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Highlights • An efficient way of genome editing using CRISPR-Cas9 in microalgae. • Knockout of a key effector gene in Lands cycle have increased in TAG accumulation. • Increase in overall lipid productivity of the green alga, Chlamydomonas reinhardtii. • Upstream process for reducing the production cost of the microalgal biofuel. Abstract Biofuel derived from microalgae have several advantages over other oleaginous crops, however, still needs to be improved with its cost aspect and can be achieved by developing of a strain with improved lipid productivity. In this study, the CRISPR-Cas9 system was incorporated to carry out a target-specific knockout of the phospholipase A 2 gene in Chlamydomonas reinhardtii. The targeted gene encodes a key enzyme in the Lands cycle. As a result, the mutants showed a characteristic of increased diacylglycerol pool, followed by a higher accumulation of triacylglycerol without being significantly compensated with the cell growth. As a result, the overall lipid productivities of phospholipase A 2 knockout mutants have increased by up to 64.25% (to 80.92 g L−1 d−1). This study can provide crucial information for the biodiesel industry. [ABSTRACT FROM AUTHOR]

Graphical abstract Highlights • Microalgae incubation after PEF-treatment improves water-soluble components release. • Microalgae incubation after PEF-treatment improves lipid extraction yields. • With 20 h of incubation, energy of PEF-treatment can be reduced to 0.25 MJ/kg DW. • Incubation is also efficient at 4 °C but less than at 25 °C. Abstract Pulsed Electric Field (PEF) pre-treatment, applied on fresh microalgae Auxenochlorella protothecoides, induces spontaneous release of a substantial water fraction and enables subsequent lipid extraction using ethanol-hexane blends. In this study, fresh microalgae suspensions were treated with PEF and incubated under inert conditions. Incubation promotes the release of ions and carbohydrates and increases the yields of subsequent lipid extraction thus enabling a considerable reduction of PEF-treatment energy. With a 20 h incubation period at 25 °C, almost total lipid extraction is achieved with a specific PEF-treatment energy of only 0.25 MJ/kg DW. Incubation on ice remains beneficial but less efficient than at 25 °C. Additionally, incubating microalgae cells in suspension at 100 g DW /L or in a dense paste, was almost equally efficient. Correlation between the different results suggests that spontaneous release of ions and carbohydrates facilitates more successful lipid extraction. A direct causality between the two phenomena remains to be demonstrated. [ABSTRACT FROM AUTHOR]

Acetate and ammonium were used as organic carbon and nitrogen sources, respectively, during mixotrophic cultivation of Chlorella pyrenoidosa . Cell growth, content of neutral lipid (NL), productivity of biomass and total lipid, and fatty acid profiles were investigated. Results showed that C. pyrenoidosa could endure high concentrations of NH 4 + -N (100–200 mg/L) and immediately entered logarithmic growth, when the culture media contained 2.0–10.0 g/L NaAc. The 2.0–10.0 g/L NaAc in the media also resulted in the NL content of 1.87–3.05 mg/10 9 cells, much higher than 0.5 mg/10 9 cells of the controls. The maximum productivities of biomass and total lipid were achieved under 50 and 10 mg/L NH 4 + -N respectively when the 2.0 g/L NaAc was dosed. The fatty acids were mainly composed of C16:0, C16:1, C18:0, and C18:1 under the mixotrophic cultivation, with the higher saturation compared to the controls. [ABSTRACT FROM AUTHOR]

Heterotrophic microalgae, capable of converting organic carbons to biofuel, as well as assimilating nutrients, have a great prospective in wastewater treatment. Meanwhile, the knowledge about heterotrophic microalgae is still far less than the autotrophic conterpart. Hence, in this study, 20 heterotrophic microalgal strains were isolated from a domestic wastewater treatment plant, and identified according to morphology and partial 18S and 23S rRNA gene sequences. Further, their biological traits were assessed in terms of N, P, TOC removal efficiencies, growth parameters, self-settleability and lipids production, expressed through a comprehensive selection index. By such, the optimal strains were chosen and applied back to treat the real wastewater, with or without pretreatment of sterilization. An organic-adaptable strain, i.e., Botryococcus sp. NJD-1, was ultimately recommended to achieve the concurrent biofuel production (up to 61.7% lipid content) and pollutants removal (up to 64.5%, 89.8% and 67.9% for N, P and TOC) in pristine wastewater. [ABSTRACT FROM AUTHOR]

The present study was conducted with microalga Nannochloropsis sp. to evaluate its tolerance responses and biofuel perspectives under different arsenic regime (0–1000 μM As(III)). Results showed that optimal biomass (61.00 ± 3.72 mg/L/d) and lipid productivity (20.27 mg/L/d) were obtained in culture treated with 100 μM As(III) in comparison to other treatments. In addition, fatty acid profile of alga was in accordance with European biodiesel standards (EN 14214), which reflects good oxidative stability of oil. High antioxidants viz., ascorbic acid, GSH and cysteine tolerance responses as well as lipid yield at 100 μM As(III), opens a new insight in the field of algal biology. Thus, microalgae Nannochloropsis sp. may be employ in remediation as well as biodiesel production. [ABSTRACT FROM AUTHOR]

High moisture content in wet algal biomass hinders effective performance of current lipid extraction methods. An improved aqueous extraction method combing thermal and enzymatic lysis was proposed and performed in algal slurry of Nannochloropsis oceanica (96.0% moisture) in this study. In general, cell-wall of N. oceanica was disrupted via thermal lysis and enzymatic lysis and lipid extraction was performed using aqueous surfactant solution. At the optimal conditions, high extraction efficiencies for both lipid (88.3%) and protein (62.4%) were obtained, which were significantly higher than those of traditional hexane extraction and other methods for wet algal biomass. Furthermore, an excessive extraction of polar lipid was found for wet biomass compared with dry biomass. The advantage of this method is to efficiently extract lipids from high moisture content algal biomass and avoid using organic solvent, indicating immense potential for commercial microalgae-based biofuel production. [ABSTRACT FROM AUTHOR]

High moisture content in wet algal biomass hinders effective performance of current lipid extraction methods. An improved aqueous extraction method combing thermal and enzymatic lysis was proposed and performed in algal slurry of Nannochloropsis oceanica (96.0% moisture) in this study. In general, cell-wall of N. oceanica was disrupted via thermal lysis and enzymatic lysis and lipid extraction was performed using aqueous surfactant solution. At the optimal conditions, high extraction efficiencies for both lipid (88.3%) and protein (62.4%) were obtained, which were significantly higher than those of traditional hexane extraction and other methods for wet algal biomass. Furthermore, an excessive extraction of polar lipid was found for wet biomass compared with dry biomass. The advantage of this method is to efficiently extract lipids from high moisture content algal biomass and avoid using organic solvent, indicating immense potential for commercial microalgae-based biofuel production. [ABSTRACT FROM AUTHOR]

Taxonomy and phylogeny of twenty two microalgal isolates were examined using both universal and newly designed molecular primers. Among the isolates, Scenedesmus bijugus , Coelastrella sp., Auxenochlorella protothecoides , and Chlorella sp. were particularly promising in terms of producing lipids as measured by fatty acid methyl esters (FAME) analysis and significant concentration of carotenoids. A comparative experiment showed that S. bijugus and Chlorella sp. were the most promising candidates (L − 1 d − 1 , with biomass) 174.77 ± 6.75, 169.81 ± 5.22 mg, lipids 40.14 ± 3.31, 39.72 ± 3.89 mg, lutein 0.47, 0.36 mg, and astaxanthin 0.27, 0.18 mg respectively. The fatty acids produced by these microalgal isolates were mainly palmitic, stearic, oleic, linoleic, and linolenic acid. The freshwater microalgal isolate S. bijugus be the most suitable isolate for producing biodiesel and carotenoids, due to high productivity of biomass, lipids, metabolites, and its suitable fatty acid profile. [ABSTRACT FROM AUTHOR]

Biofuel production from microalgae requires energy efficient processes for extracting and converting triacylglyceride lipids to fuel, compatible with coproduction of protein feeds and nutraceuticals. Wet solvent extraction involves mechanical cell rupture, lipid extraction via solvent contacting, physical phase separation, thermal solvent recovery, and transesterification. A detailed analysis of the effect of key process parameters on the parasitic energy demand of this process was performed. On a well-to-pump basis, between 16% and 320% of the resultant biodiesel energy was consumed depending solely on the process parameters. Highly positive energy balances can be achieved, but only if a correctly designed process is used. This requires processing concentrated biomass ( ca 25% w/w) with a high triacylglyceride content ( ca 30% w/w), and an efficient extraction process employing a non-polar solvent, low solvent-to-paste ratio, and efficient energy recovery. These requirements preclude many laboratory scale processes and polar co-solvents as viable options for large-scale biofuel production. [ABSTRACT FROM AUTHOR]

This study presents gravimetric enrichment of mixed culture to screen starch and lipid producing species separately in a sequencing batch reactor. In the enriched starch-producing mixed culture photobioreactor, the starch content at the end of steady state batch became 3.42 times the beginning of depletion. Whereas in the enriched lipid-producing photobioreactor, the lipid content at the end of steady state batch became 3 times the beginning of famine phase. The obtained results revealed that the gravimetric enrichment is a suitable screening method for specific production of storage compounds in none-sterile large-scaled condition. [ABSTRACT FROM AUTHOR]

[Display omitted] • Intrinsic interactions between lipid feedstocks and lipid-based biofuels. • Advancements in lipid-associated biofuel technology. • Properties and applicability of various lipid sources for biofuel production. • Biotechnology and progress in lipid production for advanced biofuels. The expanding use of fossil fuels has caused concern in terms of both energy security and environmental issues. Therefore, attempts have been made worldwide to promote the development of renewable energy sources, among which biofuel is especially attractive. Compared to other biofuels, lipid-derived biofuels have a higher energy density and better compatibility with existing infrastructure, and their performance can be readily improved by adjusting the chemical composition of lipid feedstocks. This review thus addresses the intrinsic interactions between lipid feedstocks and lipid-based biofuels, including biodiesel, and renewable equivalents to conventional gasoline, diesel, and jet fuel. Advancements in lipid-associated biofuel technology, as well as the properties and applicability of various lipid sources in terms of biofuel production, are also discussed. Furthermore, current progress in lipid production and profile optimization in the context of plant lipids, microbial lipids, and animal fats are presented to provide a wider context of lipid-based biofuel technology. [ABSTRACT FROM AUTHOR]

This study presents experimental measurements of the biochemical methane production for whole and lipid extracted Nannochloropsis salina . Results show whole microalgae produced 430 cm 3 -CH 4 g-volatile solids −1 (g-VS) ( σ = 60), 3 times more methane than was produced by the LEA, 140 cm 3 -CH 4 g-VS −1 ( σ = 30). Results illustrate current anaerobic modeling efforts in microalgae to biofuel assessments are not reflecting the impact of lipid removal. On a systems level, the overestimation of methane production is shown to positively skew the environmental impact of the microalgae to biofuels process. Discussion focuses on a comparison results to those of previous anaerobic digestion studies and quantifies the corresponding change in greenhouse gas emissions of the microalgae to biofuels process based on results from this study. [ABSTRACT FROM AUTHOR]

Highlights: [•] Thermal hydrolysis–pyrolysis was used to produce hydrocarbon from inedible lipids. [•] Feedstock fatty acid composition did not influence organic liquid product yield. [•] Organic liquid product had compounds in the gasoline and diesel boiling range. [•] The organic liquid product mostly met the CGSB specification for diesel fuel. [ABSTRACT FROM AUTHOR]

Highlights: [•] Environmental factors were proved to be helpful to lipid accumulation. [•] NaCl, Fe3+ and nitrogen starvation together made lipid content increase to 2.5 times. [•] Both high and low temperature could promote lipid accumulation. [•] UTEX 2341 was proved to be a potential species for high-quality biodiesel production. [ABSTRACT FROM AUTHOR]

Highlights: [•] Dielectric spectroscopy (DES) was used to detect algal lipid accumulation. [•] DES could be automated for real-time industrial algal lipid monitoring. [•] A transmission line model of cell suspension permittivity was developed. [•] This initial model correctly predicts observed changes in dielectric properties. [Copyright &y& Elsevier]

[Display omitted] • Lipids in engineered cellulosic feedstocks can be source for biodiesel. • Hydrothermal pretreatment (170 °C, 20 min) was found to be optimal. • Enzymatic hydrolysis allows 79% glucose and 86% xylose recovery. • Pretreatment and enzymatic hydrolysis results in 2-fold increase in lipid. • Triglycerides and free fatty acids were observed in enzymatic residue and liquor. Lipids accumulated in the vegetative tissues of cellulosic feedstocks can be a potential raw material for biodiesel and bioethanol production. In this work, bagasse of genetically engineered sorghum was subjected to liquid hot-water pretreatment at 170, 180, and 190 °C for different reaction time. Under the optimal pretreatment condition (170 °C, 20 min), the residue was enriched in glucan (57.39 ± 2.63 % w/w) and xylan (13.38 ± 0.49 % w/w). The total lipid content of the pretreated residue was 6.81% w/w, similar to that observed in untreated bagasse (6.30% w/w). Pretreatment improved the enzymatic digestibility of bagasse, allowing a recovery of 79% w/w and 86% w/w of glucose and xylose, respectively. The pretreatment and enzymatic saccharification resulted in a 2-fold increase in total lipid in enzymatic residue compared to the original bagasse. Thus, pretreatment and enzymatic hydrolysis enabled high sugar recovery while concentrating triglycerides and free fatty acids in the residue. [ABSTRACT FROM AUTHOR]

Abstract: Nitrogen deficiency promotes lipid formation in many microalgae, but also limits growth and lipid productivity. In spite of numerous studies, there is poor understanding of the interactions of growth and lipid content, the time course of lipid accumulation and the magnitude of nitrogen deficiency required to stimulate lipid formation. These relationships were investigated in six species of oleaginous green algae, comparing high and low levels of deficiency. Nitrogen stress typically had disproportionate effects on growth and lipid content, with profound differences among species. Optimally balancing the tradeoffs required a wide range in nitrogen supply rate among species. Some species grew first and then accumulated lipids, while other species grew and accumulated lipids concurrently which resulted in increased lipid productivity. Accumulation of high lipid content generally resulted from a response to minimal stress. The data highlight the tremendous biodiversity that may be exploited to optimally produce lipids with precision nitrogen stress. [Copyright &y& Elsevier]

Abstract: A switchable hydrophilicity solvent (SHS) was studied for its effectiveness at extracting lipids from freeze-dried samples of Botryococcus braunii microalgae. The SHS N,N-dimethylcyclohexylamine extracted up to 22wt.% crude lipid relative to the freeze-dried cell weight. The solvent was removed from the extract with water saturated with carbon dioxide at atmospheric pressure and recovered from the water upon de-carbonation of the mixture. Liquid chromatography–mass spectrometry (LC–MS) showed that the extracted lipids contained high concentrations of long chain tri-, di- and mono-acylglycerols, no phospholipids, and only 4–8% of residual solvent. Unlike extractions with conventional organic solvents, this new method requires neither distillation nor the use of volatile, flammable or chlorinated organic solvents. [Copyright &y& Elsevier]

Abstract: Despite claims that microalgal biofuels are environmentally friendlier alternatives to conventional fuels, debate surrounding its ecological benefits or drawbacks still exists. LCA is used to analyze various biofuel production technologies from ‘cradle to gate’. Energy and CO2 balances are carried out for a hypothetical integrated PBR-raceway microalgae-to-biodiesel production in Singapore. Based on a functional unit of 1MJ biofuel, the total energy demands are 4.44MJ with 13% from biomass production, 85% from lipid extraction, and 2% from biodiesel production. Sensitivity analysis was carried out for adjustments in energy requirements, percentage lipid contents, and lower/higher heating product value. An ‘Optimistic Case’ was projected with estimates of: 45% lipid content; reduced energy needs for lipid extraction (1.3MJ per MJ biodiesel); and heating value of biodiesel (42MJ/kg). The life cycle energy requirements dropped significantly by about 60%. The results are compared with other published case studies from other countries. [Copyright &y& Elsevier]

Abstract: The scalability of microalgae growth systems is a primary research topic in anticipation of the commercialization of microalgae-based biofuels. To date, there is little published data on the productivity of microalgae in growth systems that are scalable to commercially viable footprints. To inform the development of more detailed assessments of industrial-scale microalgae biofuel processes, this paper presents the construction and validation of a model of microalgae biomass and lipid accumulation in an outdoor, industrial-scale photobioreactor. The model incorporates a time-resolved simulation of microalgae growth and lipid accumulation based on solar irradiation, species specific characteristics, and photobioreactor geometry. The model is validated with 9weeks of growth data from an industrially-scaled outdoor photobioreactor. Discussion focuses on the sensitivity of the model input parameters, a comparison of predicted microalgae productivity to the literature, and an analysis of the implications of this more detailed growth model on microalgae biofuels lifecycle assessment studies. [Copyright &y& Elsevier]

Abstract: A 1.2kW, 2450MHz resonant continuous microwave processing system was designed and optimized for oil extraction from green algae (Scenedesmus obliquus). Algae-water suspension (1:1 w/w) was heated to 80 and 95°C, and subjected to extraction for up to 30min. Maximum oil yield was achieved at 95°C and 30min. The microwave system extracted 76–77% of total recoverable oil at 20–30min and 95°C, compared to only 43–47% for water bath control. Extraction time and temperature had significant influence (p <0.0001) on extraction yield. Oil analysis indicated that microwaves extracted oil containing higher percentages of unsaturated and essential fatty acids (indicating higher quality). This study validates for the first time the efficiency of a continuous microwave system for extraction of lipids from algae. Higher oil yields, faster extraction rates and superior oil quality demonstrate this system’s feasibility for oil extraction from a variety of feedstock. [ABSTRACT FROM AUTHOR]

Abstract: In vivo determination of neutral lipids with Nile red fluorescence has been used as a rapid screening method for certain types of microalgae, but has been unsuccessful in others, particularly those with thick, rigid cell walls that prevent penetration of the fluorescence dye into the cell. To solve the problem, a microwave-assisted Nile red staining method for microalgal lipid determination was developed. In a two-step staining protocol, 50 and 60s were selected as the optimal microwave times for the pretreatment and staining process, respectively. Moreover, several calibration methods for quantitative analysis of neutral lipids in microalgae were investigated and compared with conventional gravimetric methods. Factors that affected the in vivo quantification of cellular neutral lipids were also investigated. Application of the new method for detection and quantification of neutral lipids in a number of green microalgae was demonstrated. [ABSTRACT FROM AUTHOR]

Abstract: This article proposes a dynamical model of microalgal lipid production under nitrogen limitation. In this model, intracellular carbon is divided between a functional pool and two storage pools (sugars and neutral lipids). The various intracellular carbon flows between these pools lead to a complex dynamic with a strong discrepancy between synthesis and mobilization of neutral lipids. The model has been validated with experiments of Isochrysis aff. galbana (clone T-iso) culture under various nitrogen limitation conditions and under nitrogen starvation. The hysteresis behavior of the neutral lipid quota observed experimentally is accurately predicted. [ABSTRACT FROM AUTHOR]

Abstract: Photosynthetic carbon partitioning into starch and neutral lipid was investigated in the oleaginous green microalga Pseudochlorococcum sp. When grown under low light and nitrogen-replete conditions, the algal cells possessed a basal level of starch. When grown under high light and nitrogen-limited conditions, starch synthesis was transiently up-regulated. After nitrogen depletion, starch content decreased while neutral lipid rapidly increased to 52.1% of cell dry weight, with a maximum neutral lipid productivity of 0.35gL−1 D−1. These results suggest that Pseudochlorococcum used starch as a primary carbon and energy storage product. As nitrogen was depleted for an extended period of time, cells shift the carbon partitioning into neutral lipid as a secondary storage product. Partial inhibition of starch synthesis and degradation enzymes resulted in a decrease in neutral lipid content, indicating that conversion of starch to neutral lipid may contribute to overall neutral lipid accumulation. Biotechnological application of Pseudochlorococcum sp. as a production strain for biofuel was assessed. [ABSTRACT FROM AUTHOR]

Abstract: Global petroleum reserves are shrinking at a fast pace, increasing the demand for alternate fuels. Microalgae have the ability to grow rapidly, and synthesize and accumulate large amounts (approximately 20–50% of dry weight) of neutral lipid stored in cytosolic lipid bodies. A successful and economically viable algae based biofuel industry mainly depends on the selection of appropriate algal strains. The main focus of bioprospecting for microalgae is to identify unique high lipid producing microalgae from different habitats. Indigenous species of microalgae with high lipid yields are especially valuable in the biofuel industry. Isolation, purification and identification of natural microalgal assemblages using conventional techniques is generally time consuming. However, the recent use of micromanipulation as a rapid isolating tool allows for a higher screening throughput. The appropriate media and growth conditions are also important for successful microalgal proliferation. Environmental parameters recorded at the sampling site are necessary to optimize in vitro growth. Identification of species generally requires a combination of morphological and genetic characterization. The selected microalgal strains are grown in upscale systems such as raceway ponds or photobireactors for biomass and lipid production. This paper reviews the recent methodologies adopted for site selection, sampling, strain selection and identification, optimization of cultural conditions for superior lipid yield for biofuel production. Energy generation routes of microalgal lipids and biomass are discussed in detail. [ABSTRACT FROM AUTHOR]

Abstract: The potential of microalgae as a source of renewable energy has received considerable interest, but if microalgal biofuel production is to be economically viable and sustainable, further optimization of mass culture conditions are needed. Wastewaters derived from municipal, agricultural and industrial activities potentially provide cost-effective and sustainable means of algal growth for biofuels. In addition, there is also potential for combining wastewater treatment by algae, such as nutrient removal, with biofuel production. Here we will review the current research on this topic and discuss the potential benefits and limitations of using wastewaters as resources for cost-effective microalgal biofuel production. [ABSTRACT FROM AUTHOR]

Abstract: Biofuels made from algal biomass are being considered as the most suitable alternative energy in current global and economical scenario. Microalgae are known to produce and accumulate lipids within their cell mass which is similar to those found in many vegetable oils. The efficient lipid producer algae cell mass has been reported to contain more than 30% of their cell weight as lipids. According to US DOE microalgae have the potential to produce 100 times more oil per acre land than any terrestrial plants. This article reviews up to date literature on the composition of algae, mechanism of oil droplets, triacylglycerol (TAG) production in algal biomass, research and development made in the cultivation of algal biomass, harvesting strategies, and recovery of lipids from algal mass. The economical challenges in the production of biofuels from algal biomass have been discussed in view of the future prospects in the commercialisation of algal fuels. [ABSTRACT FROM AUTHOR]

Abstract: In this study Fourier transform infrared micro-spectroscopy (FTIR) was used to determine lipid and carbohydrate content over time in the freshwater microalgae Chlamydomonas reinhardtii and Scenedesmus subspicatus grown in batch culture in limiting concentrations of nitrogen (N). Both algae exhibited restricted cell division and increased cell size following N-limitation. FTIR spectra of cells in N-limited media showed increasing lipid:amide I and carbohydrate:amide I ratios over time. The use of lipid- and starch-staining dyes confirmed that the observed ratio changes were due to increased lipid and carbohydrate synthesis. These results demonstrate rapid metabolic responses of C. reinhardtii and S. subspicatus to changing nutrient availability, and indicate the efficiency of FTIR as a reliable method for high-throughput determination of lipid induction. [Copyright &y& Elsevier]

Abstract: Lipid extraction is a critical step in the development of biofuels from microalgae. Here a new procedure was proposed to extract hydrocarbons from dried and water-suspended samples of the microalga Botryococcus braunii by using switchable-polarity solvents (SPS) based on 1,8-diazabicyclo-[5.4.0]-undec-7-ene (DBU) and an alcohol. The high affinity of the non-ionic form of DBU/alcohol SPS towards non-polar compounds was exploited to extract hydrocarbons from algae, while the ionic character of the DBU-alkyl carbonate form, obtained by the addition of CO2, was used to recover hydrocarbons from the SPS. DBU/octanol and DBU/ethanol SPS were tested for the extraction efficiency of lipids from freeze-dried B. braunii samples and compared with n-hexane and chloroform/methanol. The DBU/octanol system was further evaluated for the extraction of hydrocarbons directly from algal culture samples. DBU/octanol exhibited the highest yields of extracted hydrocarbons from both freeze-dried and liquid algal samples (16% and 8.2% respectively against 7.8% and 5.6% with n-hexane). [Copyright &y& Elsevier]

• The highest lipid accumulation of C. hoffmannii was obtained as 52.0%. • The maximum total C16 and C18 FAME rate were detected as 96.3%. • The first report about usage of C. hoffmannii lipids for biodiesel production. • Using carrot pomace for biolipid production is a promising approach. • Lignocellulosic materials have the highest energy return on investment value. Coniochaeta hoffmannii was isolated from soils contaminated with biscuit factory wastes showed the maximum lipid accumulation capacity in the study. Lipid production was optimized in terms of pH, carrot pomace loading, nitrogen type and amount, incubation time. Solvent, alcohol type and catalyst concentration, dried/wet biomass concentration, reaction approaches and time were optimized for lipid extraction and transesterification. The highest lipid accumulation was found as 52.0% at pH 4 in the presence of 10% carrot pomace, 0.5 g/L cheese whey at the end of the 48 h incubation. The maximum total C16 and C18 FAME rates were detected at the 25 °C, in the presence of 4 g/L dried C. hoffmannii biomass, methanol and 3% NaOH by using the in-situ transesterification process at the end of the 0.5 h as 96.3%. This is the first report about the usage of C. hoffmannii lipids obtained from carrot pomace for sustainable biodiesel production. [ABSTRACT FROM AUTHOR]