Microbial Biodiesel Production Research Articles

Petroleum Sludge as a Feedstock for the Microbial Biodiesel Production: Emerging Prospects

Petroleum Sludge as a Feedstock for the Microbial Biodiesel Production: Emerging Prospects

Microorganisms-promoted biodiesel production from biomass: A review

Biodiesel is considered as a potential substitute for fossil fuel due to its renewability, sustainability, environmentally friendliness, and biodegradability, especially with comparable fuel properties to diesel. The chemocatalytic production of biodiesel from plant oils is widely used in industrial production due to its low cost and high conversion rate. However, the disadvantages are high energy consumption and environmentally unfriendly processing such as chemical catalysts, downstream technology and simultaneously produced waste. Therefore, in the past decade, enzyme-catalyzed biodiesel has attracted more attentions due to its sustainability and environmental friendliness. High-cost, enzyme stability and reusability are the main obstacles to the large-scale industrial development of microbial biodiesel. This review first showcases the state-of-the-art of microbial biodiesel production, including (1) lipid accumulation of oleaginous microorganisms from pretreated lignocellulose biomass, and (2) production of biodiesel from microbial oils via transesterification by immobilized lipase. Also, the technological challenges and future developmental trends are discussed, with the goal of providing the possibility of more economical large-scale industrial production. This paper provides opportunities for the sustainable and eco-friendly production of enzymatic biodiesel in the future.

Volatile Fatty Acids from Lipid-Extracted Yeast Provide Additional Feedstock for Microbial Lipid Production

Microbial lipid production from oleaginous yeasts is a promising process for the sustainable development of the microbial biodiesel industry. However, the feedstock cost poses an economic problem for the production of microbial biodiesel. After lipid extraction, yeast biomass can be used as an organic source for microbial biodiesel production. In this study, volatile fatty acids (VFAs), produced via anaerobic digestion of a lipid-extracted yeast (LEY) residue, were utilized as a carbon source for the yeast Cryptococcus curvatus. The response surface methodology was used to determine the initial pH and inoculum volume for the optimal VFA production. The experimental result for VFA concentration was 4.51 g/L at an initial pH of 9 and an inoculation 25%. The optimization results from the response surface methodology showed that the maximal VFA concentration was 4.58 g/L at an initial pH of 8.40 and an inoculation of 39.49%. This study indicates that VFAs from LEY can be used as a carbon source for microbial biodiesel production, with the potential to significantly reduce feedstock costs.

Multi-objective optimization for biomass and lipid production by oleaginous bacteria using vegetable waste as feedstock

In this study, pretreated organic wastes such as waste paper cups, cardboard waste, and vegetable waste were screened for the growth and lipid production of oleaginous bacteria DS-7 (isolated from the dairy effluent scum). The pretreated vegetable waste was found to be the best feedstock for biomass and lipid production by the DS-7. Further, process parameters such as inoculation time, substrate concentration (w/v) (amount of pretreated vegetable waste), pH, and inoculum size were optimized using a multi-objective optimization technique to enhance the biomass and lipid productions. The optimization study successfully enhanced the biomass concentration (g/L) and lipid content (%) by 47.9% and 15.84%, respectively in comparison with the unoptimized state. The biomass and lipid productivities were 42% (1.449 g/L/d) and 51% (1.267 g/L/d) greater than unoptimized conditions. The characteristics of the biodiesel obtained from the valorization of vegetable waste were comparable to the standard. Thus, the vegetable waste can be utilized as a potential feedstock for microbial biodiesel production.

Mixed glycerol and orange peel-based substrate for fed-batch microbial biodiesel production

The aqueous extraction of orange peel waste (OPW), the byproduct of the juice extraction process generated annually in massive amounts (21 Mton), yields a carbohydrate-rich liquid fraction, termed orange peel extract (OPE). Several studies highlight that the combination of glycerol, a biodiesel byproduct, with carbohydrate mixtures might boost microbial lipid production. This study performed first a shaken flask screening of 15 oleaginous yeast strains based on their growth and lipid-producing abilities on OPE- and glycerol-based media. This screening enabled the selection of R. toruloides NRRL 1091 for the assessment of the process transfer in a stirred tank reactor (STR). This assessment relied, in particular, on either single- and double-stage feeding fed-batch (SSF-FB and DSF-FB, respectively) processes where OPE served as the primary medium and nitrogen-containing glycerol-OPE mixtures as the feeding one. The continuous supply mode at low dilution rates (0.02 and 0.01 h−1 for SSF-FB and DSF-FB, respectively) starting from the end of the exponential growth of the initial batch phase enabled the temporal extension of biomass and lipid production. The SSF-FB and DSF-FB processes attained high biomass and lipid volumetric productions (LVP) and ensured significant lipid accumulation on a dry cell basis (YL/X). The SSF-FB process led to LVP of 20.6 g L−1 after 104 h with volumetric productivity (rL) of 0.20 g L−1 h−1 and YL/X of 0.80; the DSF-FB process yielded LVP, rL and YL/X values equal to 15.92 g L−1, 0.11 g L−1 h−1 and 0.65, respectively. The fatty acid profiles of lipids from both fed-batch processes were not significantly different and resembled that of Jatropha oil, a vastly used feedstock for biodiesel production. These results suggest that OPE constitutes an excellent basis for the fed-batch production of R. toruloides lipids, and this process might afford a further option in OPW-based biorefinery.

Microbial biodiesel production from industrial organic wastes by oleaginous microorganisms: Current status and prospects

This review aims to encourage the technical development of microbial biodiesel production from industrial-organic-wastes-derived volatile fatty acids (VFAs). To this end, this article summarizes the current status of several key technical steps during microbial biodiesel production, including (1) acidogenic fermentation of bio-wastes for VFA collection, (2) lipid accumulation in oleaginous microorganisms, (3) microbial lipid extraction, (4) transesterification of microbial lipids into crude biodiesel, and (5) crude biodiesel purification. The emerging membrane-based bioprocesses such as electrodialysis, forward osmosis and membrane distillation, are promising approaches as they could help tackle technical challenges related to the separation and recovery of VFAs from the fermentation broth. The genetic engineering and metabolic engineering approaches could be applied to design microbial species with higher lipid productivity and rapid growth rate for enhanced fatty acids synthesis. The enhanced in situ transesterification technologies aided by microwave, ultrasound and supercritical solvents are also recommended for future research. Technical limitations and cost-effectiveness of microbial biodiesel production from bio-wastes are also discussed, in regard to its potential industrial development. Based on the overview on microbial biodiesel technologies, an integrated biodiesel production line incorporating all the critical technical steps is proposed for unified management and continuous optimization for highly efficient biodiesel production.

Microbial biodiesel production: novel method of utilizing sago wastewater for lipid production using oleaginous yeast, Candida tropicalis ASY1

A study was undertaken to determine the physico-chemical characteristicsof sago wastewater (SWW) from various sago industries in Salem andNamakkal districts of India. Besides, different SWW (raw wastewater,aerobic wastewater, treated wastewater and starch wastewater) from withinthe industry were evaluated. The results concluded that effluents from thisregion are whitish and greyish brown in color with majority supra-colloidalor settable suspended solids with high TS, TSS, and TDS. The ammoniacal,nitrate, and nitrite nitrogen ranged from 2.7 to 6.5, 4.5 to 19.1, and 0.4to 2.8 mg.L-1. The total nitrogen content of SWW samples was ranged from180- 650 mg.L-1. The phosphate level of SWW was very high (640-1096 mg.L-1). The COD and BOD content of SWW from all the industries ranged from2560 to 3200 mg.L-1 and 34000 to 76000 mg.L-1 indicates its high organicnature. SWW effluents are acidic in nature (pH 4.3-5.2). The free cyanide(CN) content was ranged from 1.2 to 5.2 mg.L-1. The treated wastewater hadneutral pH (pH - 6.2), and had a reduced level of EC, starch content, TSS,COD and BOD.

Cost, energy and GHG emission assessment for microbial biodiesel production through valorization of municipal sludge and crude glycerol.

In this study, cost simulations were made based on 20 million L blended biodiesel B-10 production per year using INRS and conventional process. In case of INRS process, microbial lipid was produced by T. oleaginosus using washed municipal secondary sludge fortified with crude glycerol while lipid was extracted from wet biomass using biodegradable surfactant and petroleum-diesel (PD). The conventional process uses commercial substrates for lipid production and organic solvents for lipid extraction from dry biomass. The unit B-10 production cost of INRS process was estimated to be $ 0.72/L for an annual capacity of 20 million L, which is 9.5 times more economical than conventional biodiesel production process. For INRS process, the unit B-10 biodiesel production cost was sensitive to plant capacity and lipid productivity during the fermentation. INRS process exhibited positive net energy gain and positive GHG capture, which proves to be energetically and environmentally viable.

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

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

Microbial biodiesel production: From sucrose-based carbon sources to alkyl esters via enzymatic transesterification

Abstract Sucrose, in contrast to glucose, is a lower-cost carbon source. This manuscript discloses that its use in submerged fungal growth promotes lipids more suitable to biodiesel production due to lower unsaturation indexes. The wild strain of Mucor circinelloides URM 4182, a potential source of microbial lipid, was grown on three sucrose-based carbon sources: commercial brown sugar, sugarcane juice, and sugarcane syrup, achieving biomass accumulation productivities up to 3.62 ± 0.04 g L−1 day−1, and lipid productivities as high as 0.93 ± 0.01 g L−1 day−1. Lipid extraction was performed using ethanol, a green alternative to petroleum-derived solvent, and characterized according to their fatty acid distribution, from which C16:0 and C18:1 stand as the most abundant fatty acids. Extracted lipids were converted to ethyl esters via lipase-catalyzed reactions achieving ester contents up to 97.4% and low contents of mono- and diacylglycerols (1.9% and 1.0%, respectively). The interest in utilizing sucrose-based sources is due to the wide abundance of such feedstock in sugarcane-producing regions, and due to the potential of increasing value to these low-cost carbon sources. The results presented herein demonstrate a simple, yet efficient, process of converting sucrose and its derivatives to microbial biodiesel using green-chemistry processes, such as lipid extraction using ethanol, avoiding the use of harsh and toxic chemicals commonly utilized, and enzyme-catalyzed reactions.

Microbial Biodiesel Production by Direct Transesterification of Rhodotorula glutinis Biomass

(1) Background: Lipids derived from oleaginous microbes have become promising alternative feedstocks for biodiesel. This is mainly because the lipid production rate from microbes is one to two orders of magnitude higher than those of energy crops. However, the conventional process for converting these lipids to biodiesel still requires a large amount of energy and organic solvents; (2) Methods: In this study, an oleaginous yeast, Rhodotorula glutinis, was used for direct transesterification without lipid pre-extraction to produce biodiesel, using sulfuric acid or sodium hydroxide as a catalyst. Such processes decreased the amount of energy and organic solvents required simultaneously; (3) Results: When 1 g of dry R. glutinis biomass was subject to direct transesterification in 20 mL of methanol catalyzed by 0.6 M H2SO4 at 70 °C for 20 h, the fatty acid methyl ester (FAME) yield reached 111%. Using the same amount of biomass and methanol loading but catalyzed by 1 g/L NaOH at 70 °C for 10 h, the FAME yield reached 102%. The acid-catalyzed process showed a superior moisture tolerance; when the biomass contained 70% moisture, the FAME yield was 43% as opposed to 34% of the base-catalyzed counterpart; (4) Conclusions: Compared to conventional transesterification, which requires lipid pre-extraction, direct transesterification not only simplifies the process and shortens the reaction time, but also improves the FAME yield.

Direct Methanolysis of Oleaginous Yeast Biomass (<i>Pseudozyma parantarctica</i>) to Microbial Biodiesel

In this study, intracellular lipids of a novel oleaginous biomass of P. parantarctica were converted to biodiesel directly using simple acid catalyst methanolysis. The optimum condition of this method was investigated. Under optimum conditions (0.1 M H2SO4, 10 h reaction time, 65°C reaction temperature, and 1:20 (w/v) biomass-to-methanol ratio), the yield of crude biodiesel was 93.18 ± 2.09% based on total cellular lipids. The composition of crude biodiesel was C16:C18 fatty acid methyl esters (FAMEs) for 91.91%. Especially, the C18:1 methyl ester was the main FAME (47.10%). In addition, the result showed that this technique could produce the microbial biodiesel from biomass containing high free fatty acids (FFAs) without soap formation. The predicted cetane number and kinematic viscosity of biodiesel were characterized according to ASTM D6751 and EN 14214 standards. Our results indicated that this process produces a good quality biodiesel. Moreover, it can decrease the manufacturing costs of microbial biodiesel production from oleaginous yeast biomass without cell disruption and lipid extraction.

Microbial biodiesel production from oil palm biomass hydrolysate using marine Rhodococcus sp. YHY01

The effect of various biomass derived inhibitors (i.e. furfural, hydroxymethylfurfural (HMF), vanillin, 4-hydroxy benzaldehyde (4-HB) and acetate) was investigated for fatty acid accumulation in Rhodococcus sp. YHY 01. Rhodococcus sp. YHY01 was able to utilize acetate, vanillin, and 4-HB for biomass production and fatty acid accumulation. The IC50 value for furfural (3.1mM), HMF (3.2mM), vanillin (2.0mM), 4-HB (2.7mM) and acetate (3.7mM) was calculated. HMF and vanillin affect fatty acid composition and increase saturated fatty acid content. Rhodococcus sp. YHY 01 cultured with empty fruit bunch hydrolysate (EFBH) as the main carbon source resulted in enhanced biomass (20%) and fatty acid productivity (37%), in compression to glucose as a carbon source. Overall, this study showed the beneficial effects of inhibitory molecules on growth and fatty acid production, and support the idea of biomass hydrolysate utilization for biodiesel production by avoiding complex efforts to remove inhibitory compounds.

A sustainable use of Ricotta Cheese Whey for microbial biodiesel production.

The increasing demand of plant oils for biodiesel production has highlighted the need for alternative strategies based either on non-food crops or agro-industrial wastes that do not compete with food and feed production. In this context, the combined use of wastewater and oleaginous microorganisms could be a valuable production option. Ricotta cheese whey (RCW), one of the major byproducts of the dairy industry, is produced in very high and steadily increasing amounts and, due to its high organic load, its disposal is cost-prohibitive. In the present study, in order to assess the adequacy of RCW as a growth medium for lipid production, 18 strains of oleaginous yeasts were investigated in shaken flask for their growth and lipid-producing capabilities on this substrate. Among them, Cryptococcus curvatus NRRL Y-1511 and Cryptococcus laurentii UCD 68-201 adequately grew therein producing substantial amounts of lipids (6.8 and 5.1gL-1, respectively). A high similarity between the percent fatty acid methyl esters (FAME) composition of lipids from the former and the latter strain was found with a predominance of oleic acid (52.8 vs. 48.7%) and of total saturated fatty acids (37.9 vs. 40.8%). The subsequent scale transfer of the C. laurentii UCD 68-201 lipid production process on RCW to a 3-L STR led to significantly improved biomass and total lipid productions (14.4 and 9.9gL-1, respectively) with the biodiesel yield amounting to 32.6%. Although the C. laurentii FAME profile was modified upon process transfer, it resembled that of the Jatropha oil, a well established feedstock for biodiesel production. In conclusion, C. laurentii UCD 68-201, for which there is very limited amount of available information, turned out to be a very promising candidate for biodiesel production and wide margins of process improvement might be envisaged.

Assessing an effective feeding strategy to optimize crude glycerol utilization as sustainable carbon source for lipid accumulation in oleaginous yeasts

BackgroundMicrobial lipids can represent a valuable alternative feedstock for biodiesel production in the context of a viable bio-based economy. This production can be driven by cultivating some oleaginous microorganisms on crude-glycerol, a 10 % (w/w) by-product produced during the transesterification process from oils into biodiesel. Despite attractive, the perspective is still economically unsustainable, mainly because impurities in crude glycerol can negatively affect microbial performances. In this view, the selection of the best cell factory, together with the development of a robust and effective production process are primary requirements.ResultsThe present work compared crude versus pure glycerol as carbon sources for lipid production by three different oleaginous yeasts: Rhodosporidium toruloides (DSM 4444), Lipomyces starkeyi (DSM 70295) and Cryptococcus curvatus (DSM 70022). An efficient yet simple feeding strategy for avoiding the lag phase caused by growth on crude glycerol was developed, leading to high biomass and lipid production for all the tested yeasts. Flow-cytometry and fourier transform infrared (FTIR) microspectroscopy, supported by principal component analysis (PCA), were used as non-invasive and quick techniques to monitor, compare and analyze the lipid production over time. Gas chromatography (GC) analysis completed the quali-quantitative description. Under these operative conditions, the highest lipid content (up to 60.9 % wt/wt) was measured in R. toruloides, while L. starkeyi showed the fastest glycerol consumption rate (1.05 g L−1 h−1). Being productivity the most industrially relevant feature to be pursued, under the presented optimized conditions R. toruloides showed the best lipid productivity (0.13 and 0.15 g L−1 h−1 on pure and crude glycerol, respectively).ConclusionsHere we demonstrated that the development of an efficient feeding strategy is sufficient in preventing the inhibitory effect of crude glycerol, and robust enough to ensure high lipid accumulation by three different oleaginous yeasts. Single cell and in situ analyses allowed depicting and comparing the transition between growth and lipid accumulation occurring differently for the three different yeasts. These data provide novel information that can be exploited for screening the best cell factory, moving towards a sustainable microbial biodiesel production.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-016-0467-x) contains supplementary material, which is available to authorized users.

Structural diversity and possible functional roles of free fatty acids of the novel soil isolate Streptomyces sp. NP10.

Herein, a novel soil bacterium Streptomyces sp. NP10 able to grow outside usual streptomycetes optimum conditions (e.g., at 4°C, pH 9 and high NaCl concentration), exhibiting atypical hemolytic, DNAse, and cellulolytic activities, is described. This strain produces and excretes into the growth medium large amounts of free long-chain fatty acids (FAs). A concurrent lipidomics study revealed a large structural diversity of FAs with over 50 different n- and branched-chain, (un)saturated, and cyclopropane FAs (C7-C30) produced by this strain. Two of these, i-17:0cy9-10 and a-18:0cy9-10, represent new natural products and the first ever identified branched cyclopropane FAs. Both free and bound lipid profiles of Streptomyces sp. NP10 were dominated by saturated branched chain FAs (i-14:0, a-15:0, and i-16:0). Although these free FAs showed only a moderate antimicrobial activity, our results suggest that they could have an ecophysiological role in interspecies signaling with another soil microorganism Pseudomonas aeruginosa. This work represents the first comprehensive report on the structural diversity and complexity of the free FA pool in Streptomyces. A naturally occurring streptomycete, such as Streptomyces sp. NP10, which secretes significant amounts of free long-chain FAs (non-cytotoxic) into the medium, could be useful in microbial biodiesel production.

Bioconversion of lignocellulosic pretreatment effluent via oleaginous Rhodococcus opacus DSM 1069

Rhodococcus opacus DSM 1069 utilized pine organosolv pretreatment effluent as a sole carbon and energy source for 120 h at 1.5 w/v% solids concentration and accumulated a maximum of 26.99 ± 2.88% of its cellular dry weight in oils composed of oleic, palmitic, and stearic fatty acids. These results establish the potential for lignocellulosic pretreatment effluent as a feedstock for microbial biodiesel production via oleaginous R. opacus and an interesting route for biorefinery waste stream optimization.

Economic assessment of microbial biodiesel production using heterotrophic yeasts

ABSTRACTThe production of biodiesel using oleaginous microorganisms is investigated as promising alternative to produce a truly sustainable and renewable transportation fuel. While the feasibility of this approach has been shown on the laboratory scale, a commercial scale implementation is to date inhibited due to economic restraints. In order to evaluate the current cost situation and to develop suggestions to reduce production related costs, a simple cost analysis of the proposed microbial oil production process has been carried out. For closed fermentation in large-scale fermenters a break-even price of 2,350 US$ t–1 for microbial oil was calculated. In the context of a sensitivity analysis it was shown that especially alterations in capital cost can lead to overall cost reductions. Accordingly, an open pond cultivation approach was designed, cutting the cost for equipment almost in half and decreasing the break-even price to 1,723 US$ t–1. However, these reductions are only feasible when stable biomass and lipid yields can be ensured in open-pond systems, because the sensitivity analysis identified these yield parameters as leading factors influencing the break-even price. Even under very optimistic assumptions, it was not possible to reduce the break-even price below that of conventional plant oils as competitive products. Therefore, economic feasibility of the process will probably only occur if on one hand considerable technical development and efficiency improvements of the production process are made while on the other hand plant and crude oil prices are continuously increasing.

Characterization of Different Biomasses Based on Their Sugar Profile with Focus on Their Utilization for Microbial Biodiesel Production

The investigation and characterization of different biomasses as potential feedstocks for microbial lipid production was the aim of the present work. Twenty-eight biomasses were analyzed for total sugar content (glucose and xylose) and C/N-ratio as two major criteria for a suitable lipid production by oleaginous microorganisms. Considering both parameters, 9 out of 28 biomasses seemed to be suitable feedstocks for microbial lipid production. To allow for a broad evaluation of the suitability of the potential feedstocks, the estimated sugar and potential energy yield per area were calculated and the production costs and energy demand of the feedstock production were considered.

Microbial biodiesel production by direct methanolysis of oleaginous biomass

Biodiesel is usually produced by the transesterification of vegetable oils and animal fats with methanol, catalyzed by strong acids or bases. This study introduces a novel biodiesel production method that features direct base-catalyzed methanolysis of the cellular biomass of oleaginous yeast Rhodosporidium toruloides Y4. NaOH was used as catalyst for transesterification reactions and the variables affecting the esterification level including catalyst concentration, reaction temperature, reaction time, solvent loading (methanol) and moisture content were investigated using the oleaginous yeast biomass. The most suitable pretreatment condition was found to be 4gL(-1) NaOH and 1:20 (w/v) dried biomass to methanol ratio for 10h at 50°C and under ambient pressure. Under these conditions, the fatty acid methyl ester (FAME) yield was 97.7%. Therefore, the novel method of direct base-catalyzed methanolysis of R. toruloides is a much simpler, less tedious and time-consuming, process than the conventional processes with higher FAME (biodiesel) conversion yield.