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ABSTRACT Oleaginous microorganisms have the unique ability to accumulate lipids that can exceed 20% of their dry cell weight under certain conditions. Despite their potential for efficient lipid production, the metabolic pathways involved are not yet fully understood, largely due to the complexity of intracellular processes and the challenges in phenotypic prediction. This review synthesizes the latest research on the application of Genome‐scale Metabolic Network Models (GSMMs) to study oleaginous microorganisms, including bacteria, cyanobacteria, yeast, microalgae, and fungi, and provides a comprehensive analysis of how GSMMs have been utilized to decipher the metabolic mechanisms behind lipid accumulation and to identify key genes involved in lipid synthesis. The review highlights the role of GSMMs in predicting cellular behavior, optimizing metabolic engineering strategies, and discusses the future directions and potential of GSMMs in enhancing lipid production in microorganisms. This comprehensive overview not only summarizes the current state of research but also identifies gaps and opportunities for further investigation in the field.

Research on enhancing the production of lipids, particularly polyunsaturated fatty acids that are considered important for health, has focused on improvement of metabolism as well as heterologous expression of biosynthetic genes in the oleaginous fungus Aspergillus oryzae. To date, the productivity and production yield of free fatty acids have been enhanced by 10-fold to 90-fold via improvements in metabolism and optimization of culture conditions. Moreover, the productivity of ester-type fatty acids present in triacylglycerols could be enhanced via metabolic improvement. Culturing A. oryzae in a liquid medium supplemented with non-ionic surfactants could also lead to the effective release of free fatty acids from the cells. The current review highlights the advancements made in this field so far and discusses the future outlook for research on lipid production using A. oryzae. I hope the contents are useful for researchers in this field to consider the strategy of increasing production of various valuable metabolites as well as lipids in A. oryzae.

Microalgae are considered valuable bioresources due to their ability to produce high lipid content and grow under a variety of environmental conditions, making them strong candidates for sustainable biofuel production. However, the economic feasibility of microalgae-based biofuels depends on optimizing growth conditions in large-scale cultivation systems. This study investigates the effects of varying inoculum cell concentrations on the growth, lipid yield, and fatty acid composition of the locally isolated microalga Chlorella sp. SW5 in 2 L and 5 L cultivation systems. The results indicate that higher inoculum concentrations generally enhance biomass accumulation, with the 2 L system achieving the highest growth rate of 0.42 ± 0.01 day⁻1 at an inoculum concentration of 10⁶ cells/mL. Interestingly, while higher inoculum concentrations reduced lipid production in the 2 L system, the 5 L system showed the highest lipid yield (51.23% ± 4.71% dry weight) at the highest inoculum concentration (10⁷ cells/mL). Despite its moderate growth rate, the 5 L culture with a starting inoculum concentration of 10⁷ cells/mL was selected for fatty acid profiling due to its superior lipid yield and productivity. Gas chromatography-mass spectrometry (GC-MS) analysis revealed that the culture produced a total of 93.18% C14-C18 fatty acids, with a profile dominated by saturated (56.33%) and monounsaturated (16.85%) fatty acids, which are essential for biodiesel quality. These findings provide valuable insights into the potential for scaling up microalgal systems for commercial biofuel production, highlighting strategies to optimize productivity.

Currently, microalgae have become a marvelous and resource-friendly alternative source of advantageous bioproducts, such as lipids, carbohydrates, proteins, or other bioactive compounds. Because of the richness of microalgae in these high-value-added metabolites, still, it is an underdeveloped source of sustainable energy and food. There are some hurdles to profitable production, such as culture contamination and costly harvesting techniques. In the current work, a chrysophyte was isolated from dairy wastewater, identified as Poterioochromonas malhamensis based on its morphology and partial 18S rRNA gene sequences. This isolate was used to remediate dairy waste water (DWW) and to obtain neutral lipids (fatty acids) from microalgae. Microalgal growth was optimized by using different concentrations of DWW, supplemented with all the nutritive requirements for better progression and flourishment. Maximum biomass yield 1.478 g L−1 was achieved by optimized cultural conditions (different concentrations of DWW with BBM media). This strain showed high nitrate and phosphate removal efficiency (87.45% and 88.96%), respectively in 15 days. The experimental results highlighted that the lipid content and the chemical oxygen demand (COD) removal were 31.60% and 88.84%, respectively, and the lipid profile of isolated microalga was C16:0, C16:1, C18:0, C18:1, and C18:2 fatty acids. For growth and treatment purposes, 75% DWW with Bold’s Basal Medium (BBM) media showed better results. This is the first report of DWW treatment using the microalga Poterioochromonas malhamensis, as far as we are aware. Its cultivation prevented the spread of pollution of freshwater sources, remedied the DWW, and generated important lipids for industry.

Microalgal lipids hold significant potential for the production of biodiesel and dietary supplements. To enhance their cost-effectiveness and commercial competitiveness, it is imperative to improve microalgal lipid productivity. Metabolic engineering that targets the key enzymes of the fatty acid synthesis pathway, along with transcription factor engineering, are effective strategies for improving lipid productivity in microalgae. This review provides a summary of the advancements made in the past 5 years in engineering the fatty acid biosynthetic pathway in eukaryotic microalgae. Furthermore, this review offers insights into transcriptional regulatory mechanisms and transcription factor engineering aimed at enhancing lipid production in eukaryotic microalgae. Finally, the review discusses the challenges and future perspectives associated with utilizing microalgae for the efficient production of lipids.

Mucor circinelloides has the ability to use a wide range of carbon sources to accumulate lipids. The different carbon sources have significantly influenced the total lipids through different metabolic pathways in oleaginous organisms. In the present study, the effects of exogenous oil (soybean oil) at different concentrations as an alternative carbon source on its fungal growth and lipid accumulation were investigated without changing fermentation culture conditions and the original C/N ratio, while using 100% glucose as a control. Total lipids and their fatty acid composition were determined by gas chromatography (GC). Moreover, the expression levels of desaturases genes involved in fatty acid biosynthesis were investigated in the studied strains using real-time fluorescence quantitative PCR. The results showed that a concentration of 35 g/L of glucose mixed with 24.3 g/L of soybean oil were the optimal carbon sources with constant carbon to nitrogen ratio. The biomass, lipid content and lipid yield of M. circinelloides grown in this medium increased rapidly in the early stage of fermentation, and the growth trend gradually slowed down in the late stage of fermentation, decreasing slightly after 72 h of fermentation. It was found that the highest biomass, total lipids, and lipid yields were significantly increased at 19.60 g/L, 42.62%, and 8.35 g/L (33.15%, 23.8%, and 65.10%), respectively. In contrast, the control group (those cultured on glucose as a single carbon source) was only 14.72 g/L, 34.41%, and 5.06 g/L, respectively. The critical step was in which α-linolenic acid (ALA) was stored from the exogenous oil in fungal cells supplying enough soybean oil with mixed substrates for fatty acids synthesis. The overexpression of the △12-desaturase gene in WJ11 significantly (P

Abstract Background Microalgae, with their high adaptability to various stress conditions and rapid growth, are considered excellent biomass resources for lipid production and biodiesel feedstocks. However, lipid yield and productivity of the natural strains are common bottlenecks in their large-scale use for lipid production, which can be overcome by evolving new strains using conventional and advanced mutagenic techniques. It is challenging to generate microalgae strains capable of high lipid synthesis through natural selection. As a result, random mutagenesis is currently considered a viable option in many scenarios. The objective of this study was to explore atmospheric and room temperature plasma (ARTP) as a random mutagenesis technique to obtain high lipid-accumulating mutants of a green microalga for improved biodiesel production. Results A green microalgal species was isolated from the Chinese Yellow Sea and identified as Parachlorella kessleri (OM758328). The isolated microalga was subsequently mutated by ARTP to obtain high lipid-accumulating mutants. Based on the growth rate and lipid content, 5 mutants (named M1, M2, M4, M5, and M8) were selected from 15 pre-selected mutants. These five mutants varied in their growth rate from 0.33 to 0.68 day−1, with the lipid content varying between 0.25 g/L in M2 to 0.30 g/L in M8 at 10th day of cultivation. Among the mutants, M8 showed the maximum biomass productivity (0.046 g/L/day) and lipid productivity (20.19 mg/L/day), which were 75% and 44% higher than the wild strain, respectively. The triglyceride (TAG) content of M8 was found to be 0.56 g/L at 16th day of cultivation, which was 1.77-fold higher than that of the wild strain. Furthermore, M8 had the highest saturated fatty acids (C16-18) with the lowermost polyunsaturated fatty acid content, which are favorable properties of a biodiesel feedstock according to international standards. Conclusion The mutant strain of P. kessleri developed by the ARTP technique exhibited significant improvements in biomass productivity, lipid content, and biodiesel quality. Therefore, the biomass of this mutant microalga could be a potential feedstock for biodiesel production.

Abstract Background Schizochytrium sp. is a heterotrophic, oil-producing microorganism that can efficiently produce lipids. However, the industrial production of bulk chemicals using Schizochytrium sp. is still not economically viable due to high-cost culture medium. Replacing glucose with cheap and renewable lignocellulose is a highly promising approach to reduce production costs, but Schizochytrium sp. cannot efficiently metabolize xylose, a major pentose in lignocellulosic biomass. Results In order to improve the utilization of lignocellulose by Schizochytrium sp., we cloned and functionally characterized the genes encoding enzymes involved in the xylose metabolism. The results showed that the endogenous xylose reductase and xylulose kinase genes possess corresponding functional activities. Additionally, attempts were made to construct a strain of Schizochytrium sp. that can effectively use xylose by using genetic engineering techniques to introduce exogenous xylitol dehydrogenase/xylose isomerase; however, the introduction of heterologous xylitol dehydrogenase did not produce a xylose-utilizing engineered strain, whereas the introduction of xylose isomerase did. The results showed that the engineered strain 308-XI with an exogenous xylose isomerase could consume 8.2 g/L xylose over 60 h of cultivation. Xylose consumption was further elevated to 11.1 g/L when heterologous xylose isomerase and xylulose kinase were overexpressed simultaneously. Furthermore, cultivation of 308-XI-XK(S) using lignocellulosic hydrolysates, which contained glucose and xylose, yielded a 22.4 g/L of dry cell weight and 5.3 g/L of total lipid titer, respectively, representing 42.7 and 30.4% increases compared to the wild type. Conclusion This study shows that engineering of Schizochytrium sp. to efficiently utilize xylose is conducive to improve its utilization of lignocellulose, which can reduce the costs of industrial lipid production.

The study evaluated the use of Chlorella vulgaris for bioremediating hydroponic wastewater and producing biomass under different cultivation modes and to explore the economic implications of microalgal biofuels. Total nitrogen (TN) removal efficiency was 98.5% in mixotrophic conditions and 96% in heterotrophic conditions, and total phosphorus (TP) was completely removed (>99%) in both cultivation conditions. TN removal was higher for that which was cultivated under the mixotrophic mode of cultivation. The maximum biomass production (1.26 g/L) and biomass productivity (0.1108 g/L/day) were also reported for mixotrophic conditions. Lipid content was slightly higher for that which was cultivated under heterotrophic conditions: 33 wt% on an ash-free dry weight (AFDW) basis. The highest lipid production was obtained under mixotrophic growth (0.341 g/L). Higher net profit was obtained for both mixotrophic and heterotrophic cultivations: 30.6 million $/year for a plant capacity of 3.29 × 104 tone/year and 30.12 million $/year for a plant capacity of 3.17 × 104 tone/year respectively. Sensitivity analysis showed that biodiesel and nutritious supplements from soluble protein have the greatest impact on the process economics regarding mixotrophic cultivation, while biodiesel and feeds from insoluble protein have the largest effect on the process economics regarding heterotrophic and autotrophic cultivations. HIGHLIGHTS C. vulgaris was cultivated in hydroponic wastewater to remove nutrients and to produce lipid and biomass.; C. vulgaris showed higher TN removal efficiency under mixotrophic growth (TN: 98.5%).; Maximum lipid content was reported for heterotrophic growth (33 wt% AFDW).; Mixotrophic cultivation showed maximum biomass production (1.26 g/L) and biomass productivity (0.1108 g/L/day).; The best economic benefits were obtained for mixotrophic (net profit: 30.6 million $/year) and heterotrophic (net profit: 30.12 million $/year) cultivations.;

Bioenergy production from algae has gained significant attention due to its potential as a sustainable and renewable energy source. This research investigates the optimization of bioenergy production by focusing on bioreactor design to enhance the efficiency of algal biomass growth and lipid extraction. The study involves analyzing key bioreactor parameters such as light intensity, nutrient concentration, pH levels, and carbon dioxide (CO₂) supply. Experimental results demonstrated a 25% increase in lipid production through optimized conditions, showing promising improvements in biofuel yield. The findings of this research provide valuable insights into the potential scalability of algae-based bioenergy systems.

Abstract Rhodosporidium toruloides is a useful oleaginous yeast, but lipids production is affected by various factors including nutrients in the culture medium. Herein, the R-ZL2 high-yield mutant strain was used to investigate the effects of different carbon sources (sucrose, glucose, xylose), nitrogen sources (ammonium sulphate, ammonium nitrate), and C/N ratio on lipids production capacity, get the following conclusion (1) Compared with glucose and xylose, sucrose was a superior carbon source for lipids production; (2) When using ammonium sulphate as the nitrogen source, a C/N ratio of 200:1 achieved the highest biomass, lipids production and lipids content (10.7 g/L, 6.32 g/L and 59%, respectively), and lipids produced under different C/N conditions have potential for biodiesel production (except for C/N = 40 and C/N = 80); (3) When using ammonium nitrate as the nitrogen source, a C/N ratio of 200:1 achieved the highest biomass, lipids production and lipids content (12.1 g/L, 8.25 g/L and 65%, respectively), and lipids produced under different C/N ratio conditions have potential for biodiesel production. Thus, a combination of sucrose and ammonium nitrate was optimal for the lipid accumulation in R-ZL2. The findings will lay a foundation for further improving lipids yields.

In this work, microbial lipid production from non-detoxified Eucalyptus bark hydrolysate (EBH) with oleaginous xylose-utilizing Ashbya gossypii strains was explored. The best producing strain from a set of engineered strains was identified in synthetic media mimicking the composition of the non-detoxified EBH (SM), the lipid profile was characterized, and yeast extract and corn steep liquor (CSL) were pinpointed as supplements enabling a good balance between lipid accumulation, biomass production, and autolysis by A. gossypii. The potential of the engineered A. gossypii A877 strain to produce lipids was further validated and optimized with minimally processed inhibitor-containing hydrolysate and high sugar concentration, and scaled up in a 2 L bioreactor. Lipid production from non-detoxified EBH supplemented with CSL reached a lipid titer of 1.42 g/L, paving the way for sustainable single-cell oil production within the concept of circular economy and placing lipids as an alternative by-product within microbial biorefineries.

AimOur aim was to identify the relationship between several surrogate insulin resistance (IR) indices based on lipid products and the development of hypertension.Materials and methodsA total of 3,281 participants aged ≥ 18 years enrolled in the China Health and Nutrition Survey from 2009 to 2015 and who were followed up for 6 years were included in the final analysis. Logistic regression was used to analyze the association between different IR indices and incident hypertension.ResultsThere were 882 (28.9%) hypertensive participants in 2015. With regard to the homeostasis model assessment of insulin resistance (HOMA-IR) based on insulin level, subjects in the highest quartile of HOMA-IR values were more likely to develop hypertension [RR = 1.58 (1.26–1.98), P < 0.001] after being adjusted by sex and age, smoke habits, alcohol consumption, community type, married status, and education years in 2009. Subjects in the highest quartile of the triglyceride-glucose index (TyG) combined with body mass index (BMI) and waist circumference (WC) had more than two times the risk of hypertension after full adjustment compared with individuals in the lowest quartile (both P < 0.001), and the trend continued when adjusted for the HOMA-IR. Compared with those in the lowest quartile of TyG-BMI values, females in the highest quartile had a higher risk of developing hypertension than males [2.82 (2.01–3.97) vs. 2.56 (1.80–3.64)] after the full adjustment, and the trend existed independent of IR. Young participants in the highest quartile of the HOMA-IR had significantly higher risks of hypertension compared with subjects in the lowest quartile [1.67 (1.31–2.14), P < 0.005], and this trend was not significant in the elderly participants.ConclusionThe results from our large-scale study elucidate the superiority of the TyG-BMI and TyG-WC compared with the HOMA-IR in the prediction of hypertension, which may be related to lipid deposition. The sex-specific predictive value is distinct for different IR indicators.

Chlamydomonas reinhardtii is a model strain to explore algal lipid metabolism mechanism, and exhibits great potentials in large-scale production of lipids. Completely lacking nitrogen is an efficient strategy to trigger the lipid synthesis in microalgal cells, while it always leads to the obvious reduction in the biomass. To illustrate the optimal culture substrate carbon (C) and nitrogen (N) levels to simultaneously stimulate the growth and lipid production of C. reinhardtii, cells were cultivated under altered C and N concentrations. Results showed that replenishing 6 g/L sodium acetate (NaAc) could increase 1.50 and 1.53 times biomass and lipid productivity compared with 0 g/L NaAc treatment (the control), but total lipid content slightly decreased. Reducing 75% of basic medium (TAP) N level (0 g/L NaAc + 0.09 g/L NH4Cl treatment) could promote 21.57% total lipid content in comparison with the control (containing 0.38 g/L NH4Cl), but decrease 44.45% biomass and 34.15% lipid productivity. The result of the central composite design (CCD) experiment suggested the optimum total lipid content together with higher biomass and lipid productivity could be obtained under the condition of 4.12 g/L NaAc and 0.20 g/L NH4Cl. They reached 32.14%, 1.68 g/L and 108.21 mg/L/d, and increased by 36.77%, 93.10% and 1.75 times compared with the control, respectively. It suggests moderately increasing C supply and decreasing N levels could synchronously improve the biomass and lipid content of C. reinhardtii.

Oleic acid is increasingly required in many industries, causing the indiscriminate extension of land for the cultivation of certain agricultural products to extract their oil. The current contribution aimed to cultivate Candida wangnamkhiaoensis (CW) for the production of lipids and determine the profile of fatty acids in these lipids. The lipid yield was compared in the yeast when using glucose or glycerol as the substrate, in both cases being over 24%. The main fatty acids in the oil derived from CW were oleic, palmitic, stearic, and linoleic acid. The fatty acid composition of the oil from CW was very similar to that of avocado oil and resembled that of olive oil and palm oil. The advantages of cultivating CW include its relatively high percentage of oleic acid and the balance of other fatty acids, its capacity to generate lipids in a short time (48–72 h), the controlled environment of production (versus the variability of the cultivation of agricultural products), and the relatively limited surface area required. CW shows potential as an alternative and economical source of oleic acid for the food, drug, cosmetics, lubricant, and biofuel industries, and does not require the alteration of large extensions of land.

Low biomass yield and nutrient removal efficiency are problems challenging the employment of microorganisms for wastewater remediation. Starch processing effluent (SPE) was used as a fermentation substrate to co-culture Chlorella vulgaris and Rhodotorula glutinis for biofuel feedstock production. Co-culture options were compared, and the optimal conditions were identified. The result shows that microalgae and yeast should be inoculated simultaneously at the beginning of SPE-based fermentation to achieve high biomass yield and the optimal inoculation ratio, light intensity, and temperature should be 2:1, 150 μmol/m2/s, and 25 °C, respectively. Under the optimal conditions, the lipid yield of microorganisms was 1.81 g/L and the carbon–conversion ratio reached 82.53% while lipid yield and the carbon–conversion ratio in a monoculture fell in the range of 0.79–0.81 g/L and 55.93–62.61%, respectively. Therefore, compared to the monoculture model, the co-fermentation of Chlorella vulgaris and Rhodotorula glutinis in starch processing effluent could convert nutrients to single-cell oil in a more efficient way. It should be noted that with the reduced concentration of residual organic carbon in effluent and the increased carbon–conversion ratio, co-fermentation of microalgae and yeast can be regarded as a promising and applicable strategy for starch processing effluent remediation and low-cost biofuel feedstock production.

Abstract Background Converting wastewater sludge to lipid is considered as one of the best strategies of sludge management. The current problem of lipid production from wastewater sludge is the low yield (0.10–0.16 g lipid/g dry sludge) due to the low availability of easily uptaken materials (such as soluble monosaccharide and oligosaccharide) in sludge to oleaginous microorganism (Rhodotorula glutinis, Trichosporon oleaginosus, Lipomyces starkeyi). Pretreatments are efficient methods to improve sludge bioavailability. This study is aimed to achieve high lipid production from sludge and high sludge reduction. Results In this study, it was observed that the soluble chemical oxygen demand (SCOD) had significantly increased after different pretreatment. The SCOD in the supernatant was increased from 32.64 to 180.25 mg/L, 924.16 mg/L, 1029.89 mg/L and 3708.31 mg/L after acidic (pH 2 for 2 h), alkaline (pH 12 for 2 h), microwave irradiation (15 min with 5 min interval), and ultrasonication (30 min at 450 W and 20 kHz frequency with 5 s on and 2 s off mode) pretreatment, respectively. Pretreatments have also increased the release of total nitrogen (TN) and total phosphorus (TP) from solids. The sludge after different pretreatments were used as a medium for lipid production, and the highest lipid content (36.67% g/g) was obtained in the fermentation with ultrasonication pretreatment sludge, and the sludge reduction was 63.10%. For other pretreatments, the lipid content and sludge reduction were 18.42% and 32.63% in acid pretreatment case, 21.08% and 36.44% in alkaline pretreatment case, and 26.31% and 43.03% in microwave pretreatment case, respectively. Conclusion It was found that ultrasonication pretreatment was the most efficient way to increase the sludge biodegradability (SCOD) and to release TN and TP from solid phase to liquid phase. Pretreated sludge for lipid production achieved significant improvement in lipid yield and sludge reduction. Lipids produced from pretreated sludge were transesterified to biodiesel and the analysis showed that biodiesel had a similar composition as commercial biodiesel. The study reveals that pretreatment on sludge is a promising method for enhancing biological sludge management efficiency.

Abstract Lignocellulosic biomass shows high potential as a renewable feedstock for use in biodiesel production via microbial fermentation. Yarrowia lipolytica, an emerging oleaginous yeast, has been engineered to efficiently convert xylose, the second most abundant sugar in lignocellulosic biomass, into lipids for lignocellulosic biodiesel production. Yet, the lipid yield from xylose or lignocellulosic biomass remains far lower than that from glucose. Here we developed an efficient xylose‐utilizing Y. lipolytica strain, expressing an isomerase‐based pathway, to achieve high‐yield lipid production from lignocellulosic biomass. The newly developed xylose‐utilizing Y. lipolytica, YSXID, produced 12.01 g/L lipids with a maximum yield of 0.16 g/g, the highest ever reported, from lignocellulosic hydrolysates. Consequently, this study shows the potential of isomerase‐based xylose‐utilizing Y. lipolytica for economical and sustainable production of biodiesel and oleochemicals from lignocellulosic biomass.

Abstract Background Without sufficient alternatives to crude oil, as demand continues to rise, the global economy will undergo a drastic decline as oil prices explode. Dependence on crude oil and growing environmental impairment must eventually be overcome by creating a sustainable and profitable alternative based on renewable and accessible feedstock. One of the promising solutions for the current and near-future is the substitution of fossil fuels with sustainable liquid feedstock for biofuel production. Among the different renewable liquid feedstock’s studied, wastewater is the least explored one for biodiesel production. Sago wastewater is the byproduct of the cassava processing industry and has starch content ranging from 4 to 7%. The present investigation was aimed to produce microbial lipids from oleaginous yeast, Candida tropicalis ASY2 for use as biodiesel feedstock and simultaneously decontaminate the sago processing wastewater for reuse. Initial screening of oleaginous yeast to find an efficient amylolytic with maximum lipid productivity resulted in a potent oleaginous yeast strain, C. tropicalis ASY2, that utilizes SWW as a substrate. Shake flask experiments are conducted over a fermentation time of 240 h to determine a suitable fatty acid composition using GC-FID for biodiesel production with simultaneous removal of SWW pollutants using ASY2. Results The maximum biomass of 0.021 g L−1 h−1 and lipid productivity of 0.010 g L−1 h−1 was recorded in SWW with lipid content of 49%. The yeast strain degraded cyanide in SWW (79%) and also removed chemical oxygen demand (COD), biological oxygen demand (BOD), nitrate (NO3), ammoniacal (NH4), and phosphate (PO4) ions (84%, 92%, 100%, 98%, and 85%, respectively). GC-FID analysis of fatty acid methyl esters (FAME) revealed high oleic acid content (41.33%), which is one of the primary fatty acids for biodiesel production. Conclusions It is evident that the present study provides an innovative and ecologically sustainable technology that generates valuable fuel, biodiesel using SWW as a substrate and decontaminates for reuse.

Continued reliance on diesel from fossil sources is not sustainable and detrimental to human, animal and plant existence owing to discharge of toxics and poisonous gasses. The present target is the new strains of microalgal from fish farm for diesel production through experimental screening processes. Water samples were collected from fish ponds in Owode-ede, Osun State Nigeria from which microalgae were isolated using algae culturing techniques. Isolates were screened for Lipid Production (LP) potential using Volumetric Lipid Productivity (VLP). Isolates with the higher LP were identified using morphological methods. Filtration, solvent extraction and gravimetric methods were employed for determination of biomass concentration, lipid extraction and quantification. Data recorded were analyzed using appropriate statistics. A total of 12 microalgal were obtained. They include Scenedesmus, Chlorella Crucigenia, Spirogyra. Oscillatoria, Chlamydomonas, Euglena. Cladophora, Closterium, Microcystis, Ankistrodesmus,and Pediastrum. The first two mentioned genera are the most promising isolates with high LP (51.43±0.84 and 45.38±1.67%) and VLP (257.25±5.29 and 181.52±0.89 mg/L/day). They are ideal isolates for production of microbial fuel.

Lipid extraction from microbial and microalgae biomass requires the separation of oil-rich cells from the production media. This downstream procedure represents a major bottleneck in biodiesel production, increasing the cost of the final product. Flocculation is a rapid and cheap system for removing solid particles from a suspension. This natural characteristic is displayed by some microorganisms due to the presence of lectin-like proteins (called flocculins/adhesins) in the cell wall. In this work, we showed, for the first time, that the heterologous expression of the adhesin Cfl1p endows the oleaginous species Cutaneotrichosporon oleaginosus with the capacity of cell flocculation. We used Helm’s test to demonstrate that the acquisition of this trait allows for reducing the time required for the separation of lipid-rich cells from liquid culture by centrifugation without altering the productivity. This improves the lipid production process remarkably by providing a more efficient downstream.

The interest in microorganisms to produce microbial lipids at large-scale processes has increased during the last decades. Rhodosporidium toruloides-1588 could be an efficient option for its ability to simultaneously utilize five- and six-carbon sugars. Nevertheless, one of the most important characteristics that any strain needs to be considered or used at an industrial scale is its capacity to grow in substrates with high sugar concentrations. In this study, the effect of high sugar concentrations and the effect of ammonium sulfate were tested on R. toruloides-1588 and its capacity to grow and accumulate lipids using undetoxified wood hydrolysates. Batch fermentations showed a catabolic repression effect on R. toruloides-1588 growth at sugar concentrations of 120 g/L. The maximum lipid accumulation was 8.2 g/L with palmitic, stearic, oleic, linoleic, and lignoceric acids as predominant fatty acids in the produced lipids. Furthermore, R. toruloides-1588 was able to utilize up to 80% of the total xylose content. Additionally, this study is the first to report the effect of using high xylose concentrations on the growth, sugar utilization, and lipid accumulation by R. toruloides-1588.

Nanoparticle application in microalgae for enhanced lipid production is an ongoing work that leads towards the contribution in biodiesel production. During this decade, metal nanoparticles are constantly being reported to have numerous applications in diverse fields, because of their unique optical, electrical, and magnetic properties. They can interact with the biomolecules of cells and thereby alters cellular metabolisms, which in turn reflects their ability to regulate some primary or secondary metabolic pathways. Nanoparticles derived from metals like Fe, Cu, and Se are taking part in redox processes and their presence in many enzymes may modulate algal metabolisms. Besides by upregulating or downregulating the expression of several genes, nanoparticle exposure can alter gene expressions in many organisms. In microalgae such as Chlorella vulgaris, C. pyrenoidosa, Scenedesmus obliquus, S. rubescens, Trachydiscus minutus, Parachlorella kessleri, and Tetraselmis suecica; metal nanoparticle exposure in different environmental conditions have impacts on various physiological or molecular changes, thereby increasing the growth rate, biomass and lipid production. The present mini-review gives an insight into the various advantages and a future outlook on the application of nanoparticles in microalgae for biofuel production. Also, it can be proposed that nanoparticles could be useful in blocking or deactivating the AGPase enzyme (involved in the glucose to starch conversion pathway), binding to its active site, thereby increasing lipid production in microalgae that could be utilized for enhanced biodiesel production.

Abstract Background Oleaginous yeasts are able to accumulate very high levels of neutral lipids especially under condition of excess of carbon and nitrogen limitation (medium with high C/N ratio). This makes necessary the use of two-steps processes in order to achieve high level of biomass and lipid. To simplify the process, the decoupling of lipid synthesis from nitrogen starvation, by establishing a cytosolic acetyl-CoA formation pathway alternative to the one catalysed by ATP-citrate lyase, can be useful. Results In this work, we introduced a new cytoplasmic route for acetyl-CoA (AcCoA) formation in Rhodosporidium azoricum by overexpressing genes encoding for homologous phosphoketolase (Xfpk) and heterologous phosphotransacetylase (Pta). The engineered strain PTAPK4 exhibits higher lipid content and produces higher lipid concentration than the wild type strain when it was cultivated in media containing different C/N ratios. In a bioreactor process performed on glucose/xylose mixture, to simulate an industrial process for lipid production from lignocellulosic materials, we obtained an increase of 89% in final lipid concentration by the engineered strain in comparison to the wild type. This indicates that the transformed strain can produce higher cellular biomass with a high lipid content than the wild type. The transformed strain furthermore evidenced the advantage over the wild type in performing this process, being the lipid yields 0.13 and 0.05, respectively. Conclusion Our results show that the overexpression of homologous Xfpk and heterologous Pta activities in R. azoricum creates a new cytosolic AcCoA supply that decouples lipid production from nitrogen starvation. This metabolic modification allows improving lipid production in cultural conditions that can be suitable for the development of industrial bioprocesses using lignocellulosic hydrolysates.

Abstract The use of fossil fuels has been strongly related to critical problems currently affecting society, such as: global warming, global greenhouse effects and pollution. These problems have affected the homeostasis of living organisms worldwide at an alarming rate. Due to this, it is imperative to look for alternatives to the use of fossil fuels and one of the relevant substitutes are biofuels. There are different types of biofuels (categories and generations) that have been previously explored, but recently, the use of microalgae has been strongly considered for the production of biofuels since they present a series of advantages over other biofuel production sources: (a) they don’t need arable land to grow and therefore do not compete with food crops (like biofuels produced from corn, sugar cane and other plants) and; (b) they exhibit rapid biomass production containing high oil contents, at least 15 to 20 times higher than land based oleaginous crops. Hence, these unicellular photosynthetic microorganisms have received great attention from researches to use them in the large-scale production of biofuels. However, one disadvantage of using microalgae is the high economic cost due to the low-yields of lipid content in the microalgae biomass. Thus, development of different methods to enhance microalgae biomass, as well as lipid content in the microalgae cells, would lead to the development of a sustainable low-cost process to produce biofuels. Within the last 10 years, many studies have reported different methods and strategies to induce lipid production to obtain higher lipid accumulation in the biomass of microalgae cells; however, there is not a comprehensive review in the literature that highlights, compares and discusses these strategies. Here, we review these strategies which include modulating light intensity in cultures, controlling and varying CO2 levels and temperature, inducing nutrient starvation in the culture, the implementation of stress by incorporating heavy metal or inducing a high salinity condition, and the use of metabolic and genetic engineering techniques coupled with nanotechnology.

Abstract Background Euglena gracilis, a unicellular flagellated microalga, is regarded as one of the most promising species as microalgal feedstock for biofuels. Its lipids (mainly wax esters) are suitable for biodiesel and jet fuel. Culture of E. gracilis using wastewater effluent will improve the economics of E. gracilis biofuel production. Enhancement of the productivity of E. gracilis biomass is critical to creating a highly efficient biofuels production system. Certain bacteria have been found to promote microalgal growth by creating a favorable microenvironment. These bacteria have been characterized as microalgae growth-promoting bacteria (MGPB). Co-culture of microalgae with MGPB might offer an effective strategy to enhance microalgal biomass production in wastewater effluent culture systems. However, no MGPB has been identified to enhance the growth of E. gracilis. The objectives of this study were, therefore, to isolate and characterize the MGPB effective for E. gracilis and to demonstrate that the isolated MGPB indeed enhances the production of biomass and lipids by E. gracilis in wastewater effluent culture system. Results A bacterium, Emticicia sp. EG3, which is capable of promoting the growth of microalga E. gracilis, was isolated from an E. gracilis-municipal wastewater effluent culture. Biomass production rate of E. gracilis was enhanced 3.5-fold and 3.1-fold by EG3 in the co-culture system using a medium of heat-sterilized and non-sterilized wastewater effluent, respectively, compared to growth in the same effluent culture but without EG3. Two-step culture system was examined as follows: E. gracilis was cultured with or without EG3 in wastewater effluent in the first step and was further grown in wastewater effluent in the second step. Production yields of biomass and lipids by E. gracilis were enhanced 3.2-fold and 2.9-fold, respectively, in the second step of the system in which E. gracilis was co-cultured with EG3 in the first step. Conclusion Emticicia sp. EG3 is the first MGPB for E. gracilis. Growth-promoting bacteria such as EG3 will be promising agents for enhancing E. gracilis biomass/biofuel productivities.

Abstract Background Microalgae as a viable biodiesel feedstock show great potential to approach the challenges of energy shortage and environment pollution, but their economic feasibility was seriously hampered by high production cost. Thus, it is in urgent need to reduce the cost of cultivation and improve the biomass and lipid production of microalgae. In this work, anaerobic digestion effluent from cattle manure combined with municipal wastewater was used as a cost-effective medium for cultivating microalgae and expected to obtain high biomass. The pretreatment of anaerobic digested effluent containing dilution rate, sterilization and nutrient optimization was investigated. Then, initial pH and light intensity for algal growth, lipid production and wastewater purification were optimized in this study. Results Scenedesmus sp. could grow rapidly in 10% anaerobic digestion effluent from cattle manure combined with secondary sedimentation tank effluent without sterilization. Optimum nutrient additives for higher biomass were as follows: glucose 10 g/L, NaNO3 0.3 g/L, K2HPO4·3H2O 0.01 g/L, MgSO4·7H2O 0.075 g/L and trace element A5 solution 1 mL/L. Biomass of 4.65 g/L and lipid productivity of 81.90 mg/L/day were achieved during 7-day cultivation accompanying over 90% of COD, NO3 −-N, NH4 +-N, and 79–88% of PO4 3−-P removal with optimized initial pH of 7.0 and light intensity of 5000 l×. The FAME profile in ADEC growth medium consisted in saturated (39.48%) and monounsaturated (60.52%) fatty acids with the 16- to 18-chain-length fatty acids constituting over 98% of total FAME. Conclusions This study proves the potential of anaerobic digested effluent combined with municipal wastewater for microalgae culture, and provides an effective avenue for simultaneous microalgal lipid production and treatment of two kinds of wastewater.

Abstract Background Rhodotorula toruloides is a promising platform organism for production of lipids from lignocellulosic substrates. Little is known about the metabolic aspects of lipid production from the lignocellolosic sugar xylose by oleaginous yeasts in general and R. toruloides in particular. This study presents the first proteome analysis of the metabolism of R. toruloides during conversion of xylose to lipids. Results Rhodotorula toruloides cultivated on either glucose or xylose was subjected to comparative analysis of its growth dynamics, lipid composition, fatty acid profiles and proteome. The maximum growth and sugar uptake rate of glucose-grown R. toruloides cells were almost twice that of xylose-grown cells. Cultivation on xylose medium resulted in a lower final biomass yield although final cellular lipid content was similar between glucose- and xylose-grown cells. Analysis of lipid classes revealed the presence of monoacylglycerol in the early exponential growth phase as well as a high proportion of free fatty acids. Carbon source-specific changes in lipid profiles were only observed at early exponential growth phase, where C18 fatty acids were more saturated in xylose-grown cells. Proteins involved in sugar transport, initial steps of xylose assimilation and NADPH regeneration were among the proteins whose levels increased the most in xylose-grown cells across all time points. The levels of enzymes involved in the mevalonate pathway, phospholipid biosynthesis and amino acids biosynthesis differed in response to carbon source. In addition, xylose-grown cells contained higher levels of enzymes involved in peroxisomal beta-oxidation and oxidative stress response compared to cells cultivated on glucose. Conclusions The results obtained in the present study suggest that sugar import is the limiting step during xylose conversion by R. toruloides into lipids. NADPH appeared to be regenerated primarily through pentose phosphate pathway although it may also involve malic enzyme as well as alcohol and aldehyde dehydrogenases. Increases in enzyme levels of both fatty acid biosynthesis and beta-oxidation in xylose-grown cells was predicted to result in a futile cycle. The results presented here are valuable for the development of lipid production processes employing R. toruloides on xylose-containing substrates.

As an interesting alternative microbial platform for the sustainable synthesis of oleochemical building blocks and biofuels, oleaginous yeasts are increasing in both quantity and diversity. In this study, oleaginous yeast species from northern Thailand were discovered to add to the topology. A total of 127 yeast strains were isolated from 22 forest soil samples collected from mountainous areas. They were identified by an analysis of the D1/D2 domain of the large subunit rRNA (LSU rRNA) gene sequences to be 13 species. The most frequently isolated species were Lipomyces tetrasporus and Lipomyces starkeyi. Based on the cellular lipid content determination, 78 strains of ten yeast species, and two potential new yeast that which accumulated over 20% of dry biomass, were found to be oleaginous yeast strains. Among the oleaginous species detected, Papiliotrema terrestris and Papiliotrema flavescens have never been reported as oleaginous yeast before. In addition, none of the species in the genera Piskurozyma and Hannaella were found to be oleaginous yeast. L. tetrasporus SWU-NGP 2-5 accumulated the highest lipid content of 74.26% dry biomass, whereas Lipomyces mesembrius SWU-NGP 14-6 revealed the highest lipid quantity at 5.20 ± 0.03 g L−1. The fatty acid profiles of the selected oleaginous yeasts varied depending on the strain and suitability for biodiesel production.

Environmental pollution, greenhouse gas emissions, depletion of fossil fuels, and a growing population have sparked a search for new and renewable energy sources such as biodiesel. The use of waste or residues as substrates for microbial growth can favor the implementation of a biorefinery concept with reduced environmental footprint. Cyanobacteria constitute microorganisms with enhanced ability to use industrial effluents, wastewaters, forest residues for growth, and concomitant production of added-value compounds. In this study, a recently isolated cyanobacterium strain of Pseudanabaena sp. was cultivated on hydrolysates from pretreated forest biomass (silver birch and Norway spruce), and the production of biodiesel-grade lipids was assessed. Optimizing carbon source concentration and the (C/N) carbon-to-nitrogen ratio resulted in 66.45% w/w lipid content when microalgae were grown on glucose, compared to 62.95% and 63.79% w/w when grown on spruce and birch hydrolysate, respectively. Importantly, the lipid profile was suitable for the production of high-quality biodiesel. The present study demonstrates how this new cyanobacterial strain could be used as a biofactory, converting residual resources into green biofuel.

This paper studied the bifurcation phenomena that can occur in a continuous bioreactor for lipid-rich biomass production using Rhodococcus opacus that feeds on refinery wastewater. An unstructured model of the bioreactor was developed based on experimentally validated studies reported in the literature. The analysis of the unsteady-state model was carried out both analytically and through numerical simulations. It was shown that the inhibition effect of the biomass growth rate can lead to the occurrence of static bistability in the model. Periodic behavior was also found for some range of model parameters. The effect of model parameters on the productivity of the bioreactor was also investigated. The analysis carried out in this paper allowed for the detection of unsafe operating regions in the bioreactor which, would help in the attenuation of these operational problems in the early stage of process design.

Adding organic compounds to wastewater can improve the carbon/nitrogen ratio and benefit microalgae growth. We studied microalgal growth, nutrient removal and lipid accumulation of Chlorella pyrenoidosa cultured in a mixture of swine wastewater (SW) and furfural wastewater (FW). The mix ratio of SW:DFW (diluted furfural wastewater) had a significant effect on microalgae growth. As the mix ratio of SW:DFW decreased from 1:0.5 to 1:19, the maximum microalgal biomass increased, while the specific growth rate initially increased and then decreased. The efficiency of nutrient removal also depended on the mix ratio of wastewater. The highest chemical oxygen demand (COD) removal efficiency (57.30%) occurred at the mix ratio of SW:DFW = 1:3. The highest removal efficiencies of total phosphorous (TP) reached 61.93% when the mix ratio of SW:DFW was 1:9. Wastewater at the mix ratio of SW:DFW = 1:19 had a maximum lipid productivity of 49.48 mg L−1 d−1, which was 4.9 times higher than that at a mix ratio of SW:DFW = 1:0.5. These results showed that C. pyrenoidosa can be used to remove nutrients from mixed wastewater sources and simultaneously produce algal lipids.

Microalgal heterotrophic cultivation is an emerging technology that can enable producing high cell-density algal cell cultures, which can be coupled with photoautotrophic cultivation for valuable chemicals such as lipids manufacturing. However, how the heterotrophically grown algal cells respond to the lipid-inducing conditions has not been fully elucidated so far. In this study, when the heterotrophically grown Scenedesmus acuminatus cells were subjected to the high light (HL) and nitrogen-limited (NL) conditions, both the biomass and lipid productivity were enhanced as compared to that of the photoautotrophically grown counterparts. The chlorophyll a fluorometry analysis showed that the Fv/Fm and Y(II) of the heterotrophically grown cells subjected to the HL and NL conditions was recovered to the maximum value of 0.75 and 0.43, respectively, much higher than those of the photoautotrophically grown cells under the same stress conditions. Transcriptomic analysis revealed that heterotrophically grown cells fully expressed the genes coding for the photosystems proteins, including the key photoprotective proteins D1, PsbS, light-harvesting-complex (LHC) I and LHC II. Meanwhile, downregulation of the carotenoid biosynthesis and upregulation of the glycolysis/gluconeogenesis, tricarboxylic acid (TCA) cycle and oxidative phosphorylation pathways were observed when the heterotrophically grown cells were subjected to the HL and N-limited conditions for lipid production. It was deduced that regulation of these pathways not only enhanced the light utilization but also provided the reducing power and ATP by which the biomass accumulation was significantly elevated. Besides, upregulation of the acetyl-CoA carboxylase/biotin carboxylase, digalactosyl diacylglycerol synthase and diacylglycerol acyltransferase 2 encoding genes may be attributable to the enhanced lipid production. Understanding the cellular responses during the trophic transition process could guide improvement of the strength of trophic transition enhancing microalgal biomass and lipid production.

With the aim of developing bioprocesses for waste valorization and a reduced water footprint, we optimized a two-step fermentation process that employs the oleaginous yeast Cutaneotrichosporon oleaginosus for the production of oil from liquid cheese whey permeate. For the first step, the addition of urea as a cost-effective nitrogen source allowed an increase in yeast biomass production. In the second step, a syrup from candied fruit processing, another food waste supplied as carbon feeding, triggered lipid accumulation. Consequently, yeast lipids were produced at a final concentration and productivity of 38 g/L and 0.57 g/L/h respectively, which are among the highest reported values. Through this strategy, based on the valorization of liquid food wastes (WP and mango syrup) and by recovering not only nutritional compounds but also the water necessary for yeast growth and lipid production, we addressed one of the main goals of the circular economy. In addition, we set up an accurate and fast-flow cytometer method to quantify the lipid content, avoiding the extraction step and the use of solvents. This can represent an analytical improvement to screening lipids in different yeast strains and to monitoring the process at the single-cell level.

Oleaginous fungi have recently gained increasing attention among different microorganisms due to their ability for lipid production for the preparation of biofuel. In the present study, a locally isolated fungus E45, identified genetically as Aspergillus sp. strain EM2018, was found to produce 25.2% of the total lipids content of its dry cell weight (DCW). Optimization of culture conditions was performed and lipid accumula­tion increased by about 2.4 fold (from 25.2% to 60.1% of DCW) when the fungus was grown for seven days in the potato dextrose (50 g/L) liquid medium at pH 5.0, incubation temperature at 30 ºC and inoculum size of 2 × 106 spore/mL. Supplementation of the medium with yeast extract and NaNO3 at a concentration of 0.05% as organic and inorganic nitrogen sources, respectively, increased lipid production (53.3% lipid/dry biomass). Gas chromatography analysis of fungal lipids revealed the presence of saturated (mainly palmitic acid C16:0 (33%) and lignoceric acid C24:0 (15%)) and unsaturated fatty acids in different proportions (mainly linoleic acid C18:2 (24.4%), oleica cid C18:1 (14%) and arachidonic C20:4 (7.4%). These findings suggest this new oleaginous fungus as a promising feedstock for various industrial applications and for the preparation of biodiesel.

Microbial oil triacylglycerol (TAG) from the renewable feedstock attract much attention. The oleaginous yeast Yarrowia lipolytica has become the most studied for lipid biosynthesis. Fatty acid desaturases catalyze the introduction of a double bond into fatty-acid hydrocarbon chains to produce unsaturated fatty acids. Desaturases are known to enhance lipid accumulation. In this study, we have achieved a significant increase in lipid production and increase the unsaturated fatty acids content in Y. lipolytica. By comparing the expression of the native genes of △-9 stearoyl-CoA desaturase (SCD) and △12 desaturase (△12D), and an exogenous △15 desaturase (△15D) from flax in the strain with deleted peroxisomal biogenesis factor 10 (PEX10) and overexpressed diacylglyceride acyl-transferase (DGA1), we found that the strain with overexpressed △15 desaturase accumulated 30.7% lipid. Simultaneously, we explored the effect of two copies of desaturase genes (12D-SCD, 15D-SCD, 12D-15D) on lipid production, and found co-expression of △12D and △15D accumulated 42.6% lipid. The lipid content was further increased by 56.3% through the deletion of the multifunctional enzyme (MFE1) and the overexpression of acetyl-CoA carboxylase (ACC1). Finally, the lipid productivity of 50 g/L and maximal lipid content of 77.8% DCW are obtained using a 5-L stirred-tank bioreactor during the stationary phase in the engineered YL-10. Our result demonstrated that the △12 and △15 desaturases play an important role in lipid production in Y. lipolytica and provides an effective strategy for biodiesel development.

Abstract Microalgae have great potential for the production of biofuels due to the ability of the organism to accumulate large quantities of storage lipids under stress conditions. Mitogen activated protein kinase (MAPK) signaling cascades are widely recognized for their role in stress response signal transduction in eukaryotes. To assess the correlation between MAPK activation and lipid productivity, Chlamydomonas reinhardtii was studied under various concentrations of NaCl. The results demonstrated that C. reinhardtii exhibits elevated levels of extracellular-signal regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) activities after undergoing osmotic stress, as well as an increase in cellular lipid content. To establish a more direct causal link between both kinases and lipid productivity, C. reinhardtii was subjected to biochemically induced regulation of ERK and JNK pathways. Activating the MEK-ERK pathway via C6 ceramide treatment increased ERK activation and lipid production simultaneously, while PD98059 mediated inhibition of the pathway yielded opposite results. Interestingly, suppression of the JNK pathway with SP600125 resulted in a substantial decrease in cell viability under osmotic stress. These results suggest that ERK and JNK MAP kinases have important roles in microalgal lipid accumulation and cell growth under osmotic stress, respectively.

Abstract Production of biofuels and bioenergy precursors by phototrophic microorganisms, such as microalgae and cyanobacteria, is a promising alternative to conventional fuels obtained from non-renewable resources. Several species of microalgae have been investigated as potential candidates for the production of biofuels, for the most part due to their exceptional metabolic capability to accumulate large quantities of lipids. Constraint-based modeling, a systems biology approach that accurately predicts the metabolic phenotype of phototrophs, has been deployed to identify suitable culture conditions as well as to explore genetic enhancement strategies for bioproduction. Core metabolic models were employed to gain insight into the central carbon metabolism in photosynthetic microorganisms. More recently, comprehensive genome-scale models, including organelle-specific information at high resolution, have been developed to gain new insight into the metabolism of phototrophic cell factories. Here, we review the current state of the art of constraint-based modeling and computational method development and discuss how advanced models led to increased prediction accuracy and thus improved lipid production in microalgae.

Abstract Background The aim of this work was to establish a process for the heterotrophic growth of green microalgae using forest biomass hydrolysates. To provide a carbon source for the growth of the green microalgae, two forest biomasses (Norway spruce and silver birch) were pretreated with a hybrid organosolv-steam explosion method, resulting in inhibitor-free pretreated solids with a high cellulose content of 77.9% w/w (birch) and 72% w/w (spruce). Pretreated solids were hydrolyzed using commercial cellulolytic enzymes to produce hydrolysate for the culture of algae. Results The heterotrophic growth of A. protothecoides was assessed using synthetic medium with glucose as carbon source, where the effect of sugar concentration and the carbon-to-nitrogen ratio were optimized, resulting in accumulation of lipids at 5.42 ± 0.32 g/L (64.52 ± 0.53% lipid content) after 5 days of culture on glucose at 20 g/L. The use of birch and spruce hydrolysates was favorable for the growth and lipid accumulation of the algae, resulting in lipid production of 5.65 ± 0.21 g/L (66 ± 0.33% lipid content) and 5.28 ± 0.17 g/L (63.08 ± 0.71% lipid content) when grown on birch and spruce, respectively, after only 120 h of cultivation. Conclusions To the best of our knowledge, this is the first report of using organosolv pretreated wood biomass hydrolysates for the growth and lipid production of microalgae in the literature. The pretreatment process used in this study provided high saccharification of biomass without the presence of inhibitors. Moreover, the lipid profile of this microalga showed similar contents to vegetable oils which improve the biodiesel properties.

Abstract Background Compared with other general energy crops, microalgae are more compatible with desert conditions. In addition, microalgae cultivated in desert regions can be used to develop biodiesel. Therefore, screening oil-rich microalgae, and researching the algae growth, CO2 fixation and oil yield in desert areas not only effectively utilize the idle desertification lands and other resources, but also reduce CO2 emission. Results Monoraphidium dybowskii LB50 can be efficiently cultured in the desert area using light resources, and lipid yield can be effectively improved using two-stage induction and semi-continuous culture modes in open raceway ponds (ORPs). Lipid content (LC) and lipid productivity (LP) were increased by 20% under two-stage industrial salt induction, whereas biomass productivity (BP) increased by 80% to enhance LP under semi-continuous mode in 5 m2 ORPs. After 3 years of operation, M. dybowskii LB50 was successfully and stably cultivated under semi-continuous mode for a month during five cycles of repeated culture in a 200 m2 ORP in the desert area. This culture mode reduced the supply of the original species. The BP and CO2 fixation rate were maintained at 18 and 33 g m−2 day−1, respectively. Moreover, LC decreased only during the fifth cycle of repeated culture. Evaporation occurred at 0.9–1.8 L m−2 day−1, which corresponded to 6.5–13% of evaporation loss rate. Semi-continuous and two-stage salt induction culture modes can reduce energy consumption and increase energy balance through the energy consumption analysis of life cycle. Conclusion This study demonstrates the feasibility of combining biodiesel production and CO2 fixation using microalgae grown as feedstock under culture modes with ORPs by using the resources in the desert area. The understanding of evaporation loss and the sustainability of semi-continuous culture render this approach practically viable. The novel strategy may be a promising alternative to existing technology for CO2 emission reduction and biofuel production.

In this study, magnetic Fe3O4 nanoparticles (NPs) were used as an effective enhancer to increase the biomass and total lipid production of Chlorella sp. UJ-3. It was found that the biomass of algal cells increased significantly when they were exposed to low concentrations of Fe3O4 NPs (20 mg/L), while the best total lipid content of algal cells was achieved when they were exposed to high concentrations of Fe3O4 NPs (100 mg/L). Therefore, we established a strategy to promote the growth and lipid accumulation of microalgae by initially exposing the algal cells to low concentrations of Fe3O4 NPs and then treating them with an increased concentration of Fe3O4 NPs after 12 days of culture. For this strategy, the biomass and total lipid production of algal cells increased by 50% and 108.7%, respectively, compared to the untreated control. The increase in lipid production and change in the fatty acid composition of Chlorella cells were found to help them to cope with the increased number of reactive oxygen species produced due to oxidative stress in alga cells after the addition of Fe3O4 NPs. This study provided a highly efficient way to improve the lipid production of microalgae using nanoparticles.

Microbial lipid production with cost effectiveness is a prerequisite for the oleochemical sector. In this work, genome-wide transcriptional responses on the utilization of xylose and glucose in oleaginous Aspergillus oryzae were studied with relation to growth and lipid phenotypic traits. Comparative analysis of the active growth (t1) and lipid-accumulating (t2) stages showed that the C5 cultures efficiently consumed carbon sources for biomass and lipid production comparable to the C6 cultures. By pairwise comparison, 599 and 917 differentially expressed genes (DEGs) were identified in the t1 and t2 groups, respectively, in which the consensus DEGs were categorized into polysaccharide-degrading enzymes, membrane transports, and cellular processes. A discrimination in transcriptional responses of DEGs set was also found in various metabolic genes, mostly in carbohydrate, amino acid, lipid, cofactors, and vitamin metabolisms. Although central carbohydrate metabolism was shared among the C5 and C6 cultures, the metabolic functions in acetyl-CoA and NADPH generation, and biosynthesis of terpenoid backbone, fatty acid, sterol, and amino acids were allocated for leveraging biomass and lipid production through at least transcriptional control. This study revealed robust metabolic networks in the oleaginicity of A. oryzae governing glucose/xylose flux toward lipid biosynthesis that provides meaningful hints for further process developments of microbial lipid production using cellulosic sugar feedstocks.

Abstract Background The light/dark cycle is one of the most important factors affecting the microalgal growth and lipid accumulation. Biomass concentration and lipid productivity could be enhanced by optimization of light/dark cycles, and this is considered an effective control strategy for microalgal cultivation. Currently, most research on effects of light/dark cycles on algae is carried out under autotrophic conditions and little information is about the effects under mixotrophic cultivation. At the same time, many studies related to mixotrophic cultivation of microalgal strains, even at large scale, have been performed to obtain satisfactory biomass and lipid production. Therefore, it is necessary to investigate cellular metabolism under autotrophic and mixotrophic conditions at different light/dark cycles. Even though microalgal lipid production under optimal environmental factors has been reported by some researchers, the light/dark cycle and temperature are regarded as separate parameters in their studies. In practical cases, light/dark cycling and temperature variation during the day occur simultaneously. Therefore, studies about the combined effects of light/dark cycles and temperature variation on microalgal lipid production are of practical value, potentially providing significant guidelines for large-scale microalgal cultivation under natural conditions. Results In this work, cell growth and lipid accumulation of an oleaginous microalgal mutant, Scenedesmus sp. Z-4, were investigated at five light/dark cycles (0 h/24 h, 8 h/16 h, 12 h/12 h, 16 h/8 h, and 24 h/0 h) in batch culture. The results showed that the optimal light/dark cycle was 12 h/12 h, when maximum lipid productivity rates of 56.8 and 182.6 mg L−1 day−1 were obtained under autotrophic and mixotrophic cultivation, respectively. Poor microalgal growth and lipid accumulation appeared in the light/dark cycles of 0 h/24 h and 24 h/0 h under autotrophic condition. Prolonging the light duration was unfavorable to the production of chlorophyll a and b, which was mainly due to photooxidation effect. Polysaccharide was converted into lipid and protein when the light irradiation time increased from 0 to 12 h; however, further increasing irradiation time had a negative effect on lipid accumulation. Due to the dependence of autotrophically cultured cells on light energy, the light/dark cycle has a more remarkable influence on cellular metabolism under autotrophic conditions. Furthermore, the combined effects of temperature variation and light/dark cycle of 12 h/12 h on cell growth and lipid accumulation of microalgal mutant Z-4 were investigated under mixotrophic cultivation, and the results showed that biomass was mainly produced at higher temperatures during the day, and a portion of biomass was converted into lipid under dark condition. Conclusions The extension of irradiation time was beneficial to biomass accumulation, but not in favor of lipid production. Even though effects of light/dark cycles on autotrophic and mixotrophic cells were not exactly the same, the optimal lipid productivities of Scenedesmus sp. Z-4 under both cultivation conditions were achieved at the light/dark of 12 h/12 h. This may be attributed to its long-term acclimation in natural environment. By combining temperature variation with optimal light/dark cycle of 12 h/12 h, this study will be of great significance for practical microalgae-biodiesel production in the outdoor conditions.

Abstract Background Oleaginous yeasts are fast emerging as a possible feedstock for biodiesel production. Yarrowia lipolytica, a model oleaginous yeast is known to utilize a variety of hydrophobic substrates for lipid accumulation including waste cooking oil (WCO). Approaches to increase lipid content in this yeast include metabolic engineering which requires manipulation of multiple genes in the lipid biosynthesis pathway. A classical and cost-effective approach, namely, random chemical mutagenesis on the yeast can lead to increased production of biodiesel as is explored here. Results In this study, chemical mutagenesis using the alkylating agent, N- methyl-N′-nitro-N-nitrosoguanidine (MNNG) as well as an additional treatment with cerulenin, a fatty acid synthase inhibitor generated 800 mutants of Y. lipolytica NCIM 3589 (761 MNNG treated and 39 MNNG + cerulenin treated). A three-stage screening using Sudan Black B plate technique, Nile red fluorimetry and total lipid extraction using solvent was performed, which enabled selection of ten high lipid yielding mutants. Time course studies of all the ten mutants were further undertaken in terms of biomass, lipid yield and lipid content to select three stable mutants (YlB6, YlC7 and YlE1) capable of growing and accumulating lipid on WCO, with lipid contents of 55, 60 and 67% as compared to 45% for the wild type. The mutants demonstrated increased volumetric lipid productivities (0.062, 0.044 and 0.041 g L−1 h−1) as compared to the wild type (0.033 g L−1 h−1). The fatty acid profile of the three mutants consisted of a high content of C16 and C18 saturated and monounsaturated fatty acids and was found to be suitable for biodiesel production. The fuel properties, namely, density, kinematic viscosity, total acid number, iodine value of the three mutants were evaluated and found to lie within the limits specified by internationally accepted standards. Additionally, it was noted that the mutants demonstrated better cetane numbers and higher heating values than the wild type strain. Conclusion The chemical mutagenesis strategy adopted in this study resulted in the successful isolation of three stable high SCO yielding mutants. The mutants, namely, YlB6, YlC7 and YlE1 exhibited a 1.22, 1.33 and 1.49-fold increase in lipid contents when grown on 100 g L−1 waste cooking oil than the parental yeast strain. The fatty acid methyl ester (FAME) profiles of all the three mutants was determined to be suitable for biodiesel suggesting their potential applicability while simultaneously addressing the management of waste cooking oil.

Abstract Background Simultaneous wastewater treatment and lipid production by oleaginous microalgae show great potential to alleviate energy shortage and environmental pollution, because they exhibit tremendous advantages over traditional activated sludge. Currently, most research on wastewater treatment by microalgal are carried out at optimized temperature conditions (25–35 °C), but no information about simultaneous wastewater treatment and lipid production by microalgae at low temperatures has been reported. Microalgal growth and metabolism will be inhibited at low temperature conditions, and satisfactory wastewater treatment performance will be not obtained. Therefore, it is critical to domesticate and screen superior microalgal strains with low temperature adaptability, which is of great importance for wastewater treatment and biodiesel production. Results In this work, simultaneous wastewater treatment and lipid production were achieved by a microalgal mutant Scenedesmus sp. Z-4 at the low temperature conditions (4, 10, and 15 °C). The results showed that algal growth was inhibited at 4, 10, and 15 °C compared to that at the optimal temperature of 25 °C. However, decreased temperature had no significant effect on the total cellular lipid content of algae. Importantly, lipid productivity at 10 °C was compromised by more net energy output relevant to biodiesel production, which demonstrated that the low temperature of 10 °C was favorable to wastewater treatment and energy recovery by Scenedesmus sp. Z-4. When molasses wastewater with optimal COD concentration of 8000 mg L−1, initial inoculation ratio of 15%, and C/N ratio of 15 was used to cultivate microalgae, the maximum removal rate of COD, TN, and TP at 10 °C reached 87.2, 90.5, and 88.6%, respectively. In addition, lipid content of 28.9% and lipid productivity of 94.4 mg L−1 day−1 were obtained. Conclusions Scenedesmus sp. Z-4 had good adaptability to low temperature conditions, and showed great potential to realize simultaneous wastewater treatment and lipid production at low temperatures. The proposed approach in the study was simple compared to other wastewater treatment methods, and this potential novel process was still efficient to remove COD, N, and P at low temperatures. Thus, it had a vital significance for the wastewater treatment in low temperature regions.

Abstract Background Microalgae grown under different nutrient deficient conditions present a good source of natural lipids with applications for several types of biofuels. The expression of acetyl-CoA carboxylase gene can further provide an insight to the mechanisms leading to enhanced lipid production under such stresses. In this study, two nutrients viz. nitrogen and phosphorus were modulated to see its effect on lipid productivity in selected cyanobacteria and its correlation with Accase followed by molecular dynamics simulation. Results Selected cyanobacteria viz. Oscillatoria sp. (SP8), Anabaena sp. (SP12), Anabaena sp. (SP13), Microcoleus sp. (SP18), and Nostoc sp. (SP20) varied in their ability to accumulate lipids which ranged from a lowest of 0.13% in Anabaena sp. (SP13) to the maximum of 7.24% in Microcoleus sp. (SP18). Microcoleus sp. (SP18) also recorded highest lipid accumulation at both N (6 mM NaNO3) and P (0.20 mM K2HPO4) limiting conditions. The overall expression of accD was found to be upregulated in both Oscillatoria sp. (SP8) and Microcoleus sp. (SP18) for all nitrogen concentrations but was differentially regulated with both positive and negative induction under phosphorus stress conditions. Maximum induction was observed in Microcoleus sp. (SP18) at 0.20 mM K2HPO4. The obtained 3D structure of SP8 protein (21.8 kDa) showed six alpha helices, while SP18 protein (16.7 kDa) exhibited four alpha helices and four beta sheets. The phi (ϕ)/psi(ψ) angles of the amino acid residues observed in Ramachandran plot analysis showed that both SP8 and SP18 proteins were highly stable with more than 90% amino acids in allowed regions. The molecular dynamics simulation results also indicated the stability of ligand-bound protein complexes. Conclusion It has been demonstrated that cyanobacterial isolates are affected differently by nutrient limitation leading to variation in their lipid productivity. The same has been revealed by the behavior of accD gene expression which was regulated more by nutrients concentrations rather than the organism. However, the ligand-bound protein complexes were stable throughout MD simulations.

The genome-scale metabolic model of a lipid-overproducing strain of Mucor circinelloides WJ11 was developed. The model (iNI1159) contained 1,159 genes, 648 EC numbers, 1,537 metabolites, and 1,355 metabolic reactions, which were localized in different compartments of the cell. Using flux balance analysis (FBA), the iNI1159 model was validated by predicting the specific growth rate. The metabolic traits investigated by phenotypic phase plane analysis (PhPP) showed a relationship between the nutrient uptake rate, cell growth, and the triacylglycerol production rate, demonstrating the strength of the model. A putative set of metabolic reactions affecting the lipid-accumulation process was identified when the metabolic flux distributions under nitrogen-limited conditions were altered by performing fast flux variability analysis (fastFVA) and relative flux change. Comparative analysis of the metabolic models of the lipid-overproducing strain WJ11 (iNI1159) and the reference strain CBS277.49 (iWV1213) using both fastFVA and coordinate hit-and-run with rounding (CHRR) showed that the flux distributions between these two models were significantly different. Notably, a higher flux distribution through lipid metabolisms such as lanosterol, zymosterol, glycerolipid and fatty acids biosynthesis in iNI1159 was observed, leading to an increased lipid production when compared to iWV1213. In contrast, iWV1213 exhibited a higher flux distribution across carbohydrate and amino acid metabolisms and thus generated a high flux for biomass production. This study demonstrated that iNI1159 is an effective predictive tool for the pathway engineering of oleaginous strains for the production of diversified oleochemicals with industrial relevance.