability to accumulate considerable amounts of lipid. A primary screening with Nile red staining was followed by sulfo-phosphovanillin reaction. The most potent isolate was identified employing both morphological and molecular methods as the Zygomycetous fungus, Cunninghamella echinulata AUMC14396. The lipid of this fungus was extracted, transesterified, and analyzed via GC-MS to elucidate the fatty acid (FA) composition. The fatty acid profile revealed the presence of various fatty acids with different concentrations. Of the total fatty acids, saturated FAs comprised 44.49%, while monounsaturated FAs were 19.91%, and polyunsaturated fatty acids (PUFAs) were 35.61%. The most predominant species was linoleic acid (13.79%) followed by stearic (12.78%) and oleic (12.50%) acids. The polyunsaturated fatty acid, gamma linolenic acid was present in a considerable amount (8.02%). Other important PUFAs were also found in the fatty acid profile such as arachidonic acid, eicosapentaenoic acid, and docosahexaenoic acid. The results of this study were discussed in respect to the important applications in the context of production of pharmaceuticals and nutraceuticals.

Tricarboxylate Citrate Transporter of an Oleaginous Fungus Mucor circinelloides WJ11: From Function to Structure and Role in Lipid Production.

The citrate transporter protein (CTP) plays an important role in citrate efflux from the mitochondrial matrix to cytosol that has great importance in oleaginous fungi. The cytoplasmic citrate produced after citrate efflux serves as the primary carbon source for the triacylglycerol and cholesterol biosynthetic pathways. Because of the CTP's importance, our laboratory has extensively studied its structure/function relationships in Mucor circinelloides to comprehend its molecular mechanism. In the present study, the tricarboxylate citrate transporter (Tct) of M. circinelloides WJ11 has been cloned, overexpressed, purified, kinetically, and structurally characterized. The Tct protein of WJ11 was expressed in Escherichia coli, isolated, and functionally reconstituted in a liposomal system for kinetic studies. Our results showed that Tct has a high affinity for citrate with Km 0.018 mM. Furthermore, the tct overexpression and knockout plasmids were created and transformed into M. circinelloides WJ11. The mitochondria of the tct-overexpressing transformant of M. circinelloides WJ11 showed a 49% increase in citrate efflux, whereas the mitochondria of the tct-knockout transformant showed a 39% decrease in citrate efflux compared to the mitochondria of wild-type WJ11. To elucidate the structure-function relationship of this biologically important transporter a 3D model of the mitochondrial Tct protein was constructed using homology modeling. The overall structure of the protein is V-shaped and its 3D structure is dimeric. The transport stability of the structure was also assessed by molecular dynamics simulation studies. The activity domain was identified to form hydrogen bond and stacking interaction with citrate and malate upon docking. Tricarboxylate citrate transporter has shown high binding energy of −4.87 kcal/mol to citric acid, while −3.80 kcal/mol to malic acid. This is the first report of unraveling the structural characteristics of WJ11 mitochondrial Tct protein and understanding the approach of the transporting toward its substrate. In conclusion, the present findings support our efforts to combine functional and structural data to better understand the Tct of M. circinelloides at the molecular level and its role in lipid accumulation.

Submerged Fermentation of Animal Fat By-Products by Oleaginous Filamentous Fungi for the Production of Unsaturated Single Cell Oil

Animal waste fats were explored as a fermentation substrate for the production of high-value unsaturated single cell oil (SCO) using oleaginous fungi, Mucor circinelloides and Mortierella alpina. Both strains showed good growth and lipid accumulation when using animal fat as a single carbon source. The biomass concentration of 16.7 ± 2.2 gDCW/L and lipid content of 54.1%wt (of dry cell weight) were obtained for Mucor circinelloides in shake flask experiments, surpassing the biomass yield achieved in batch and fed-batch fermentation. In contrast, Mortierella alpina gave the highest biomass concentration (8.3 ± 0.3 gDCW/L) and lipid content (55.8%wt) in fed-batch fermentation. Fat grown Mortierella alpina was able to produce arachidonic acid (ARA), and the highest ARA content of 23.8%wt (of total lipid weight) was in fed-batch fermentation. Gamma-linolenic acid (GLA) was produced by both fungal strains. At the end of fed-batch fermentation, the GLA yields obtained for Mucor circinelloides and Mortierella alpina were 4.51%wt and 2.77%wt (of total lipid weight), respectively. This study demonstrates the production of unsaturated SCO-rich fungal biomass from animal fat by fermentation.

Functional characterization of two diacylglycerol acyltransferase 1 genes in Mortierella alpina.

Diacylglycerol acyltransferase (DGAT) is a crucial enzyme in the triacylglycerol (TAG) biosynthesis pathway. The oleaginous fungus Mortierella alpina can accumulate large amounts of arachidonic acid (ARA, C20:4) in the form of TAG. Therefore, it is important to study the functional characteristics of its DGAT. Two putative genes MaDGAT1A/1B encoding DGAT1 were identified in M. alpina ATCC 32222 genome by sequence alignment. Sequence alignment with identified DGAT1 homologs showed that MaDGAT1A/1B contain seven conserved motifs that are characteristic of the DGAT1 subfamily. Conserved domain analysis showed that both MaDGAT1A and MaDGAT1B belong to the Membrane-bound O-acyltransferases superfamily. The transforming with MaDGAT1A/1B genes could increase the accumulation of TAG in Saccharomyces cerevisiae to 4·47 and 7·48% of dry cell weight, which was 7·3-fold and 12·3-fold of the control group, respectively, but has no effect on the proportion of fatty acids in TAG. This study showed that MaDGAT1A/1B could effectively promote the accumulation of TAG and therefore may be used in metabolic engineering aimed to increase TAG production of oleaginous fungi.

Heterologous Expression of Two Malate Transporters From an Oleaginous Fungus Mucor circinelloides Improved the Lipid Accumulation in Mucor lusitanicus.

The fungus, Mucor lusitanicus, is of great interest for microbial lipids, because of its ability to accumulate intracellular lipid using various carbon sources. The biosynthesis of fatty acid requires the reducing power NADPH, and acetyl-CoA, which is produced by the cleavage of citrate in cytosol. In this study, we employed different strategies to increase lipid accumulation in the low lipid-producing fungi via metabolic engineering technology. Hence, we constructed the engineered strain of M. lusitanicus CBS 277.49 by using malate transporter (mt) and 2-oxoglutarate: malate antiporter (sodit) from M. circinelloides WJ11. In comparison with the control strain, the lipid content of the overexpressed strains of mt and sodit genes were increased by 24.6 and 33.8%, respectively. These results showed that mt and sodit can affect the distribution of malate in mitochondria and cytosol, provide the substrates for the synthesis of citrate in the mitochondria, and accelerate the transfer of citrate from mitochondria to cytosol, which could play a significant regulatory role in fatty acid synthesis leading to lipids over accumulation.

SNF1β-Modulated Glucose Uptake and the Balance between Polyunsaturated Fatty Acids and Carbohydrates in Mortierella alpina.

A sucrose nonfermenting protein kinase 1 (SNF1) complex is an important metabolic regulator in fungi that is critical to cell metabolism and stress response. In this study, the role of an SNF1 β-subunit in the oleaginous fungus Mortierella alpina (MaSip2) was investigated. The MaSip2 contained a glycogen-binding domain and a conserved SNF1-complex interaction region; its transcriptional level during lipogenesis shared high consistency with a previously reported SNF1 γ-subunit (MaSnf4). Overexpression of MaSip2 in M. alpina significantly promoted glucose uptake and resulted in 34.1% increased total biomass, leading to 44.8% increased arachidonic acid yield after 7 day fermentation. MaSip2 also regulated the balance between polyunsaturated fatty acids and carbohydrates in M. alpina. Intracellular metabolite analysis revealed increased carbohydrate-related metabolite accumulation in MaSip2 overexpression strains. On the contrary, knockdown of MaSip2 increased the total fatty acid unsaturation degree, especially under low-temperature conditions. This research improved our knowledge of SNF1 complex in M. alpina and provided a target gene for enhancing glucose utilization and modulating fatty acid composition for better application of oleaginous fungi.

Enhancement of oil productivity of Mortierella alpine and investigation into the potential of Pickering oil-in-water emulsions to improve its oxidative stability.

Mortierella alpine is an oleaginous fungi known for its tendency to produce oil and polyunsaturated fatty acid. Initial experiment indicated that magnesium oxide nanoparticles (MgONPs) accelerated glucose consumption and, consequently, oil production. After enhancement of Mortierella alpine CBS 754.68' oil production, the oxidative stability of the oil rich in long‐chain polyunsaturated fatty acids (arachidonic acid) encapsulated by modified chitosan (CS) was assayed. To confirm the modification of CS, Fourier transform infrared spectroscopy (FTIR) spectrum indicated that the connection between CS and capric acid (CA) as well as stearic acid (SA) was well formed, leading to a considerable improvement in nanoparticle formation, measured by the SEM photographs, and physical and oxidative stability of emulsions. The oxidative stability of Mortierella alpine' oil emulsion in a period of 20 days at ambient temperature was monitored. Of all treated media, CS‐SA nanoparticles were of the most oxidative stability. The rheological tests showed that viscosity behaviors were dominated by elastic behaviors in the impregnating emulsion with unmodified CS at the applied frequencies, and the elastic behavior of the emulsion sample prepared with CS‐SA was slightly higher than that of the emulsion prepared with CS‐CA. The results of redispersibility indicated that the powdered emulsion stabilized by CS‐SA had the lowest water absorption.

Characterization of NAD+/NADP+-Specific Isocitrate Dehydrogenases From Oleaginous Fungus Mortierella alpina Involved in Lipid Accumulation.

Mortierella alpina has a strong capacity for lipid accumulation. Isocitrate dehydrogenase (IDH) plays an important role in affecting the flow of intracellular carbon sources and reducing power NADPH for lipid biosynthesis. In this study, the effect of various IDHs (NAD+- and NADP+-specific) in M. alpina on the lipid accumulation was investigated through homologous overexpression. The results showed that the transcription level and enzyme activity of the IDHs from M. alpina (MaIDHs) in homologous overexpressing strains were higher than those of the control strain, but that their biomass was not significantly different. Among the various NAD+-specific MaIDH1/2/3 overexpression, NAD+-MaIDH3 reduced total lipid content by 12.5%, whereas overexpression NAD+-MaIDH1 and NAD+-MaIDH2 had no effect on fatty acid content. Intracellular metabolites analysis indicated that the overexpression NAD+-MaIDH3 strain had reduced the fatty acid accumulation, due to its greater carbon flux with the tricarboxylic acid cycle and less carbon flux with fatty acid biosynthesis. For the NADP+-MaIDH4/5/6 recombinant strains overexpressing only NADP+-MaIDH4 enhanced the total fatty acid content by 8.2%. NADPH analysis suggested that this increase in lipid accumulation may have been due to the great reducing power NADPH is produced in this recombinant strain. This study provides theoretical basis and guidance for the analysis of the mechanism of IDH function and the potential to improve lipid production in M. alpina.

Optimization of culture conditions for biomass and lipid production by oleaginous fungus Penicillium citrinum PKB20 using response surface methodology (RSM)

Penicillium citrinum is a naturally occurring filamentous fungus with immense potential for biomass-based diesel production because they are prolific producers of cellulose-degrading enzymes and also accumulate significant amount of lipids. In this study, four media components, namely, carbon source, nitrogen (organic and inorganic) source and metal salts, and three fermentation parameters viz. Incubation time, temperature and pH were first screened for their effect on the biomass and lipid production by P. citrinum PKB20 using ‘one variable at a time’ (OVAT) approach. OVAT based experiments revealed that glucose ( X G ), yeast extract ( X Y ), ammonium sulphate ( X A ) and magnesium ion ( X M ) had the greatest impact on biomass yield and lipid accumulation when grown under batch cultivation mode at pH 7, temperature 30 °C and incubation time of 6 days. A central composite design based response surface methodology (RSM) was then adopted to model and optimize the ideal concentrations of the media constituents for maximum production of biomass and lipid from oleaginous fungus Penicillium citrinum PKB20.The optimization condition was found to be 10% of X G , 0.75 g/L of X Y , 0.38 g/L of X A and 0.06 g/L of X M and under optimum condition, the biomass and lipid production was 7.9 g/L and 1.73 g/L respectively. The study thus presents the optimum concentrations of key media components and culture conditions for maximum biomass and lipid production by Penicillium citrinum PKB20 under the studied cultivation and harvesting conditions. • Glucose, yeast extract, ammonium sulphate, magnesium significantly affect growth and lipid production by P. citrinum PKB20. • RSM was used to optimize the concentration of media constituents for maximum biomass (7.9 g/L) and lipid (1.73 g/L) production. • Influence of interaction of media components on growth and lipid biosynthesis were analyzed using RSM.

Bioconversion of pomegranate residues into biofuels and bioactive lipids

Pomegranate residues (PRs), the solid residues comprising peels and seeds after juice extraction, are generated in abundance in Greece and contain a variety of carbon sources. Therefore, these residues can be regarded as a potential feedstock for chemical and biotechnological processes. The polysaccharides contained in PRs were extracted and hydrolyzed in a one-step process (not involving the use of chemical reagents), and the resulting broth was used as substrate in biotechnological applications, including ethanol and single cell oil (SCO) production. The yeasts Meyerozyma guilliermondii, Scheffersomyces coipomoensis, Sugiyamaella paludigena and especially Saccharomyces cerevisiae were able to efficiently convert reducing sugars derived from PRs into bioethanol. Ethanol production in static cultures (ensuring nearly anaerobic conditions) ranged from 3.6 to 12.5 g/L. In addition, the oleaginous yeasts Lipomyces lipofer and Yarrowia lipolytica as well as M. guilliermondii, S. coipomoensis and S. paludigena were tested for their ability to produce lipids suitable as feedstock for biodiesel production. Lipids were accumulated at concentrations up to 18% and were rich in palmitic and oleic acids. Finally, the oleaginous fungus Cunnichamella echinulata was cultivated on PR-based solid substrates for γ-linolenic acid (GLA) production. The fermented bio-products (i.e. fermented substrate plus fungal mycelia) contained up to 4.8 mg GLA/g of dry weight. Phenolic removal (up to 30%) was achieved by several of the above-mentioned microorganisms, including C. echinulata, L. lipofer, M. guilliermondii, S. paludigena and Y. lipolytica. In conclusion, the findings of this study demonstrate that PRs can be used as raw material for microbial growth, ethanol and SCO production, which is of economic and environmental importance.