Microbial lipids from cellulolytic oleaginous fungus Penicillium citrinum PKB20 as a potential feedstock for biodiesel production

Microbial lipids derived from oleaginous fungi are considered as an alternative feedstock for biodiesel production. We attempt to isolate a cellulolytic oleaginous fungi as a potential feedstock for biodiesel production. The fungus was identified by 5.8 S-ITS rRNA gene sequencing. The extracellular enzyme activities were recorded after every 24 h for 7 days. Nile red staining and fluorescence microscopy was used to visualise the lipid bodies within the fungal hyphae. A renewable heterogeneous base catalyst derived from Musa balbisiana cola peels was used for the transesterification of Penicillium citrinum PKB20 derived oil into biodiesel. GC-MS analysis was used to analyse the fatty acid methyl esters (FAME) profile of the transesterified lipids. Penicillium citrinum PKB20 was isolated from detritus rich soil of Assam, India. The endoglucanase, xylanase and β-glucosidase enzyme activities were found to be 292.83 ± 0.29, 111.72 ± 0.45 and 6.54 ± 0.13 U/mg respectively. The specific enzyme activity for extracellular lipase was found to be 3.12 ± 0.16 U/mg. It could accumulate up to 60.61% of lipids in nitrogen-limited medium (7.34 ± 0.45 g/L biomass production). The extracted lipids were converted to biodiesel with 89.3% conversion efficiency. The predominant fatty acids were oleic acid (30.09%), palmitic acid (20.25%) and linoleic acid (33.14%) suggesting a balance between oxidative stability and cold flow properties for suitable biodiesel quality. Penicillium citrinum PKB20 was found to be a potential feedstock for biodiesel production with desirable fuel properties. The cellulolytic nature could be utilised for simultaneous lipid production directly on cellulosic substrates.     

Arachidonic acid production by Mortierella alpina grown on solid substrates

Mortierella alpina grown in solid state fermentations on cereal substrates gave up to 16% lipid in the final biomass. Arachidonic was at 50% of total fatty acids, with a yield of 36 mg/g of original substrate. Microbial lipid production was successfully scaled up to use 5-kg dry substrate batches.     

Direct bioconversion of rice residue from canteen waste into lipids by new amylolytic oleaginous yeast Sporidiobolus pararoseus KX709872

The new amylolytic oleaginous red yeast, Sporidiobolus pararoseus KX709872, produced both α-amylase (540?±?0.09?mU/mL) and amyloglucosidase (23?±?0.00?mU/mL) and showed good ability to directly convert rice residue from canteen waste to biomass and lipids. Effects of medium composition and cultivation conditions on growth and lipid accumulation for strain KX709872 were investigated under shaking flask and upscaling levels. At C?:?N ratio of 25?:?1, pH 5.45, 22.36°C, and 199.40?rpm for 7 days, volumetric production of biomass and lipids, lipid content, and lipid productivity reached 17.69?±?0.44, 8.35?±?0.19?g/L, 49.48?±?0.41% (w/w), and 1.67?±?0.11?g/L/day, respectively. Production of lipids was also implemented in 5.0-L stirred tank bioreactor with 2.5?L of optimized medium at 300?rpm and 3.0 vvm for 5 days. Volumetric production of biomass and lipids, lipid content, and lipid productivity were 16.33?±?0.49, 8.75?±?0.13?g/L, 56.61?±?0.04% (w/w), and 2.19?±?0.03?g/L/day, respectively. Meanwhile, the fatty acids of lipids from strain KX709872 had high oleic acid content (60?62%) which was similar to those of vegetable oils, indicating that these lipids are promising as an alternative biodiesel feedstock. Moreover, the biodiesel derived from lipids of strain KX709872 had properties satisfying the criteria of ASTM D6751 and EN 14214 standards.     

Production of algae-based biodiesel using the continuous catalytic Mcgyan® process

This study demonstrates the production of algal biodiesel from Dunaliella tertiolecta, Nannochloropsis oculata, wild freshwater microalgae, and macroalgae lipids using a highly efficient continuous catalytic process. The heterogeneous catalytic process uses supercritical methanol and porous titania microspheres in a fixed bed reactor to catalyze the simultaneous transesterification and esterification of triacylglycerides and free fatty acids, respectively, to fatty acid methyl esters (biodiesel). Triacylglycerides and free fatty acids were converted to alkyl esters with up to 85% efficiency as measured by 300 MHz ¹H NMR spectroscopy. The lipid composition of the different algae was studied gravimetrically and by gas chromatography. The analysis showed that even though total lipids comprised upwards of 19% of algal dry weight the saponifiable lipids, and resulting biodiesel, comprised only 1% of dry weight. Thus highlighting the need to determine the triacylglyceride and free fatty acid content when considering microalgae for biodiesel production.     

利用平板反应器大量培养高产油绿藻——尖状栅藻的生长和油脂积累规律

以新近分离的淡水绿藻--尖状栅藻(Scenedesmus acuminatus)为研究对象,将改良的BG-11培养基中的初始NaNO₃浓度降低为6.0mmol/L和3.6mmol/L,利用新设计的内置拉筋平板式光生物反应器对尖状栅藻(S. acuminatus)进行大量培养。测定不同时相的生物量、总脂含量、脂组分含量及脂肪酸组成和含量,分析尖状栅藻(S. acuminatus)大量培养时的生长和油脂积累规律。当初始NaNO₃浓度为6mmol/L时其最高生物量(6.27g/L)明显高于初始NaNO₃浓度为3.6mmol/L时的生物量(5.30g/L);而最高的总脂含量在初始NaNO₃浓度为3.6mmol/L时获得为干重的56.6%,高于初始NaNO₃浓度为6mmol/L时的总脂含量(51.6%)。总脂经硅胶柱层析分级后得到三种类型的脂组分:中性脂、糖脂和磷脂,随着培养时间的延长中性脂含量逐渐增加,培养至18d后,中性脂的含量分别达到总脂的 90.9%(6 mmol/L NaNO₃)和 92.0%(3.6 mmol/L NaNO₃)及干重的 47.5%(6.0 mmol/L NaNO₃)和 51.4%(3.6 mmol/L NaNO₃)。主要脂肪酸组成为棕榈酸、棕榈油酸、硬脂酸、油酸、亚麻油酸和亚麻酸,这六种脂肪酸在不同时相的含量变化范围分别为89.92%~96.18%(占总脂肪酸)和12.5%~50.7%(占细胞干重)。总脂、中性脂及总脂肪酸单位体积产率分别为:0.18 g/L/d,0.16 g/L/d和0.15 g/L/d(6.0 mmol/L NaNO₃)及0.16 g/L/d,0.15 g/L/d和0.15 g/L/d(3.6 mmol/L NaNO₃)。研究结果表明,尖状栅藻(S. acuminatus)是一株易于规模化培养、脂肪酸组成适合于生物柴油生产的高产油微藻。     

Molecular Identification and Comparative Evaluation of Tropical Marine Microalgae for Biodiesel Production

Marine microalgae have emerged as important feedstock for liquid biofuel production. The identification of lipid-rich native microalgal species with high growth rate and optimal fatty acid profile and biodiesel properties is the most challenging step in microalgae-based biodiesel production. In this study, attempts have been made to bio-prospect the biodiesel production potential of marine and brackish water microalgal isolates from the west coast of India. A total of 14 microalgal species were isolated, identified using specific molecular markers and based on the lipid content; seven species with total lipid content above 20% of dry cell weight were selected for assessing biodiesel production potential in terms of lipid and biomass productivities, nile red fluorescence, fatty acid profile and biodiesel properties. On comparative analysis, the diatoms were proven to be promising based on the overall desirable properties for biodiesel production. The most potential strain Navicula phyllepta MACC8 with a total lipid content of 26.54 % of dry weight of biomass, the highest growth rate (0.58 day^?1) and lipid and biomass productivities of 114 and 431 mgL^?1 day^?1, respectively, was rich in fatty acids mainly of C16:0, C16:1 and C18:0 in the neutral lipid fraction, the most favoured fatty acids for ideal biodiesel properties. The biodiesel properties met the requirements of fuel quality standards based on empirical estimation. The marine diatoms hold a great promise as feedstock for large-scale biodiesel production along with valuable by-products in a biorefinery perspective, after augmenting lipid and biomass production through biochemical and genetic engineering approaches.     

Further increased production of free fatty acids by overexpressing a predicted transketolase gene of the pentose phosphate pathway in Aspergillus oryzae faaA disruptant

Free fatty acids are useful as source materials for the production of biodiesel fuel and various chemicals such as pharmaceuticals and dietary supplements. Previously, we attained a 9.2-fold increase in free fatty acid productivity by disrupting a predicted acyl-CoA synthetase gene (faaA, AO090011000642) in Aspergillus oryzae. In this study, we achieved further increase in the productivity by overexpressing a predicted transketolase gene of the pentose phosphate pathway in the faaA disruptant. The A. oryzae genome is predicted to have three transketolase genes and overexpression of AO090023000345, one of the three genes, resulted in phenotypic change and further increase (corresponding to an increased production of 0.38 mmol/g dry cell weight) in free fatty acids at 1.4-fold compared to the faaA disruptant. Additionally, the biomass of hyphae increased at 1.2-fold by the overexpression. As a result, free fatty acid production yield per liter of liquid culture increased at 1.7-fold by the overexpression.     

Production of eicosapentaenoic acid (EPA) in Monodus subterraneus grown in a helical tubular photobioreactor as affected by cell density and light intensity

The effect of cell density (1–4.5 g L^-1) and light intensity (44 and 82 mol m^-2 s^-1) on fatty acid composition andeicosapentaenoic acid (EPA, 20:5 3) production was studied ina semi-continuous culture of Monodus subterraneus grown in a helicaltubular photobioreactor (`Biocoil') under laboratory conditions. Under lowlight, the highest proportion of EPA (31.5% of total fatty acids) and EPAcontent (3.5% of dry weight), biomass productivity (1.3 g L^-124 h^-1) and EPA productivity (44 mg L^-1 24 h^-1)occurred at optimal cell density of about 1.7 g L^-1. Cell densityhad no effect on the total fatty acid (TFA) content and was maintained atca. 11% of dry weight. Under high light, the highest proportion ofEPA to fatty acids (31.8%), the total fatty acids content (13.4%) andEPA content (4.3% of dry weight) occurred at cell density of about 3.4gL^-1. But the highest biomass productivity (1.7 g L^-124 h^-1) and EPA productivity (56 mg L^-1 24 h^-1) wereobtained at a cell density of 1.6 and 2.6g L^-1, respectively. Ourresults suggest that manipulating the cell density and light intensity canmodify the composition of fatty acid and production of eicosapentaenoicacid (EPA) in M. subterraneus.     

Effects of medium composition on the growth and lipid production of microplasmodia of Physarum polycephalum

Physarum polycephalum is a plasmodial slime mold. One of the trophic stages in the life cycle of this organism is a plasmodium. In submerged culture, plasmodia are fragmented into microplasmodia. The latter both lack cell walls and are capable of rapid growth. There has been limited information on the effects of medium composition on the growth and lipid accumulation of microplasmodia. In this study, optimization of medium components by response surface methodology showed that tryptone and yeast extract concentrations had the most significant effects on lipid and biomass production; significant synergistic interactions between glucose and tryptone concentration on these responses were also recorded. The optimal medium was composed of 20 g/L of glucose, 6.59 g/L of tryptone, and 3.0 g/L of yeast extract. This medium yielded 13.86 g/L of dry biomass and 1.97 g/L of lipids. These amounts are threefold higher than those of the American Type Culture Collection (ATCC) medium. In addition, biomass and lipid production reached maximal values between only 4 and 5 days. Fatty acid compositions analysis by gas chromatography-mass spectrometer (GC–MS) revealed that P. polycephalum lipids consisted mainly of oleic acid (40.5%), linoleic acid (10%), and octadecynoic (15.8%). This is the first report on the fatty acid composition of P. polycephalum microplasmodia. These results suggest that the biomass of microplasmodia could be used as a source of material for direct conversion into biodiesel because of the absence of cell walls or it could also be used as a supplemental source of beneficial fatty acids for humans, albeit with some further evaluation needed.     

Comparison of Trophic Modes to Maximize Biomass and Lipid Productivity of <Emphasis Type="Italic">Micractinium inermum</Emphasis> NLP-F014

An optimum trophic mode condition was investigated to maximize biomass and lipid productivity of Micractinium inermum NLP-F014, which grown successfully in blended wastewater medium. In this study, four trophic modes were used, including photoautotrophic, photoheterotrophic, heterotrophic and mixotrophic modes. Mixotrophic mode showed the highest biomass and lipid productivity. However, a high concentration of organics resulted the negative effect on the growth of M. inermum NLP-F014. Mixotrophic cultivation using glucose below 500 mg/L was able to produce maximum biomass productivity up to 0.90 ± 0.03 g/L/day as well as maximum lipid productivity up to 129.31 ± 0.10 mg/L/day. From lipid analysis on mixotrophic mode using glucose, the major fatty acids are oleic acid (C18:1), linoleic acid (C18:2) and palmitic acid (C16:0). These results suggest that mixotrophic mode cultivation with wastewater containing chemical oxygen demand (COD) below 500 mg/L could be applicable for biodiesel production of M. inermum NLP-F014.     

Optimization of microwave-assisted transesterification of dry algal biomass using response surface methodology

The effect of microwave irradiation on the simultaneous extraction and transesterification (in situ transesterification) of dry algal biomass to biodiesel was investigated. A high degree of oil/lipid extraction from dry algal biomass and an efficient conversion of the oils/lipids to biodiesel were demonstrated in a set of well-designed experimental runs. A response surface methodology (RSM) was used to analyze the influence of the process variables (dry algae to methanol (wt/vol) ratio, catalyst concentration, and reaction time) on the fatty acid methyl ester conversion. Based on the experimental results and RSM analysis, the optimal conditions for this process were determined as: dry algae to methanol (wt/vol) ratio of around 1:12, catalyst concentration about 2 wt.%, and reaction time of 4 min. The algal biodiesel samples were analyzed with GC-MS and thin layer chromatography (TLC) methods. Transmission electron microscopy (TEM) images of the algal biomass samples before and after the extraction/transesterification reaction are also presented.     

The augmented lipid productivity in an emerging oleaginous model alga <Emphasis Type="Italic">Coccomyxa subellipsoidea</Emphasis> by nitrogen manipulation strategy

The lipid productivity controlled by both of biomass and lipid content was really crucial for economic-feasibility of microalgae-based biofuels production. This study attempted at augmenting lipid productivity in an emerging oleaginous model alga Coccomyxa subellipsoidea by different nitrogen manipulation including one-stage continuous N-sufficiency (OCNS), N-deprivation (OCND), N-limitation (OCNL), and also two-stage batch N-starvation (TBNS). Amongst four tested nitrogen manipulation strategies, OCNS performed remarkable promoting effect on cell metabolic growth and the maximum biomass was achieved by 7.39 g/L. Whereas TBNS regime induced the highest lipid content (over 50.5%). Only OCNL treatment augmented the lipid productivity by 232.37 mg/L/day, representing 1.25-fold more than TBNS and even as much as 5.06-fold more than that of OCND strategy. OCNL also strengthened the proportions of saturated (C16:0 and C18:0) and monounsaturated fatty acid (C18:1) which were inclined to high-quality biofuels-making. This might be due to that most part of energy and metabolic flux (e.g. acetyl-CoA) derived from TCA cycle and glycolysis flowed into fatty acids biosynthesis pathway (especially C18:1) response to OCNL manipulation. This study represented a pioneering work of utilizing OCNL for lipids production by C. subellipsoidea and clearly implied that OCNL might be a feasible way for algal lipid production on a commercial scale and also promoted the potential of C. subellipsoidea as an ideal biodiesel feedstock.     

Lipid and total fatty acid productivity in photoautotrophic fresh water microalgae: screening studies towards biodiesel production

Microalgae are considered as a promising feedstock for biomass production. The selection of the most suitable species is based on several key parameters such as lipid and fatty acid productivity. In the present study, the growth of different microalgae strains was examined in freshwater media for photoautotrophs suited for large-scale applications to identify the most suitable medium for each species. In the optimal medium, Scenedesmus obliquus showed the highest biomass productivity measured as increase of cell dry weight (0.25 g cellu dry weight (CDW) L^?1 day^?1), while Botryococcus braunii showed the highest lipid and total fatty acid content (430 and 270 mg g^?1 CDW, respectively) among the tested species. Regarding lipid and total fatty acid productivity, S. obliquus was the most lipid and total fatty acid productive strain with 41 and 18 mg L^?1 day^?1 during the exponential phase, respectively. Additionally, the proportion of saturated and monounsaturated fatty acids increased with duration of the incubation in S. obliquus, while polyunsaturated fatty acids decreased. These results nominate S. obliquus as a promising microalga in order to serve as a feedstock for renewable energy production.     

Optimization of the biomass production of oil algae Chlorella minutissima UTEX2341

High production cost is a major obstacle to the extensive use of microalgae biodiesel. To cut the cost and achieve higher biomass productivity, Chlorella minutissima UTEX2341 was cultured under photoheterotrophic conditions. With the carbon, nitrogen and phosphorus concentration of 26.37, 2.61 and 0.03 g L?1 d?1 respectively, a maximum biomass productivity of 1.78 g L?1 d?1 was obtained, which was 59 times more than that cultured under autotrophic condition. The lipid productivity reached 0.29 g L?1 d?1, which was 11.9 times higher than the highest value reported by Oh et al. (2010). The conversion rate of microalgae lipids to FAME was found to be elevated from 45.65% to 62.97% and the FAME productivity increased from 1.16 to 180.68 mg L?1 d?1 after the optimization. 94% of the fatty acid of C. minutissima UTEX2341 was found to be composed of palmitic, oleic, linoleic and γ linoleic and the unsaturated fatty acids were the main parts (79.42%).     

Scenedesmus sp. NJ-1 isolated from Antarctica: a suitable renewable lipid source for biodiesel production

Microalgal lipids are promising alternative feedstocks for biodiesel production. Scenedesmus sp. NJ-1, an oil-rich freshwater microalga isolated from Antarctica, was identified to be a suitable candidate to produce biodiesel in this study. This strain could grow at temperatures ranging from 4 to 35?°C. With regular decrease in nitrate concentration in the medium, large quantities of triacylglycerols accumulated under batch culture conditions detected by thin layer chromatography and BODIPY 505/515 fluorescent staining. Scenedesmus sp. NJ-1 achieved the average biomass productivity of 0.105?g?l^?1?d^?1 (dry weight) and nearly the highest lipid content (35?% of dry cell weight) was reached at day 28 in the batch culture. Neutral lipids accounted for 78?% of total lipids, and C18:1 (n-9), C16:0 were the major fatty acids in total lipids, composing 37 and 20?% of total fatty acids of Scenedesmus sp. NJ-1 grown for 36?days, respectively. These results suggested that Scenedesmus sp. NJ-1 was a good source of microalgal oils for biodiesel production.     

Effects of some inhibitors on the growth and lipid accumulation of oleaginous yeast Rhodosporidium toruloides and preparation of biodiesel by enzymatic transesterification of the lipid

Microbial lipid produced using yeast fermentation with inexpensive carbon sources such as lignocellulosic hydrolyzate can be an alternative feedstock for biodiesel production. Several inhibitors that can be generated during acid hydrolysis of lignocellulose were added solely or together into the culture medium to study their individual inhibitory actions and their synergistic effects on the growth and lipid accumulation of oleaginous yeast Rhodosporidium toruloides. When the inhibitors were present in isolation in the medium, to obtain a high cell biomass accumulation, the concentrations of formic acid, acetic acid, furfural and vanillin should be lower than 2, 5, 0.5 and 1.5 g/L, respectively. However, the synergistic effects of these compounds could dramatically decrease the minimum critical inhibitory concentrations leading to significant growth and lipid production inhibitions. Unlike the above-cited inhibitors, sodium lignosulphonate had no negative influence on biomass accumulation when its concentration was in the range of 0.5-2.0 g/L; in effect, it was found to facilitate cell growth and sugar-to-lipid conversion. The fatty acid compositional profile of the yeast lipid was in the compositional range of various plant oils and animal tallow. Finally, the crude yeast lipid from bagasse hydrolyzate could be well converted into fatty acid methyl ester (FAME, biodiesel) by enzymatic transesterification in a tert-butanol system with biodiesel yield of 67.2% and lipid-to-biodiesel conversion of 88.4%.     

Production of microbial lipid: Effects of growth rate and oxygen on lipid synthesis and fatty acid composition of Rhodotorula gracilis

Microbial lipids produced by Rhodotorula gracilis NRRL Y-1091 grown in continuous culture under nitrogen-limiting condition were evaluated and the effects of growth rate and oxygen concentration on the degree of unsaturatoin of fatty acids studied. As the growth rate increased the protein content of the biomass increased but cell biomass, lipid content, and lipid productivity decreased; the specific lipid production rate remained constant at about 0.012 g lipid/g dry biomass/h. The maximum lipid content recorded was 49.8% (w/w) of the cell mass at a growth rate of 0.02 h(-1). The growth rate also affected fatty acid composition; polyunsaturated fatty acids (C18:2 and C18:3) increaded with growth rate while other fatty acids (C16:0, C18:0, C18:1) decreased. Increase in oxygen concentration between 5 and 234muM increased the lipid content without significantly affecting its degree of unsaturation. On the other hand, the degree of unsaturation was significantly affected by specific oxygen uptake rate for this obligate aerobe, Rh. gracilis.     

耐乙酸粘红酵母合成油脂

乙酸是木质纤维素在水解过程中的主要副产物,高浓度的乙酸严重影响产油微生物的生长和油脂合成。本文研究了粘红酵母对乙酸的耐受性及其利用乙酸合成微生物油脂的能力。结果表明,在初始葡萄糖、木糖浓度分别为6 g/L和44 g/L的混合糖培养基中,乙酸浓度低于10 g/L时,不会对菌体生长产生抑制作用,油脂合成还得到了促进。当乙酸添加量为10 g/L时,生物量、油脂产量、油脂含量较对照组分别提高了21.5%、171.2%和121.6%。进一步研究表明,粘红酵母具备利用乙酸合成油脂的能力,当以乙酸为唯一碳源,浓度为25 g/L时,油脂产量达到3.20 g/L,油脂质量得率为13%。微生物油脂成分分析表明,粘红酵母以乙酸为底物制得的油脂可以作为制备生物柴油的油脂原料,其主要成分为棕榈酸、硬脂酸、油酸、亚油酸和亚麻酸,其中饱和脂肪酸和不饱和脂肪酸含量分别为40.9%和59.1%。由于粘红酵母具有利用乙酸合成微生物油脂的能力,在以木质纤维素水解液为原料生产微生物油脂的脱毒过程中,一定浓度的乙酸可以不必脱除。     

Studies on the removal of dyes from a synthetic textile effluent using barley husk in static-batch mode and in a continuous flow,packed-bed,reactor

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.     

Biomass productivity and productivity of fatty acids and amino acids of microalgae strains as key characteristics of suitability for biodiesel production

Microalgae are discussed as an alternative source for the production of biofuels. The lipid content compared to cultivation time of used species is the main reason for any choice of a special strain. This paper reviews more analytical data of 38 screened microalgae strains. After the cultivation period, total content of lipids was analysed. The extracted fatty acids were quantified as fatty acid methyl esters by GC analysis. The amino acids were analysed by HPLC. Chlorella sp., Chlorella saccharophila, Chlorella minutissima and Chlorella vulgaris were identified as species with the highest productivity of fatty acids relevant to transesterification reactions. The components were mainly linoleic acid, palmitic acid and oleic acid. To increase productivity of highly saturated fatty acids, cultivation parameters light intensity and temperature were varied. In this manner, the ideal conditions for biodiesel production were defined in this publication.