Appl Biochem Biotechnol DOI 10.1007/s12010-015-1663-6
Implication of Industrial Waste for Biomass and Lipid Production in Chlorella minutissima Under Autotrophic, Heterotrophic, and Mixotrophic Grown Conditions Kashyap Kumar Dubey 1 & Sudhir Kumar 1 & Deepak Dixit 1 & Punit Kumar 1 & Dhirendra Kumar 1 & Arshad Jawed 2 & Shafiul Haque 2,3
Received: 9 March 2015 / Accepted: 7 May 2015 # Springer Science+Business Media New York 2015
Abstract Following the diminishing hopes from the first and second generation biofuels, mainly due to the limitations of land availability, feed stock requirements, and complicated pre-treatments, third generation biofuels from microalgae are becoming a priority in the current scenario. The present study focuses on comparison and optimization of lipid accumulation efficiency in algal strain Chlorella minutissima grown under autotrophic, heterotrophic, and mixotrophic modes of nutrition, employing various carbon sources obtained from cheap industrial wastes such as glucose, acetate, and glycerol. Other pertinent factors such as the effect of various nitrogen sources, effect of salinity on the cell growth, and lipid accumulations in the algal cells were also studied. The results suggested that C. minutissima can grow efficiently under autotrophic, heterotrophic, and mixotrophic modes of nutrition. C. minutissima cells were capable of utilizing other non-popular carbon sources such as glycerol and acetate collected as waste products from different industries along with commonly used glucose. The maximum biomass concentration (8.9 g/L) and lipid content (36.19 %) were found in heterotrophic mode of nutrition. Our findings indicated that C. minutissima can efficiently utilize these cheaper carbon sources from industrial waste products for its growth and the production cost of various bioenergy sources can be reduced significantly. Keywords Chlorella minutissima . Bioenergy . Lipid accumulation . Industrial waste
* Shafiul Haque [email protected] 1
Microbial Biotechnology Laboratory, University Institute of Engineering and Technology, M.D. University, Rohtak 124001 Haryana, India
2
Research & Scientific Studies Unit, College of Nursing and Allied Health Sciences, Jazan University, Jazan 45142, Saudi Arabia
3
Department of Biosciences, Faculty of Natural Sciences, Jamia Millia Islamia (A Central University), New Delhi 110025, India
Appl Biochem Biotechnol
Introduction Constant economic growth combined with rising population has led to a steady increase in global energy demands [1]. Fossil fuels have become indispensable for economic development [2, 3]. Biofuel is one of the prime candidates for the replacement of fossil fuels. A plethora of research has been done on first and second generation biofuels, but no significant outcomes have been achieved for its production at process level or industrial scale [4]. A prime candidate for the production of biofuels is microalgae, which are an easy and convenient resource to produce biofuels with lower impact on the environment and on the world’s food supply when compared to conventional biofuel production. Microalgae are a group of diverse marine and freshwater microscopic organisms having the capability of carrying out photosynthesis. The presence of water, CO2, and other nutrients in their habitat makes them efficient in converting solar energy into biomass [5, 6]. Their simple structure and absence of supporting structures make them a good candidate for aquaculture [7]. Microalgal biomass possesses a high caloric value that makes microalgae more suitable for biofuel than any plant (lignocellulosic)-based materials [8]. Their inherently high-lipid content, semi-steady state production, sustainability, and suitability in different culture conditions make them the right candidate for the production of biofuel [9]. A unique aspect of microalgae is the number of amenable species available for biofuel production [10]. Different species may be selected for producing biofuels with variations in the content of fatty acids present [11]. In spite of the interest in the technique, the cultivation of algae on a large scale and processes for their utilization have successfully been developed in only few countries recently, while attempts to design an economically viable process are in full swing in some of the developing countries [12]. Extensive research on different fundamental and applied aspects of microalgae has been carried out and demonstrated that algal biomass can be used for various applications such as animal feed, bio-fertilizer, soil conditioner, and as a feed in aquaculture. Having high efficiency for intracellular accumulation of lipids, microalgae have emerged as a potent candidate for research in the field of bioenergy production. Microalgae can be easily grown in a culture in which required essential nutrients such as nitrogen and phosphorus can be obtained from different sources, e.g., a waste water source, that can keep the production cost at its minimum and also prove to be environment friendly. Also, microalgae need less space to grow [13] and have high productivity in contrast with other sources of bioenergy [9]. Earlier reports indicate that under heterotrophic mode of nutrition, microalgae stimulate elevated production rate of biomass as well as a conspicuous lipid accumulation intracellularly [14]. Microalgal species such as Chlorella protothecoides can accumulate 18–25 % (w/v) and 55.2 % (w/v) lipid content under autotrophic and heterotrophic mode of cultivation, respectively [15]. Microalgae can be grown under mixotrophic mode of cultivation where the presence of light and a suitable organic carbon source, such as glucose, molasses, glycerol, acetate, biomass hydrolysate, etc., is necessary [16]. In order to produce biofuel, a reaction of triglycerides with methanol is needed, which is popularly known as transesterification reaction. Transesterification process produces methyl esters of fatty acids, which belong to the category of biodiesel, and glycerol as a byproduct. The conversion reaction is a multistep reaction where triglycerides are converted into diglycerides as a first step; afterwards, it converts into monoglycerides, and at the end, monoglycerides are converted into glycerol [17]. Biodiesel can be directly used or it can be blended with fossil fuels in a particular ratio for its utilization as a fuel. Biodiesel with a name B99 has 1 % fossil fuel, which represses the growth of molds. In addition to the production of biodiesel,
Appl Biochem Biotechnol
microalgae can also provide various types of renewable biofuels [1]. These renewable biofuels include methane produced by anaerobic digestion of the algal biomass biodiesel derived from microalgal oil and photo-biologically produced bio-hydrogen. Another advantage of using microalgae for biodiesel production is that it does not affect the existing product yield from crops. The present study focuses on comparison and optimization of the efficiency of lipid accumulation in algal strain Chlorella minutissima grown under autotrophic, heterotrophic, and mixotrophic modes of nutrition using different carbon sources such as glucose, acetate, and glycerol from cheaper industrial wastes. As glycerol and acetate are the waste products of various industrial processes, these waste products can be used as potent substrates for utilization in bioenergy production [18, 19]. Also, other relevant factors such as nitrogen source and salt concentration that affect the growth and accumulation of lipid content in the microalgal cells have been studied.
Materials and Methods Microalgal Strains A strain of unicellular green microalgae C. minutissima was obtained from the national facility for blue-green algal collections, i.e., Centre for Conservation and Utilization of Blue Green Algae (CCUBGA), Indian Agricultural Research Institute, New Delhi, India.
Culture Media Various industrial wastes were used for the growth of microalgae. The industrial wastes were collected from various industries available in nearby areas of New Delhi, India, viz. sugar industry, Rohtak (Haryana), India; sugar industry, Panipat (Haryana), India; soap industry, Rohtak (Haryana), India; Indian Oil Corporation Limited, Faridabad (Haryana), India; and Panipat refinery, Panipat (Haryana), India. The industrial wastes were clarified and added to the culture medium at a final concentration of 20 % (v/v). Concentrations of glucose, acetate, glycerol, and other components were estimated and are presented in Table 1. The effluent emerging from the final drain line was collected and employed for the study. The effluents were analyzed for physicochemical properties (Table 1). The culture media for this study was modified in order to optimize the growth of microalgae. The culture media was supplemented with different carbon sources, namely, glucose, glycerol, acetate, and their combinations, according to the experimental conditions from 0.5 to 10 % (w/v). Yeast extract was used as nitrogen source and adequate concentrations of inorganic supplements such as KNO3 (1.103 g/L), KH2PO4 (0.075 g/L), K2HPO4 (0.1 g/L), MgSO4·7H2O (0.5 g/L), Ca(NO3)3·4H2O (0.0675 g/L), FeSO4·7H2O (0.01 g/L), H3BO3 (0.00286 g/L), MnCl2·4H2O (0.00181 g/L), ZnCl2 (0.000105 g/L), Na2MoO4·2H2O (0.0000030 g/L), CuSO4·5H2O (0.000079 g/L), and CoCl2 (0.000030 g/L) were also used in the culture media [20]. Various nitrogen sources such as yeast extract (2 % w/v), soya peptone (2 % w/v), urea (0.3005 g/L), ammonium chloride (0.535 g/L), and potassium nitrate (KNO3, 1.103 g/L) were used for the study of microalgal growth and its efficiency of lipid accumulation. Glycerol assay kit (MAK 117) and acetate colorimetric assay kit (MAK 086) utilized in the study were procured from Sigma Aldrich, USA. All analytical reagents and other
Appl Biochem Biotechnol Table 1 Physicochemical properties of effluents used in this study Serial number
Parameters
Sugar refinery effluent
Soap industry effluent
Oil refinery effluent
1
Color
Dark brown
Colorless
Pale yellow
2
pH
4.29
7.81
7.06
3
Glucose concentration (w/v)
15.4 %
NA
NA
4
Acetate concentration (w/v)
0.6 %
NA
0.04 %
5
Glycerol concentration (w/v)
0.3 %
0.6 %
NA
6
BOD; COD
943 mg/L; 3205 mg/L
NA; 3.9 mg/L
11.3 mg/L; 87.3 mg/L
7
Total dissolved solids (TDS)
1380 mg/L
16 mg/L
140 mg/L
8 9
Sulfates Dissolved oxygen
274 mg/L 4.8 mg/L
350 mg/L 7.2 mg/L
0.4 mg/L NA
10
Oil and grease
12 mg/L
NA
4.4 mg/L
NA Not available
chemicals and assay kits utilized in this study were purchased from HiMedia Laboratories, Mumbai (MS), India and Sigma-Aldrich (USA).
Analytical Methods and Statistical Analysis The relative concentration of C. minutissima biomass was estimated with a UV-visible spectrophotometer (Lab India3000+) by measuring the optical density (OD) at 540 nm [19]. Dry cell weight (DCW) of C. minutissima culture broth was estimated by centrifuging at 8000 rpm for 10 min followed by washing the pellet in double distilled water twice and then drying the pellet at 90 °C temperature until the attainment of constant weight. For the estimation of glucose concentration, a method of dinitrosalicylic acid assay was employed [21]; however, acetate and glycerol present in the industrial effluents were estimated using commercial assay kits procured from Sigma-Aldrich, USA. Lipid concentration was determined by using the solvent extraction method given by Bligh and Dyer [22]. The statistical analysis was performed using one-way ANOVA along with Tukey’s method and Student’s t test. The statistical significance level was maintained as p value
Implication of Industrial Waste for Biomass and Lipid Production in Chlorella minutissima Under Autotrophic, Heterotrophic, and Mixotrophic Grown Conditions Kashyap Kumar Dubey 1 & Sudhir Kumar 1 & Deepak Dixit 1 & Punit Kumar 1 & Dhirendra Kumar 1 & Arshad Jawed 2 & Shafiul Haque 2,3
Received: 9 March 2015 / Accepted: 7 May 2015 # Springer Science+Business Media New York 2015
Abstract Following the diminishing hopes from the first and second generation biofuels, mainly due to the limitations of land availability, feed stock requirements, and complicated pre-treatments, third generation biofuels from microalgae are becoming a priority in the current scenario. The present study focuses on comparison and optimization of lipid accumulation efficiency in algal strain Chlorella minutissima grown under autotrophic, heterotrophic, and mixotrophic modes of nutrition, employing various carbon sources obtained from cheap industrial wastes such as glucose, acetate, and glycerol. Other pertinent factors such as the effect of various nitrogen sources, effect of salinity on the cell growth, and lipid accumulations in the algal cells were also studied. The results suggested that C. minutissima can grow efficiently under autotrophic, heterotrophic, and mixotrophic modes of nutrition. C. minutissima cells were capable of utilizing other non-popular carbon sources such as glycerol and acetate collected as waste products from different industries along with commonly used glucose. The maximum biomass concentration (8.9 g/L) and lipid content (36.19 %) were found in heterotrophic mode of nutrition. Our findings indicated that C. minutissima can efficiently utilize these cheaper carbon sources from industrial waste products for its growth and the production cost of various bioenergy sources can be reduced significantly. Keywords Chlorella minutissima . Bioenergy . Lipid accumulation . Industrial waste
* Shafiul Haque [email protected] 1
Microbial Biotechnology Laboratory, University Institute of Engineering and Technology, M.D. University, Rohtak 124001 Haryana, India
2
Research & Scientific Studies Unit, College of Nursing and Allied Health Sciences, Jazan University, Jazan 45142, Saudi Arabia
3
Department of Biosciences, Faculty of Natural Sciences, Jamia Millia Islamia (A Central University), New Delhi 110025, India
Appl Biochem Biotechnol
Introduction Constant economic growth combined with rising population has led to a steady increase in global energy demands [1]. Fossil fuels have become indispensable for economic development [2, 3]. Biofuel is one of the prime candidates for the replacement of fossil fuels. A plethora of research has been done on first and second generation biofuels, but no significant outcomes have been achieved for its production at process level or industrial scale [4]. A prime candidate for the production of biofuels is microalgae, which are an easy and convenient resource to produce biofuels with lower impact on the environment and on the world’s food supply when compared to conventional biofuel production. Microalgae are a group of diverse marine and freshwater microscopic organisms having the capability of carrying out photosynthesis. The presence of water, CO2, and other nutrients in their habitat makes them efficient in converting solar energy into biomass [5, 6]. Their simple structure and absence of supporting structures make them a good candidate for aquaculture [7]. Microalgal biomass possesses a high caloric value that makes microalgae more suitable for biofuel than any plant (lignocellulosic)-based materials [8]. Their inherently high-lipid content, semi-steady state production, sustainability, and suitability in different culture conditions make them the right candidate for the production of biofuel [9]. A unique aspect of microalgae is the number of amenable species available for biofuel production [10]. Different species may be selected for producing biofuels with variations in the content of fatty acids present [11]. In spite of the interest in the technique, the cultivation of algae on a large scale and processes for their utilization have successfully been developed in only few countries recently, while attempts to design an economically viable process are in full swing in some of the developing countries [12]. Extensive research on different fundamental and applied aspects of microalgae has been carried out and demonstrated that algal biomass can be used for various applications such as animal feed, bio-fertilizer, soil conditioner, and as a feed in aquaculture. Having high efficiency for intracellular accumulation of lipids, microalgae have emerged as a potent candidate for research in the field of bioenergy production. Microalgae can be easily grown in a culture in which required essential nutrients such as nitrogen and phosphorus can be obtained from different sources, e.g., a waste water source, that can keep the production cost at its minimum and also prove to be environment friendly. Also, microalgae need less space to grow [13] and have high productivity in contrast with other sources of bioenergy [9]. Earlier reports indicate that under heterotrophic mode of nutrition, microalgae stimulate elevated production rate of biomass as well as a conspicuous lipid accumulation intracellularly [14]. Microalgal species such as Chlorella protothecoides can accumulate 18–25 % (w/v) and 55.2 % (w/v) lipid content under autotrophic and heterotrophic mode of cultivation, respectively [15]. Microalgae can be grown under mixotrophic mode of cultivation where the presence of light and a suitable organic carbon source, such as glucose, molasses, glycerol, acetate, biomass hydrolysate, etc., is necessary [16]. In order to produce biofuel, a reaction of triglycerides with methanol is needed, which is popularly known as transesterification reaction. Transesterification process produces methyl esters of fatty acids, which belong to the category of biodiesel, and glycerol as a byproduct. The conversion reaction is a multistep reaction where triglycerides are converted into diglycerides as a first step; afterwards, it converts into monoglycerides, and at the end, monoglycerides are converted into glycerol [17]. Biodiesel can be directly used or it can be blended with fossil fuels in a particular ratio for its utilization as a fuel. Biodiesel with a name B99 has 1 % fossil fuel, which represses the growth of molds. In addition to the production of biodiesel,
Appl Biochem Biotechnol
microalgae can also provide various types of renewable biofuels [1]. These renewable biofuels include methane produced by anaerobic digestion of the algal biomass biodiesel derived from microalgal oil and photo-biologically produced bio-hydrogen. Another advantage of using microalgae for biodiesel production is that it does not affect the existing product yield from crops. The present study focuses on comparison and optimization of the efficiency of lipid accumulation in algal strain Chlorella minutissima grown under autotrophic, heterotrophic, and mixotrophic modes of nutrition using different carbon sources such as glucose, acetate, and glycerol from cheaper industrial wastes. As glycerol and acetate are the waste products of various industrial processes, these waste products can be used as potent substrates for utilization in bioenergy production [18, 19]. Also, other relevant factors such as nitrogen source and salt concentration that affect the growth and accumulation of lipid content in the microalgal cells have been studied.
Materials and Methods Microalgal Strains A strain of unicellular green microalgae C. minutissima was obtained from the national facility for blue-green algal collections, i.e., Centre for Conservation and Utilization of Blue Green Algae (CCUBGA), Indian Agricultural Research Institute, New Delhi, India.
Culture Media Various industrial wastes were used for the growth of microalgae. The industrial wastes were collected from various industries available in nearby areas of New Delhi, India, viz. sugar industry, Rohtak (Haryana), India; sugar industry, Panipat (Haryana), India; soap industry, Rohtak (Haryana), India; Indian Oil Corporation Limited, Faridabad (Haryana), India; and Panipat refinery, Panipat (Haryana), India. The industrial wastes were clarified and added to the culture medium at a final concentration of 20 % (v/v). Concentrations of glucose, acetate, glycerol, and other components were estimated and are presented in Table 1. The effluent emerging from the final drain line was collected and employed for the study. The effluents were analyzed for physicochemical properties (Table 1). The culture media for this study was modified in order to optimize the growth of microalgae. The culture media was supplemented with different carbon sources, namely, glucose, glycerol, acetate, and their combinations, according to the experimental conditions from 0.5 to 10 % (w/v). Yeast extract was used as nitrogen source and adequate concentrations of inorganic supplements such as KNO3 (1.103 g/L), KH2PO4 (0.075 g/L), K2HPO4 (0.1 g/L), MgSO4·7H2O (0.5 g/L), Ca(NO3)3·4H2O (0.0675 g/L), FeSO4·7H2O (0.01 g/L), H3BO3 (0.00286 g/L), MnCl2·4H2O (0.00181 g/L), ZnCl2 (0.000105 g/L), Na2MoO4·2H2O (0.0000030 g/L), CuSO4·5H2O (0.000079 g/L), and CoCl2 (0.000030 g/L) were also used in the culture media [20]. Various nitrogen sources such as yeast extract (2 % w/v), soya peptone (2 % w/v), urea (0.3005 g/L), ammonium chloride (0.535 g/L), and potassium nitrate (KNO3, 1.103 g/L) were used for the study of microalgal growth and its efficiency of lipid accumulation. Glycerol assay kit (MAK 117) and acetate colorimetric assay kit (MAK 086) utilized in the study were procured from Sigma Aldrich, USA. All analytical reagents and other
Appl Biochem Biotechnol Table 1 Physicochemical properties of effluents used in this study Serial number
Parameters
Sugar refinery effluent
Soap industry effluent
Oil refinery effluent
1
Color
Dark brown
Colorless
Pale yellow
2
pH
4.29
7.81
7.06
3
Glucose concentration (w/v)
15.4 %
NA
NA
4
Acetate concentration (w/v)
0.6 %
NA
0.04 %
5
Glycerol concentration (w/v)
0.3 %
0.6 %
NA
6
BOD; COD
943 mg/L; 3205 mg/L
NA; 3.9 mg/L
11.3 mg/L; 87.3 mg/L
7
Total dissolved solids (TDS)
1380 mg/L
16 mg/L
140 mg/L
8 9
Sulfates Dissolved oxygen
274 mg/L 4.8 mg/L
350 mg/L 7.2 mg/L
0.4 mg/L NA
10
Oil and grease
12 mg/L
NA
4.4 mg/L
NA Not available
chemicals and assay kits utilized in this study were purchased from HiMedia Laboratories, Mumbai (MS), India and Sigma-Aldrich (USA).
Analytical Methods and Statistical Analysis The relative concentration of C. minutissima biomass was estimated with a UV-visible spectrophotometer (Lab India3000+) by measuring the optical density (OD) at 540 nm [19]. Dry cell weight (DCW) of C. minutissima culture broth was estimated by centrifuging at 8000 rpm for 10 min followed by washing the pellet in double distilled water twice and then drying the pellet at 90 °C temperature until the attainment of constant weight. For the estimation of glucose concentration, a method of dinitrosalicylic acid assay was employed [21]; however, acetate and glycerol present in the industrial effluents were estimated using commercial assay kits procured from Sigma-Aldrich, USA. Lipid concentration was determined by using the solvent extraction method given by Bligh and Dyer [22]. The statistical analysis was performed using one-way ANOVA along with Tukey’s method and Student’s t test. The statistical significance level was maintained as p value
