- Open Access
Heterotrophic cultivation of Auxenochlorella protothecoides using forest biomass as a feedstock for sustainable biodiesel production
© The Author(s) 2018
Received: 25 April 2018
Accepted: 12 June 2018
Published: 20 June 2018
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.
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.
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.
Progressive depletion of fossil fuels, global climate change issues, and growing demand for energy have led to a move toward an alternative, renewable, and sustainable source of energy . Among the various alternative energy sources, biofuels are a more sustainable option for transportation fuels and for the growing energy demands of industry. The worldwide biofuel market is still in its infancy . In Europe, the policies relating to climate change and renewable energy are decided by the European Union (EU). The EU has indicated that research on biofuels production using various sources of biomass could play a vital role in replacing fossil fuels until 2045 . According to the Swedish renewable energy policy, the local transport sector (exclusive of domestic aviation) should reduce the green house gas (GHG) emissions by 70% until 2030, compared to the emissions in 2010 . There has been rapid progress in global biofuel production over the last decade. However, the first-generation biofuels have raised significant concerns due to their sustainability, as their production is based on vegetable oil and food crop sources that directly compete with sources of food for humans. The use of human food stocks for biofuel production is no longer cost-effective or ethical in the present situation . In contrast to the first-generation biofuels, non-edible lignocellulosic biomasses such as agricultural and forestry residues are used as feedstocks for the production of the second-generation biofuels . This type of fuel reduces the direct competition between food and fuel. However, this strategy has again raised an important question regarding cost-effectiveness, due to the inefficient process of conversion of biomass and the low energy efficiency . To replace the direct conversion of lignocellulosic biomass to low-value fuel for the generation of heat and electricity, conversion to high-added value-quality bio-products and energy carriers has been proposed , aimed at the establishment of a bio-refinery concept. To achieve this, it is very important to make use of all the major components of lignocellulosic biomass, namely cellulose, hemicellulose, and lignin. In this scheme, the use of hybrid organosolv-steam explosion method allows the efficient fractionation of biomass, resulting in three isolated fractions that can be used in various processes.
Microbial oil sources as feedstock for the third-generation biofuels have many advantages over their counterparts. In the last decade, microalgae have emerged as a promising source for the production of renewable biofuels, as they are efficient photosynthesizers, their culture require less growth area than terrestrial plants, and they are able to channel most of their energy into cell division, which enhance biomass productivity . They can be grown on infertile land, polluted land, or arid land, so they do not compete with the use of land for food production. Most of the algal species are oleaginous and can accumulate very high quantities of intracellular lipids (> 60% w/w lipid content) under various conditions of stress such as nutrient limitation. Moreover, they have a great ability to make use of municipal wastewater and industrial effluents, which makes them potential candidates for large-scale production . Thus, biodiesel derived from lipids stored in oleaginous microalgae can reduce environmental pollution and it is a promising substitute for the conventional diesel. In addition, the biodiesel obtained from microalgae is eco-friendly, is devoid of any harmful toxic substances, and contributes less greenhouse gas emissions than fossil diesel fuel .
The main restriction to using oleaginous microalgae as feedstock for biodiesel production is their culture techniques. There are four cultivation techniques appropriate for microalgae, which can be divided into autotrophic, heterotrophic, mixotrophic, and photoheterotrophic modes of culture . Microalgae use inorganic carbon in the form of CO2 and energy from sunlight to generate organic matter when cultured using the autotrophic mode . The microalgal oil can be produced by sequestering of atmospheric CO2, which is the major advantage of this mode . Microalgae solely depend on the exogenous organic carbon sources provided for their growth in heterotrophic cultivation, while, in mixotrophic cultivation, they use both light and exogenous organic carbon as energy sources . However, in the photoheterotrophic mode of culture light is required to metabolize the external carbon (as in mixotrophic culture), but the major difference between these two modes is that carbon dioxide cannot be absorbed and metabolized as it can be in mixotrophic mode . Heterotrophic culture has many advantages over its counterparts, as it is cost-effective and comparatively easy to operate with quite low daily maintenance. Moreover, heterotrophic cultivation can be performed in any fermenter without illumination; hence, there is no requirement of photobioreactor as in case of autotrophic cultivation which finally reduces the overall production cost . A high quantity of microalgal culture (e.g., 100,000 L) grown in heterotrophic mode can generate almost 500 tons of dry biomass of Chlorella species, which is equivalent to 50% of the total Japanese production of this algae .
Assessment of various microalgae grown on different lignocellulosic biomass
Cultivation medium (pretreatment/hydrolysis)
Cell dry weight (g/L)
Lipid content (%, w/w)
Glucose (10 g/L)
Rice straw hydrolysate (acid pretreatment and enzymatic hydrolysis)
Artificial mix sugars
Sugarcane bagasse hydrolysate (enzymatic hydrolysis)
Glucose (10 g/L)
Corn powder hydrolysate (enzymatic hydrolysis)
Glucose (40 g/L)
Cassava starch hydrolysate (two-step enzymatic hydrolysis)
Hydrolysate from Cyperus esculentus waste (two-step enzymatic hydrolysis)
Glucose (30 g/L)
Cassava starch hydrolysate (hot water treatment followed by enzymatic hydrolysis)
Corn powder hydrolysate (enzymatic hydrolysis)
Glucose (10 g/L)
Fructose (10 g/L)
Sucrose (10 g/L)
Sweet Sorghum hydrolysate (acid hydrolysis in autoclave followed by enzymatic hydrolysis)
Waste molasses hydrolysate N limited medium (enzymatic hydrolysis)
Waste molasses hydrolysate direct medium (enzymatic hydrolysis)
Sugarcane juice hydrolysate (enzymatic hydrolysis)
Glucose (20 g/L)
Jerusalem artichoke (enzymatic hydrolysis)
Glucose (20 g/L) C/N;60
OPBH (C/N; 60) (organosolv-steam explosion followed by enzymatic hydrolysis)
OPSH (C/N; 60) (organosolv-steam explosion followed by enzymatic hydrolysis)
Results and discussion
Effect of initial glucose concentration on the growth and lipid accumulation of the oleaginous microalga A. protothecoides
Among the various substrates that can be provided, glucose is a preferred carbon source for maintenance of the heterotrophic growth of oleaginous microalgae, as it has higher energy content per mol (~ 2.8 kJ/mol) than other substrates such as acetate (~ 0.8 kJ/mol) . To determine the effect of the initial concentration of glucose on the cell dry weight and lipid accumulation in the oleaginous microalga A. protothecoides, five different concentrations of glucose ranging from 20 to 100 g/L were included in the basal medium (BBM), which contained yeast extract as nitrogen source. The C/N (g/g) ratio was kept at 20 for each flask and the flasks were inoculated with a 10% volume of exponentially growing seed culture.
Growth of A. protothecoides on various initial glucose concentrations
Initial glucose concentration (g/L) in GSM
Cell dry weight (g/L)
Lipid concentration (g/L)
Lipid content (%, w/w)
Biomass yield (g/gsubstrate)
Lipid yield (g/gsubstrate)
Residual glucose concentration (g/L)
8.18 ± 0.34
2.70 ± 0.12
33.00 ± 0.52
0.456 ± 0.008
0.150 ± 0.004
2.05 ± 0.07
7.30 ± 0.48
1.68 ± 0.19
23.01 ± 0.84
0.267 ± 0.016
0.061 ± 0.009
12.66 ± 0.17
8.22 ± 0.21
1.28 ± 0.09
15.57 ± 0.59
0.296 ± 0.007
0.046 ± 0.002
32.26 ± 0.23
9.54 ± 0.19
1.58 ± 0.17
16.56 ± 0.43
0.314 ± 0.006
0.052 ± 0.001
49.65 ± 0.41
10.92 ± 0.32
2.04 ± 0.23
18.68 ± 0.76
0.319 ± 0.011
0.059 ± 0.001
65.78 ± 0.37
With the same oleaginous microalga C. protothecoides, it has been found that 3.7 g/L cell dry weight and 53.3% w/w lipid content could be obtained after 120 h of culture when the initial glucose concentration was 10 g/L . In comparison, C. protothecoides has been found to give 0.92 g/L cell dry weight with 50.3% w/w lipid content after 60 h when grown on 10 g/L glucose . In another work, this microalga produced 10.7 g/L dry biomass with 30.7% w/w of lipid content after 240 h of cultivation, when grown on 40 g/L glucose . According to the above observations, the dry biomass and lipid content of microalgal species are not only dependent on the initial concentration of sugar but also vary with the other medium components and the culture conditions provided. The selection of suitable candidate organisms and optimization of the medium for their growth and accumulation of lipid are important factors for biodiesel production from oleaginous microorganisms, as these are the very first steps to achieving this goal. Several microalgal species are well known for producing specific classes of fatty acids in their cellular compartments through simple adjustment of their culture medium . These unusual and valuable lipids from microalgal species have been shown to be a significant contribution to various industrial applications . The synthesis of different groups of fatty acids in any oleaginous microorganisms (including microalgae) depends on various factors such as culture temperature; mode of culture (autotrophic, mixotrophic, or heterotrophic); concentration, and ratio of the carbon, nitrogen, and phosphorus sources; pH, and mainly the strain of the microalga . It has been reported that C. protothecoides can synthesize four times as much lipid (57.9%, w/w) when it is grown under heterotrophic conditions than under autotrophic conditions [21, 34].
Effect of various C/N (g/g) ratios on the accumulation of lipid in A. protothecoides
Time course experiment with A. protothecoides grown on hydrolysates from organosolv-steam explosion pretreated birch and spruce biomass
Different factors, such as the crystallinity and the degree of polymerization of cellulose, the surface area accessible, and the quantity and degree of acetylation of hemicellulose and lignin severely affect the enzymatic hydrolysis of lignocellulosic biomass, so a pretreatment step is required before hydrolysis . There are usually four categories of pretreatment such as chemical, physical, biological, and physicochemical which are used to overcome the complications listed above . Organosolv is a pretreatment method, whereby the biomass is cooked at high temperature (100‒250 °C) in the presence of organic solvents (mainly ethanol), aimed at removing the lignin and hemicellulose into the liquid phase while producing a cellulose-rich solid fraction . Lignin can easily be recovered from the liquid fraction as a solid with high purity, resulting in the generation of three relatively pure fractions. An important and advantageous feature of this method is the ability to recover the organic solvent by distillation, because of the low boiling point, and to use it again for pretreatment . However, organosolv is effective for delignification of biomass but offers poor biomass deconstruction, while steam explosion is effective for the fractionation of biomass, so, in the current work, we used a hybrid organosolv—steam explosion method for the efficient fractionation and pretreatment of birch and spruce biomass .
Assessment of biomass and lipid accumulation in heterotrophically cultivated A. protothecoides
GSM (C/N, 60)
OPBH (C/N, 60)
OPSH (C/N, 60)
Cell dry weight (g/L)
8.40 ± 0.12
8.56 ± 0.21
8.37 ± 0.13
Biomass productivitya (g/L day)
1.68 ± 0.09
1.71 ± 0.07
1.67 ± 0.08
Total lipid concentration (g/L)
5.42 ± 0.27
5.65 ± 0.21
5.28 ± 0.17
Lipid content (%, w/w)
64.52 ± 0.64
66.00 ± 0.33
63.08 ± 0.71
Lipid productivitya (mg/L day)
1084 ± 14
1130 ± 24
1056 ± 21
The commercial aspect of biodiesel production from oleaginous microalgae in heterotrophic cultivation is limited by the cost of feedstocks, as this accounts for 60‒85% of the total production cost . Researchers are interested in using lignocellulosic biomass for the cultivation of microalgae, but, till now, only agricultural residues and easily hydrolysable starch-based carbon sources have been used, such as corn powder, sweet sorghum juice, and cassava starch (Table 1), and the use of forest biomass for heterotrophic cultivation of microalgae has not yet appeared in the literature . Woody lignocellulosic biomass obtained from forests remains an important feedstock for the heterotrophic cultivation of microalgae . The forest residues such as top thin branches, trimmings, small trees, and un-merchantable wood are one of the largest available feedstocks on earth; they are often left over in the forest or used for the low-cost production of energy and heating by burning .
Biochemical and morphological changes in A. protothecoides grown on various substrates
The importance of identifying the level of the pigments in oil feedstocks lies in the fact that pigments make the oil more susceptible to photo-oxidation, which further decreases the storage stability of the oil . Moreover, the presence of pigments interferes with the transesterification reaction as well as the combustion of biodiesel, decreasing their efficiency . Therefore, it is crucial to remove the chlorophyll from the microalgal oil feedstocks before processing them through transesterification reaction. There are several conventional methods already processed for the removal of chlorophyll from the oil feedstocks including physical absorption, oxidation treatment, and phosphoric acid degumming, with their use to further increase the overall production cost of biodiesel . In this context, the present study of heterotrophically cultivated microalgae at high C/N ratio showed minimum amount of pigments compared to those grown autotrophically at low C/N ratio, avoiding the time-consuming and costly removal of the pigments prior to biodiesel formation.
Estimation of the quantity of triacylglycerol (TAG) in total lipid extracted from A. protothecoides grown in GSM, OPBH, and OPSH, using TLC
Microalgae synthesize a diverse range of lipids (neutral lipids, glycolipids, and phospholipids) to perform various physiological functions. Glycolipids and phospholipids take part in the synthesis of the external membrane and the membranes of the chloroplast and the endoplasmic reticulum, while triacylglycerol is mainly neutral lipid stored in the form of lipid droplets in cellular compartments that are used to produce biodiesel by transesterification reaction. It has been reported that for various oleaginous microalgae grown under nitrogen-limiting conditions, the molecular mechanisms are shifted to accumulate large quantities of triacylglycerol instead of synthesizing cellular protein .
Estimation of fatty acid profile and biodiesel properties
Analysis of lipid profile of heterotrophically cultivated A. protothecoides by gas chromatography
GSM (C/N, 60)
OPBH (C/N, 60)
OPSH (C/N, 60)
Saturated fatty acid (SFA)
Mono unsaturated fatty acid (MUFA)
Poly unsaturated fatty acid (PUFA)
Assessment of biodiesel properties by empirical formulas
GSM; C/N 60
OPBH; C/N 60
OPSH; C/N 60
Long chain saturation factor
Oxidative stability, 110 °C
Cold filter plugging point
High heating value
Strain and culture conditions
Auxenochlorella protothecoides SAG 211-7a was obtained from the culture collection of algae (SAG) at Göttingen University, Germany, and it was maintained at 16 °C on agar plates containing Bold’s basal medium (BBM). It was initially grown autotrophically and axenically in a photobioreactor (Multi-Cultivator MC 1000-OD; Photon Systems Instruments, Czech Republic) containing BBM and yeast extract (3.35 g/L) as nitrogen source under 18/6 h light/dark regimen (intensity of 43 μmol/m2 s) at 25 ± 1 °C. Aeration was provided by bubbling air through at normal pressure. For heterotrophic culture, cells were harvested from the photobioreactor by centrifugation, washed twice with sterile distilled water, and resuspended in 0.9% sterilized saline to obtain a cell density of 6.9–9.2 × 108 cells/mL. For the preparation of inoculum, A. protothecoides was grown in 500-mL Erlenmeyer flasks containing 200 mL medium at 25 °C in an incubator in the dark with continuous shaking (180 rpm). BBM was used as basal medium supplemented with glucose (20 g/L) and yeast extract (3.35 g/L) was used as a source of nitrogen to achieve the desired C/N ratio. To determine the effect of the initial concentration of glucose on the biomass and lipid concentration of A. protothecoides, five different glucose concentrations (20, 40, 60, 80, and 100 g/L) were added with BBM in the GSM. The appropriate concentration of yeast extract was added to each flask to achieve a C/N ratio of 20. Optimization of the initial carbon concentration for maximum biomass was followed by optimization of lipid accumulation with various C/N ratios (20, 40, 60, 80, and 100) by varying the concentrations of yeast extract at the concentration of carbon (glucose) that was found to be optimal. After optimization of carbon and nitrogen concentrations for maximum biomass and lipid accumulation by A. protothecoides, OPBH- and OPSH-based media were used by adding appropriate volumes of OPBH and OPSH solutions to the basal medium (BBM) to achieve the necessary glucose concentration. Yeast extract was used as a nitrogen source and was added at a concentration that would give the desired C/N ratio. All glucose-based, OPBH-based, and OPSH-based media were adjusted to pH 6.8 before autoclaving. Each flask was inoculated with 10% of seed culture and culture was performed in an orbital shaker (180 rpm) at 25 °C in the dark. Samples were taken at regular intervals to determine the cell density, lipid content, and reducing sugar concentration.
Preparation and enzymatic hydrolysis of organosolv-steam explosion pretreated birch and spruce
Silver birch (Betula pendula L.) and Norway spruce (Picea abies L.) chips milled at less than 1 mm in size in a Retch SM 300 knife mill (Retsch GmbH, Haan, Germany) were pretreated with a hybrid organosolv-steam explosion pretreatment method that was previously developed by our group . In brief, the pretreatment conditions were as follows: birch was treated at 200 °C with 60% v/v ethanol and 1% w/wbiomass of H2SO4 for 15 min, and spruce was treated at 200 °C with 52% v/v ethanol and 1% w/wbiomass of H2SO4 for 30 min. At the end of the pretreatment, the solids were separated from the liquid by vacuum filtration, washed with ethanol, and air-dried until further use. The composition (w/w) of the pretreated birch solids was 77.9% cellulose, 8.9% hemicellulose, and 7% lignin ; that of the spruce solids was 72% cellulose, 4% hemicellulose, and 15.4% lignin.
Enzymatic hydrolysis of pretreated birch and spruce biomass took place in 500-mL Erlenmeyer flasks containing 100 g of 10% w/w biomass solution in 50-mM citrate–phosphate buffer of pH 5. Hydrolysis was performed at 50 °C for 48 h with mixing at 180 rpm. The commercial enzyme solution Cellic CTec2 (Novozymes A/S, Bagsværd, Denmark) was used at a concentration equal to 20 FPU/g of solids. At the end of enzymatic hydrolysis, the solution was centrifuged to separate the remaining solids from the liquid, and the obtained hydrolysate was used as the carbon source for algal cultivation.
Estimation of cell growth, cell dry weight (g/L), and biomass productivity, P (g/L day)
Determination of total lipid concentration (g/L), lipid content (%, w/w), and lipid productivity (mg/L day)
Determination of residual sugar
Biochemical and morphological analysis including accumulation of lipid droplets in microalgal cells
To analyze the morphological changes of A. protothecoides grown in GSM, OPBH, and OPSH, 10 µL of culture was drawn at different time intervals and pelleted. After washing three times with 0.9% w/w saline, the cells were visualized by compound light microscopy (Olympus, Germany).
Analysis of neutral lipids in extracted lipids by TLC, fatty acid profile by GC, and estimation of biodiesel properties
In this study, all experiments were conducted in triplicates. The data were expressed as mean ± standard deviation and were analyzed with one-way analysis of variance (ANOVA) using Microsoft Office Excel 2016, with p values of < 0.05 being regarded as significant.
There is an increasing research interest in the use of low-cost renewable resources (such as lignocellulosic biomass) for the cultivation of microalgae, with the purpose of producing lipids for biodiesel production. Although various sources of plant biomass have already been tried in the literature, wood biomass is an underexploited resource that, to the best of our knowledge, has not been used for the growth of microalgae. In this work, we wanted to develop a novel approach for biodiesel production using a hybrid organosolv-steam explosion pretreated birch and spruce hydrolysates and heterotrophic growth of A. protothecoides. The hybrid pretreatment method used in this study allowed the efficient fractionation of spruce and birch biomass along with production of pretreated solids with high cellulose and low lignin content that could also be applicable for other non-edible lignocellulosic biomasses. This microalga, when grown in OPBH or OPSH, synthesized high quantities of lipids (66.00 ± 0.33 and 63.08 ± 0.71%, w/w, respectively) and, to the best of our knowledge, this is the first time that the use of wood hydrolysates for the culture of microalgae has been described. Moreover, the FAME profiles of biodiesel obtained after growth on OPBH or OPSH satisfy the criteria set up by ASTM 6751-3 and EN 14214 for use as transportation fuel.
AP: performed the experimental and analytical work, analyzed the data, and drafted the manuscript. LM: conceived the study, participated in the experimental design, analyzed the data, performed the organosolv pretreatment, and drafted the manuscript. UR: conceived the study and participated in experimental design and data analysis. PC: conceived the study and participated in experimental design and data analysis. All authors read and approved the final manuscript.
We thank Sveaskog, Sweden, for providing the birch and spruce chips that were used in this study and Novozymes A/S, Denmark, for providing the Cellic® CTec2 enzyme solution.
The authors declare that they have no competing interests.
Availability of data and materials
The materials produced during the current study are available from the corresponding author on reasonable request.
Consent for publication
Ethics approval and consent to participate
The authors will like to thank Bio4Energy, a strategic research environment appointed by the Swedish government, and Kempe Foundations, for supporting this work.
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