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Chlamydomonas sp. as dynamic biorefinery feedstock for the production of methyl ester and ɛ-polylysine
Bioresource Technology 272 (2019) 281–287
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Chlamydomonas sp. as dynamic biorefinery feedstock for the production of methyl ester and ɛ-polylysine Ramachandran Sivaramakrishnana, Subramaniyam Sureshb, Aran Incharoensakdia, a b
T
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Laboratory of Cyanobacterial Biotechnology, Department of Biochemistry, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand Department of Chemistry, Ramapuram Campus, SRM Institute of Science and Technology, Chennai, India
G R A P H I C A L A B S T R A C T
A R T I C LE I N FO
A B S T R A C T
Keywords: Biorefinery Chlamydomonas sp. Flocculation ɛ-Polylysine Methyl ester Streptomyces sp.
An integrated production of methyl ester and ɛ-polylysine from Chlamydomonas sp. was studied using biorefinery approach. The harvesting efficiency of Chlamydomonas sp. was increased up to 92% by treatment with a flocculant FeCl3 at 100 mg/L for 30 min. The DMC (dimethyl carbonate) mediated enzyme catalyzed in-situ transesterification of Chlamydomonas sp. yielded the maximum methyl ester of 92% under optimized conditions. The valued-added product ɛ-polylysine was produced from hydrolysate obtained from the spent biomass of Chlamydomonas sp. using Streptomyces sp. The key components of sugar and MgSO4 used for ɛ-polysine production were optimized whereby the maximum ɛ-polylysine production was achieved at 50 g/L sugar and 0.3 g/ L MgSO4. The ɛ-polylysine production was further enhanced by supplementation of important amino acids (lysine and aspartate) and TCA cycle intermediates (citric acid and α-ketoglutaric acid). The maximum ɛpolylysine production of 2.24 g/L was found with 4 mM citric acid supplementation after 110 h.
1. Introduction Environmental problems coupled with rapid depletion of fossil fuel and its resources prompted researchers to find alternative renewable resources and its commercialization (Sivaramakrishnan and Incharoensakdi, 2017a). The biomass from microalgae with high oil content is a promising feedstock for the renewable resources. Compared
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with plants, microalgae can produce more oil per hectare with a shorter production cycle (Chew et al., 2017). The coupling of algae biofuels with high value compounds production widens the market opportunities (Barreiro et al., 2014), which fits well with a recent trend of biorefinery concept (Sivaramakrishnan and Incharoensakdi, 2018a). The biorefineries at industrial scale involve the production of multiple products through the integration of the production configuration. The
Corresponding author. E-mail address: [email protected] (A. Incharoensakdi).
https://doi.org/10.1016/j.biortech.2018.10.026 Received 27 August 2018; Received in revised form 9 October 2018; Accepted 10 October 2018 Available online 11 October 2018 0960-8524/ © 2018 Elsevier Ltd. All rights reserved.
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stone quarry pond water with the accession number of KR025878 (GenBank) (Sivaramakrishnan and Incharoensakdi, 2017c). The culture was grown and maintained in BG11, under continuous illumination (50 μmol photons/m2/s) with 100 rpm shaking at 27 ± 1 °C. The culture was regularly monitored under microscope to maintain the purity of the culture. The microalgal slurry was collected from 12 days grown culture and used for the experiments. The biochemical compositions of total lipid, carbohydrate and protein were determined as 27, 21 and 24% (w/w of biomass) respectively.
biorefinery designs employ maximum product output from a single feedstock with high profit (Moncada et al., 2015). For biorefinery approach, it is essential to select the microalgae which contain high amounts of organic matters such as lipids and carbohydrates which can be used for biorefinery approaches. Chlamydomonas sp. is one of the major and important feedstocks for the biofuel applications with mass production (Kao and Ng, 2017). Microalgae contain both transesterifiable and non-transesterifiable lipids (chlorophyll, carotenoids, etc.). Large amounts of neutral lipids suitable for methyl ester production are found in microalgae (Sivaramakrishnan and Incharoensakdi, 2017b). It is clear from the previous study that Chlamydomonas sp. is suitable for the methyl ester production (Chen et al., 2015). The methyl ester production from microalgae involves cultivation, harvesting, lipid extraction and transesterification. For the total methyl ester production, harvesting, lipid extraction and transesterification alone comprise 60% of costs (Sivaramakrishnan and Incharoensakdi, 2018b). The initial harvesting step comprises nearly 20–30% of the total production cost and may vary depending on the technology used due to the behavior or the nature of the microalgae. The efficient method to harvest microalgae can cut the cost considerably in the downstream processes. Microalgae can be harvested using flocculants, centrifugation, sedimentation or flotation processes (Lee et al., 2013). Among these methods, flocculation is an easy and cheap method compared to the centrifugation. Flocculation increases the settling efficiency of microalgae by aggregation leading to sedimentation of microalgae (Choong et al., 2016). The flocculated biomass can be used as the source for the methyl ester production via in-situ transesterification where lipids react with methanol aided by chemical or enzyme catalyst. The disadvantages of chemical catalysts are complex downstream processing such as glycerol recovery, removal of organic and inorganic chemicals from the reaction mixture and the soap formation by alkali catalysts. Hence, enzymatic transesterification offers a preferable alternative method even with high free fatty acids at lower reaction temperatures (30–50 °C) (Lee et al., 2016). The dimethyl carbonate (DMC) can be used as the acyl acceptor over alcohol like methanol or ethanol. The use of DMC for the transesterification also has important benefits such as inexpensive, non-corrosive, non-toxic, neutral, odorless and ecofriendly (Sivaramakrishnan and Muthukumar, 2013). For biorefinery, spent biomass (i.e. biomass after being used for insitu transesterification) has the carbohydrate content which can be further fermented to produce valuable compounds such as ɛ-polylysine and ethanol. ɛ-Polylysine is a basic homopolymer which contains 20–30 residues of L-lysine with an ɛ-amino group and an α-carboxyl group. This polymer has antimicrobial activity against gram-negative and gram-positive bacteria (Geornaras et al., 2007). Due to the biodegradable nature of the ɛ-polylysine, it can be used as a safe food preservative. Moreover, ɛ-polylysine has various applications, i.e. an emulsifying agent, a dietary agent, an anticancer agent enhancer, a gene delivery carrier, a biochip coating, as well as a micro or nano capsule for drug delivery (Choi et al., 2000). Currently there are numerous studies on methyl ester from microalgae; however, there are few studies on integrated production of methyl ester and value-added products. Particularly, no study was reported on combined production of methyl ester and ɛ-polylysine. This work aimed to investigate the potential of lipid and carbohydrate containing biomass from Chlamydomonas sp. for the integrated production of methyl ester and ɛ-polylysine. Moreover, the objective of the work also extends to the determination of the flocculation condition for the Chlamydomonas sp. which added the benefit towards the biorefinery approach.
2.2. Harvesting efficiency Harvesting studies using flocculants were carried out in 20 ml cylindrical glass tubes. The algal suspension was mixed and swirled with flocculant and left to sediment. Various concentrations of flocculants were used in the range of 20–140 mg/L. The harvesting efficiency was calculated using the following equation,
Harvesting efficiency (%) =
(ODi−ODf ) × 100 ODi
ODi and ODf are initial and final optical density at 730 nm of non-sedimenting portion of sample respectively. 2.3. In-situ transesterification Lipid content in Chlamydomonas sp. was determined by solvent extraction method (Sivaramakrishnan and Incharoensakdi, 2017c). In a screw-capped glass vial, 1 g of dried microalgal biomass was mixed with an appropriate amount of dimethyl carbonate. The immobilized lipase was added to the mixture as a catalyst and appropriate amount of distilled water was added to start the reaction in a temperature controlled shaking incubator (180 rpm) at 45 °C. The reaction was run up to 36 h. The lipase (Novozyme – Lipozyme CAL-B from Candida antartica, Novozymes, Denmark) as a catalyst was immobilized on Celite material with 190 U/g enzyme activity. Lipase immobilization was performed as previously described by Sivaramakrishnan and Muthukumar (2012). Various factors affecting methyl ester yield were determined as follows: solvent algae ratio (2:1, 3:1, 4:1, 5:1, 6:1, 7:1 and 8:1), catalyst concentration (2, 4, 6, 8, 10 and 12 w/w %), water addition (0.2, 0.4, 0.6, 0.8, 1 and 1.2 w/w %), reaction temperature (35, 40, 45, 50, 55, 60 and 65 °C), reaction time (6, 12, 18, 24, 30 and 36 h) and the analysis of by-product (glycerol carbonate, glycerol dicarbonate and glycerol). After the reaction was stopped, the mixture was filtered to separate methyl ester and biomass, the traces of methyl ester present in the residues were collected by solvent (DMC) wash. The recovered samples were centrifuged at 20,000g for 15 min to remove the remaining contaminants. The obtained methyl ester content was determined, and its properties were analyzed. 2.4. Fermentative production of ɛ-polylysine Streptomyces albulus (MTCC 503) (Streptomyces sp.) obtained from microbial type culture collection, Chandigarh, India was used for the production of ɛ-polylysine utilizing microalgal hydrolysate as substrate. The hydrolysate was prepared by acid hydrolysis of the spent biomass (i.e. after in-situ transesterification). The hydrolysis was optimized, and the best conditions were found to be 0.3 N H2SO4 with autoclave (120 °C and 15 psi) for 20 min (data not shown). The fermentative medium contained 50 g/L sugars, 10 g/L peptone, 8 g/L (NH4)2SO4, 0.5 g/L MgSO4·7H20, 0.03 g/L FeSO4·7H2O, 0.04 g/L ZnSO4, 0.8 g/L K2HPO4, 1.36 g/L KH2PO4 and the pH was adjusted to 6.8 using 0.1 M phosphate buffer (pH 6.8) The fermentation was initiated by adding 2.5 ml of the culture (8.0 × 108 cells/ml) and grown for 110 h at 30 °C with shaking at 180 rpm. The analysis of ɛ-polylysine production was determined by harvesting the cells after the fermentation and centrifuging at 10,000g for 10 min. One ml of 1 mM methyl orange was
2. Materials and methods 2.1. Microorganism and culture conditions The Chlamydomonas sp. used in this study was isolated from the 282
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added to 1 ml of the supernatant followed by mixing for 60 min at 37 °C. The incubated samples were centrifuged again at 10,000g for 10 min and the supernatant was measured for its absorbance at 465 nm on UV–Vis spectrophotometer (Itzhaki, 1972). 2.5. Analytical methods The determination of methyl ester and glycerol carbonate contents was done by using gas chromatography according to Sivaramakrishnan and Incharoensakdi (2016). The methyl ester properties of saponification value (SV), iodine value (IV), cetane number (CN) and degree of unsaturation (DU) were determined as described by Sivaramakrishnan and Incharoensakdi (2017d). 2.6. Statistical analysis The results obtained are mean of triplicate values and the error bars are standard deviation (means ± SD, n = 3) values. The statistical significance (p < 0.05) was analyzed by t-test comparisons using graph pad software. 3. Results and discussion 3.1. Process overview The present study involved the production of methyl ester using Chlamydomonas sp. lipids and its carbohydrate content was utilized for the ɛ-polylysine production. The overall processing steps are divided into three different stages. 1) Concentration of microalgae by flocculation method using FeCl3 as a flocculant, 2) Production of methyl ester by in-situ transesterification of Chlamydomonas sp. using immobilized lipase as a catalyst and DMC as a solvent, and 3) Spent biomass after insitu transesterification was further hydrolyzed and the hydrolysate was used for the ɛ-polylysine production. The overall scheme of the present study is depicted in Fig. 1 showing the utilization of microalgal feedstock as a biorefinery model for the methyl ester and ɛ-polylysine production.
Fig. 2. (a) Effect of flocculants concentration on harvesting. Conditions: 0.25 g/ L of biomass and 1 h. (b) Effect of flocculant (FeCl3) on harvesting at different biomass densities. Conditions: 10 mg/L of flocculant, 0.25 g/L of biomass and 1 h. Data points were mean of triplicate values with the error bar showing standard deviations.
Al2(SO4)3 are considered the best flocculants for Chlamydomonas sp. and they were used for further experiments. The concentrations of FeCl3 and Al2(SO4)3 were varied to achieve the maximum harvesting efficiency. As shown in Fig. 2a, increasing the concentration of flocculants up to 100 mg/L increased the harvesting efficiency. Comparatively, FeCl3 showed higher harvesting efficiency than the Al2(SO4)3; however, both the flocculants showed their maximum harvesting efficiency at 100 mg/L. The obtained results agreed with the previously reported study, where 200 mg/L of FeCl3 was used as a coagulant at low pH to harvest Chlorella sp. (Seo et al., 2015). Different microalgae have different properties including shape, size, specific gravity, charge and motility, all of which can affect the harvesting efficiency (Udom et al., 2013). Moreover, harvesting efficiency of algae is also affected by the constituents of the culture medium, pH and ionic strength. The use of multivalent metal cations for the flocculation has been reported as a simple method with the minimum energy requirement. Such metal ions in the growth medium coagulate negatively charged microalgal cells leading to flocculation of cells. The changes in physical properties of the microalgae also affect the flocculation (Liu et al., 2013). The suspension of cells with flocculants results in charge neutralization and polysaccharide bridging ensures maximum aggregation and agglomeration of cells. Hence it is necessary to add a flocculating agent to achieve maximum harvesting efficiency to obtain larger flocs which settle down the bottom of the vessel and thereby leaving a clear supernatant. Different algal cell concentrations were varied to determine the suitable cell concentration for the harvesting (biomass concentration – 0.25–2 g/L) and the absence of FeCl3 is set as control. Increasing biomass concentration up to 1.5 g/L increased the harvesting efficiency (Fig. 2b). Using the FeCl3 (100 mg/L) with 1.5 g/L of biomass yielded
3.2. Harvesting of microalgae by flocculation In the present study, the four different flocculants were tested for their efficiency of harvesting Chlamydomonas sp. It was observed that the flocculants greatly influence the harvesting efficiency of Chlamydomonas sp. Among the four flocculants at 80 mg/L FeCl3 and Al2(SO4)3 showed maximum harvesting efficiency of 73 and 66% respectively. Polyacrylamide showed very low harvesting efficiency (35%) which is close to the control value (28%) whereas NaOH showed moderate harvesting efficiency of 47%. From the results, FeCl3 and
Fig. 1. Schematic representation of integrated production of methyl ester and ɛpolylysine. 283
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biomass ratio is considered as the optimum and used for further studies. Lipozyme is an important enzyme and highly preferable for the lipase mediated transesterification (Wang et al., 2017). The enzyme loading is a crucial factor for successful industrial application. To study the effect of enzyme content on in-situ transesterification, different enzyme contents ranging from 2 to 12% (w/w of microalgae) were studied. The increase in the catalyst content from 2 to 8% (w/w of microalgae) resulted in an increase of the methyl ester yield (Table 1). Increasing enzyme content above 8% (w/w of microalgae) decreased the methyl ester yield. The mass transfer resistance contributes to the decrease in the yield on higher enzyme content. It is economically necessary to determine the optimum enzyme content to cut the cost of enzyme wastage. Water content is another important parameter which can affect the activity and stability of the lipases. The methyl ester yield of in-situ transesterification was increased with the increase in water addition up to 0.8% (w/w of microalgae) (Table 1). A further increase in the water addition decreased the yield due to enzyme inactivation. To maintain an interfacial area of lipase it is necessary to add essential water. Excessive water also affects the enzyme and reduces the product yield. Excess water forms hydrogen bonds with the protein functional group (Sivaramakrishnan and Incharoensakdi, 2017a). The reaction temperature also affects the in-situ transesterification of Chlamydomonas sp. lipids. Increasing temperature up to 50 °C increased the methy ester yield, whereas the yield decreased considerably at higher than 50 °C (Table 1). The optimal temperature at which the enzyme operates is very crucial. The increase of temperature increases the rate of enzyme-catalyzed reaction; however, an increased temperature damages the enzyme structure leading to a decrease of reaction rate (Huang et al., 2015). Hence, the determination of optimal reaction temperature is necessary to achieve the maximal yield with maximum reaction rate. In this study, the enzyme requires 50 °C to perform transesterification of maximum lipids into methyl esters. The influence of reaction time on in-situ transesterification of Chlamydomonas sp. lipids is shown in Table 1. The methyl ester yield was increased from 22 to 92% when the reaction time was increased from 6 to 24 h. No significant change was observed in methyl ester yield after 24 h. These results were in agreement with those reported by Shuit et al. (2010). The reaction time of 24 h was considered as an optimal reaction time since longer reaction time would add extra energy cost. Glycerol carbonate (1.6 ± 0.21%), glycerol dicarbonate (5.1 ± 0.36%) and glycerol (1.2 ± 0.12%) all of which are by products during DMC mediated transesterification were obtained at the optimized conditions (Table 1). Glycerol molecule, detached from the triglyceride during transesterification, reacts with DMC to produce glycerol carbonate molecule. Glycerol carbonate has many applications, for example, in detergents, surfactants, paint and coatings, and gas separation membrane industries (Jung et al., 2017). The glycerol carbonate production is the promising by-product with regard to biorefinery approach. Moreover, the DMC mediated glycerol carbonate synthesis in this study is more environmentally advantageous when compared to chemically synthesized glycerol carbonate. At the optimized conditions of in-situ transesterification using DMC, the highest methyl ester yield of 92% was obtained from the Chlamydomonas sp. simultaneously with 1.6% glycerol carbonate production.
Table 1 Parameters influencing in-situ transesterification of Chlamydomonas sp. Parameters DMC/biomass 2 3 4 5 6 7 8
Yield (%) ratio (ml/g) 33 ± 1.52 45 ± 1.52 54 ± 0.57 63 ± 2.08 72 ± 2.08 67 ± 3.21 64 ± 2.08
Conditions
0.5 g Chlamydomonas sp., 10% catalyst (w/w of microalgae), 1% distilled water (w/w of microalgae), 45 °C and 36 h reaction time
Catalyst content (w/w of biomass) 2 38 ± 2.08 0.5 g Chlamydomonas sp., 6 ml DMC/g microalgae, 1% 4 51 ± 1.52 distilled water (w/w of microalgae), 45 °C and 36 h 6 64 ± 1.00 reaction time 8 78 ± 1.00 10 72 ± 2.51 12 66 ± 1.52 Water content (w/w of biomass) 0.2 56 ± 2.88 0.5 g Chlamydomonas sp., 6 ml DMC/g microalgae, 8% 0.4 62 ± 0.57 catalyst (w/w of microalgae), 45 °C and 36 h reaction 0.6 71 ± 0.57 time 0.8 85 ± 1.52 1 78 ± 1.32 1.2 69 ± 1.21 Temperature (°C) 35 57 ± 1.52 40 73 ± 1.52 45 85 ± 1.52 50 92 ± 2.0 55 81 ± 0.57 60 66 ± 3.6 65 43 ± 1.52 Time (min) 6 12 18 24 30 36
22 46 71 92 92 92
± ± ± ± ± ±
0.57 0.57 1.73 1.52 0.57 0.57
0.5 g Chlamydomonas sp., 6 ml DMC/g microalgae, 8% catalyst (w/w of microalgae), 0.8% water addition (w/w of microalgae) and 36 h reaction time
0.5 g Chlamydomonas sp., 6 ml DMC/g microalgae, 8% catalyst (w/w of microalgae), 0.8% water addition (w/w of microalgae), 50 °C
highest harvesting efficiency of 92% in 30 min. The sample without FeCl3 required 180 min to achieve its maximum of 80% harvesting efficiency. Above the 1.5 g/L of biomass the harvesting efficiency decreased. The results were in agreement with those reported by Zhao et al. (2014). Hence ferric chloride was the most suitable flocculant to harvest the Chlamydomonas sp. Moreover, addition of ferric chloride did not affect the algal growth and all cell components. The growth medium with low concentrations of ferric chloride can be reused although alum compounds can affect the microalgal growth significantly (Zhao et al., 2014). The FeCl3 at 100 mg/L flocculates Chlamydomonas sp. at 1.5 g/L of biomass efficiently in a short time with minimum energy. 3.3. In-situ transesterification Various process parameters involved in the in-situ transesterification of Chlamydomonas sp. were optimized and the results are shown in Table 1. The solvent DMC was used to simultaneously extract and transesterify the Chlamydomonas sp. lipids. The optimal DMC to biomass ratio not only increases the yield, but also avoids the excess use of solvent. The lipid transformation into methyl ester increased with the increase in the DMC to biomass ratio and the maximum methyl ester yield was achieved at a ratio of 6:1 (Table 1). On the other hand, a decrease in the yield was observed with the increase of DMC to biomass ratio from 6:1 to 8:1. Below the optimized level, the DMC cannot transform all the available lipids into methyl ester (Lee et al., 2010). Addition of DMC higher than the optimum level dilutes the lipid concentration resulting in inefficient transesterification. The 6:1 DMC to
3.4. Influence of nutrient composition on ɛ-polylysine production It is necessary to enhance the ɛ-polylysine production after the methyl ester production. The improved ɛ-polylysine production increases the chance of biorefinery commercialization. Hence, the critical medium compositions were optimized to improve the ɛ-polylysine production. It is very important to obtain the optimal concentration of sugar for the maximum ɛ-polylysine yield. The sugar concentrations in the hydrolysate of spent biomass were varied from 35 to 60 g/L and tested for their effect on ɛ-polylysine production. The results clearly 284
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because the addition of amino acid above the maximum extent inhibits the enzymes involved in the ɛ-polylysine synthesis pathway, such as aspartokinase or phosphoenol pyruvate carboxylase (Bankar and Singhal, 2011). The influence of citric acid and α-ketoglutaric acid on ɛ-polylysine production is shown in Fig. 3. At 4 mM citric acid and α-ketoglutaric acid showed the highest ɛ-polylysine yields. Among the various organic acids, citric acid is the important organic acid, which can greatly influence the ɛ-polylysine production (Bankar and Singhal, 2013). Addition of α-ketoglutaric acid also showed satisfactory results, but the yield was slightly lower than that of the citric acid at optimum concentration. Hence, citric acid is the critical TCA cycle component which greatly affects the ɛ-polylysine production. Overall, the concentration of sugar plays an important role in the ɛpolylysine production and showed the maximum yield at 50 g/L of sugar. The yield was further improved after MgSO4 optimization. The addition of key amino acids and TCA cycle intermediates considerably increases the ɛ-polylysine production. Particularly, citric acid (4 mM) showed highest improvement in the ɛ-polylysine production.
indicate that the sugar concentration in the fermentation plays a vital role in the ɛ-polylysine production. The 50 g/L sugar gave the highest ɛpolylysine yield. Shukla and Mishra (2012) reported that the glucose and glycerol in a 1:1 ratio increases the ɛ-polylysine production. The yield was further increased when molasses were used as the carbon source. The increased ɛ-polylysine production was reported in Streptomyces sp. when glucose was a carbon source and when added with the intermediates in the TCA cycle (Shih and Shen, 2006). In the present study 50 g/L sugar produced a maximum of 1.54 g/L ɛ-polylysine. When testing for the effect of glucose on the yield of ɛ-polylysine, it was found that the maximum ɛ-polylysine production of 1.77 g/L was obtained at 50 g/L glucose. Hence it is clear that the 50 g/L sugar in the hydrolysate of spent biomass contained fermentable sugar producing 1.54 g/L ɛ-polylysine, which accounted for about 90% of the yield produced by 50 g/L glucose. This indicated the presence of non-fermentable sugar in the hydrolysate of spent biomass. Another critical component in the media is magnesium, which is necessary for the cell division in bacteria. Apart from the cell division, magnesium is important for the activation of the ɛ-polylysine synthetase reaction (Chheda and Vernekar, 2015). The MgSO4 concentrations were varied from 0.15 to 0.6 g/L to determine their effect on ɛ-polylysine production. The results showed that 0.15 g/L MgSO4 yielded ɛpolylysine of 1.54 g/L. Increasing MgSO4 to 0.3 g/L yielded the maximum ɛ-polylysine of 1.78 g/L. The appropriate concentration of sugar (50 g/L) and MgSO4 (0.3 g/L) is required to produce maximum yield of ɛ-polylysine.
3.6. Time-course of ɛ-polylysine and biomass content The time-course of ɛ-polylysine yield and biomass content is shown in Fig. 4. Increasing time increased both biomass and ɛ-polylysine up to 110 h, at which time the maximum ɛ-polylysine production was obtained at about 2.24 g/L. As reported in the previous studies, maximum ɛ-polylysine production by Streptomyces sp. was achieved during 48–72 h and thereafter the production rate was reduced (Hirohara et al., 2006). For Streptomyces sp., the maximum ɛ-polylysine production was achieved in 144 h (Xu et al., 2015), which was similarly observed in the study with Streptomyces albulus PD-1 (Xia et al., 2014). The decrease in the ɛ-polylysine production after 120 h is due to the ɛpolylysine degrading enzyme released by the organism during late stationary phase which changes the medium pH to acidic condition (Yamanaka et al., 2010). In the present study, the maximum product yield of ɛ-polylysine was achieved at 110 h.
3.5. Effect of critical amino acids and TCA cycle intermediates on ɛpolylysine production In the ɛ-polylysine synthesis pathway, aspartate family amino acids are primarily involved, which are L-lysine, L-aspartate, L-methonine, and L-threonine. There is no significant change of ɛ-polylysine yield with the addition of methionine and threonine because these two amino acids are not participating in the biosynthesis of ɛ-polylysine (Hirohara et al., 2006). Various TCA cycle intermediates are involved in ɛ-polylysine production, but citric acid and α-ketoglutaric acid are the two important TCA cycle intermediates which can greatly influence the ɛpolylysine production (Bankar and Singhal, 2013). The important component for the synthesis ɛ-polylysine is lysine, which is the direct amino acid influencing the ɛ-polylysine production. In general, the synthesis of lysine occurs in the aspartate pathway. Addition of 2 mM lysine resulted in the maximum ɛ-polylysine production of 1.86 g/L (Fig. 3). The increase in ɛ-polylysine production after lysine addition is due to the utilization of lysine as a precursor for ɛ-polylysine production. Addition of 2 mM aspartate also increased the ɛ-polylysine production up to 1.91 g/L whereas further increase in the aspartate concentration decreased the ɛ-polylysine production. This is
3.7. Feasibility analysis with literature data Based on the process design, raw material and many other factors greatly influence the cost of biofuel ($1.65–$33.16 Gal−1). The metaanalysis methods reduce the cost considerably in the range of $11.68–$ 14.31 Gal−1 (Quinn and Davis, 2015). The cost of biodiesel production through open ponds and closed photobioreactors were found to be $9.84 Gal−1 and $20.53 Gal−1 respectively (Davis et al., 2011). The fuel cost can also be reduced by $1.59–3.67 Gal−1 if the techno-
Fig. 3. Effect of critical amino acids and precursors on ɛ-polylysine production. Conditions: 50 g/L sugar, 0.15 g/L MgSO4, 5% (v/v) inoculum, 90 h at 30 °C with 180 rpm. Data points were mean of triplicate values with the error bar showing standard deviations.
Fig. 4. Time-course of ɛ-polylysine production and biomass content. Conditions: 50 g/L sugar, 0.15 g/L MgSO4, 4 mM citric acid, 5% (v/v) inoculum, 30 °C with 180 rpm. Data points were mean of triplicate values with the error bar showing standard deviations. 285
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the limits when compared to ASTM and EN standards. The satisfactory methyl ester properties were obtained from the direct transesterification of Chlamydomonas sp.
Table 2 Properties of Chlamydomonas sp. methyl ester. Properties
Values
EN
ASTM
SV IV CN DU
209.65 54.16 60.15 51.44
NA 120 > 51 NA
NA NA > 47 NA
4. Conclusion The present study demonstrated the effective harvesting method of microalgae and integrated production of methyl ester and ɛ-polylysine as a biorefinery approach. The DMC mediated transesterification of Chlamydomonas sp. efficiently produces the methyl ester and valuable glycerol carbonate. The sugar and MgSO4 at optimal concentration play an important role in the ɛ-polylysine production by Streptomyces sp. The addition of TCA cycle intermediates in the fermentation medium considerably increases the ɛ-polylysine production. The promising path of the biorefinery concept in the present study will help to develop the economy based sustainable fuels and value-added compounds production in the near future.
Units for properties: SV (mg KOH g−1); IV (g I2 100 g−1); CN (> 47); DU (wt %). EN – European standards, ASTM – American society for testing and materials; NA – not available.
economic analysis was done based on mass balance studies (Leung and Guo, 2006). Novel findings can improve the economic feasibility of biodiesel as alternative for crude oil. Biorefinery approach can be adopted to cut the biofuel prices by producing more than one product. Silva et al. (2014) reported that 33% of the cost can be cut by utilizing both lipid and carbohydrate. In the present study, utilization of Chlamydomonas sp. as a biorefinery approach for the production of more than one product such as methyl ester, glycerol carbonate and ɛ-polylysine can be the promising biorefinery model. The overall mass balance of methyl ester and ɛ-polylysine production from Chlamydomonas sp. is shown in Fig. 1. One gram of biomass contains 0.25 g of transesterifiable lipids which can be used to produce 0.23 g of methyl ester, and 0.18 g of fermentable sugar was obtained from hydrolysate of spent biomass, which is used for the production of 0.0080 g of ɛ-polylysine. The remaining spent biomass residues can be further used as a substrate for biogas or as fertilizer.
Acknowledgements R. Sivaramakrishnan is thankful to the Graduate School and Faculty of Science, Chulalongkorn University (CU), for senior post-doctoral fellowship from Ratchadaphiseksomphot Endowment Fund. A.I. acknowledges the research grant from CU on the Frontier Research Energy Cluster (CU-59-048-EN) and from the Thailand Research Fund (IRG 5780008). Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.biortech.2018.10.026.
3.8. Fatty acid compositions of Chlamydomonas sp. and its fuel properties The fatty acid compositions of Chlamydomonas sp. were found to be palmitic acid: 54.12 ± 0.67%, palmitoleic acid: 1.38 ± 0.04%, stearic acid: 14. 86 ± 0.77%, oleic acid: 9.22 ± 0.62%, linoleic acid: 12.31 ± 0.52% and linolenic acid: 8.11 ± 0.43%. The overall saturated fatty acid content was 68.98% and mono and poly unsaturated fatty acids were 31.02%. The saturation index (SI) is defined as the stearic to oleic acid ratio, the SI of obtained methyl ester was found to be 1.6 ± 0.2. The palmitic acid is the major component of Chlamydomonas sp. Palmitic acid is the suitable composition for the biodiesel production and ensures good cold flow property (Gua et al., 2018). Transesterification performed with palm oil using DMC improves the fuel quality (Gua et al., 2018). The combination of both saturated and unsaturated fatty acids is important for the good fuel quality (Miao et al., 2009). The Chlamydomonas sp. used in this study showed 69% saturated and 31% unsaturated fatty acids which was in agreement with the previous study (Miao et al., 2009). The methyl ester properties were determined according to the calculations mentioned in the methods section. The results were compared with the values mentioned in American society for testing and materials (ASTM D6751) (Table 2). The cetane number (CN) is an important index of the methyl ester quality which relates to combustion properties and the ignition delay time. Hence CN value influences the engine performance and high CN value (> 47) showed good engine performance as well as low smoke emission. The obtained CN value in the present study was in compliance with the standard value and found to be satisfactory. Iodine value (IV) is a measure of total unsaturation of methyl ester and the value is often used to represent the oxidative stability of fuel. Hence high IV value reduces the oxidative stability. In the present study, the obtained IV value was satisfactory (< 120) and ensures the satisfactory oxidative stability. Degree of unsaturation (DU) is a measure of oxidative stability which is calculated from the amount of monounsaturated and polyunsaturated fatty acids. The DU value in this study agreed with the results reported by Sivaramakrishnan and Incharoensakdi (2018a,b,c). The important properties of methyl ester from the present study listed in Table 2 showed that all the values are in
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Chlamydomonas sp. as dynamic biorefinery feedstock for the production of methyl ester and ɛ-polylysine Ramachandran Sivaramakrishnana, Subramaniyam Sureshb, Aran Incharoensakdia, a b
T
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Laboratory of Cyanobacterial Biotechnology, Department of Biochemistry, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand Department of Chemistry, Ramapuram Campus, SRM Institute of Science and Technology, Chennai, India
G R A P H I C A L A B S T R A C T
A R T I C LE I N FO
A B S T R A C T
Keywords: Biorefinery Chlamydomonas sp. Flocculation ɛ-Polylysine Methyl ester Streptomyces sp.
An integrated production of methyl ester and ɛ-polylysine from Chlamydomonas sp. was studied using biorefinery approach. The harvesting efficiency of Chlamydomonas sp. was increased up to 92% by treatment with a flocculant FeCl3 at 100 mg/L for 30 min. The DMC (dimethyl carbonate) mediated enzyme catalyzed in-situ transesterification of Chlamydomonas sp. yielded the maximum methyl ester of 92% under optimized conditions. The valued-added product ɛ-polylysine was produced from hydrolysate obtained from the spent biomass of Chlamydomonas sp. using Streptomyces sp. The key components of sugar and MgSO4 used for ɛ-polysine production were optimized whereby the maximum ɛ-polylysine production was achieved at 50 g/L sugar and 0.3 g/ L MgSO4. The ɛ-polylysine production was further enhanced by supplementation of important amino acids (lysine and aspartate) and TCA cycle intermediates (citric acid and α-ketoglutaric acid). The maximum ɛpolylysine production of 2.24 g/L was found with 4 mM citric acid supplementation after 110 h.
1. Introduction Environmental problems coupled with rapid depletion of fossil fuel and its resources prompted researchers to find alternative renewable resources and its commercialization (Sivaramakrishnan and Incharoensakdi, 2017a). The biomass from microalgae with high oil content is a promising feedstock for the renewable resources. Compared
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with plants, microalgae can produce more oil per hectare with a shorter production cycle (Chew et al., 2017). The coupling of algae biofuels with high value compounds production widens the market opportunities (Barreiro et al., 2014), which fits well with a recent trend of biorefinery concept (Sivaramakrishnan and Incharoensakdi, 2018a). The biorefineries at industrial scale involve the production of multiple products through the integration of the production configuration. The
Corresponding author. E-mail address: [email protected] (A. Incharoensakdi).
https://doi.org/10.1016/j.biortech.2018.10.026 Received 27 August 2018; Received in revised form 9 October 2018; Accepted 10 October 2018 Available online 11 October 2018 0960-8524/ © 2018 Elsevier Ltd. All rights reserved.
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stone quarry pond water with the accession number of KR025878 (GenBank) (Sivaramakrishnan and Incharoensakdi, 2017c). The culture was grown and maintained in BG11, under continuous illumination (50 μmol photons/m2/s) with 100 rpm shaking at 27 ± 1 °C. The culture was regularly monitored under microscope to maintain the purity of the culture. The microalgal slurry was collected from 12 days grown culture and used for the experiments. The biochemical compositions of total lipid, carbohydrate and protein were determined as 27, 21 and 24% (w/w of biomass) respectively.
biorefinery designs employ maximum product output from a single feedstock with high profit (Moncada et al., 2015). For biorefinery approach, it is essential to select the microalgae which contain high amounts of organic matters such as lipids and carbohydrates which can be used for biorefinery approaches. Chlamydomonas sp. is one of the major and important feedstocks for the biofuel applications with mass production (Kao and Ng, 2017). Microalgae contain both transesterifiable and non-transesterifiable lipids (chlorophyll, carotenoids, etc.). Large amounts of neutral lipids suitable for methyl ester production are found in microalgae (Sivaramakrishnan and Incharoensakdi, 2017b). It is clear from the previous study that Chlamydomonas sp. is suitable for the methyl ester production (Chen et al., 2015). The methyl ester production from microalgae involves cultivation, harvesting, lipid extraction and transesterification. For the total methyl ester production, harvesting, lipid extraction and transesterification alone comprise 60% of costs (Sivaramakrishnan and Incharoensakdi, 2018b). The initial harvesting step comprises nearly 20–30% of the total production cost and may vary depending on the technology used due to the behavior or the nature of the microalgae. The efficient method to harvest microalgae can cut the cost considerably in the downstream processes. Microalgae can be harvested using flocculants, centrifugation, sedimentation or flotation processes (Lee et al., 2013). Among these methods, flocculation is an easy and cheap method compared to the centrifugation. Flocculation increases the settling efficiency of microalgae by aggregation leading to sedimentation of microalgae (Choong et al., 2016). The flocculated biomass can be used as the source for the methyl ester production via in-situ transesterification where lipids react with methanol aided by chemical or enzyme catalyst. The disadvantages of chemical catalysts are complex downstream processing such as glycerol recovery, removal of organic and inorganic chemicals from the reaction mixture and the soap formation by alkali catalysts. Hence, enzymatic transesterification offers a preferable alternative method even with high free fatty acids at lower reaction temperatures (30–50 °C) (Lee et al., 2016). The dimethyl carbonate (DMC) can be used as the acyl acceptor over alcohol like methanol or ethanol. The use of DMC for the transesterification also has important benefits such as inexpensive, non-corrosive, non-toxic, neutral, odorless and ecofriendly (Sivaramakrishnan and Muthukumar, 2013). For biorefinery, spent biomass (i.e. biomass after being used for insitu transesterification) has the carbohydrate content which can be further fermented to produce valuable compounds such as ɛ-polylysine and ethanol. ɛ-Polylysine is a basic homopolymer which contains 20–30 residues of L-lysine with an ɛ-amino group and an α-carboxyl group. This polymer has antimicrobial activity against gram-negative and gram-positive bacteria (Geornaras et al., 2007). Due to the biodegradable nature of the ɛ-polylysine, it can be used as a safe food preservative. Moreover, ɛ-polylysine has various applications, i.e. an emulsifying agent, a dietary agent, an anticancer agent enhancer, a gene delivery carrier, a biochip coating, as well as a micro or nano capsule for drug delivery (Choi et al., 2000). Currently there are numerous studies on methyl ester from microalgae; however, there are few studies on integrated production of methyl ester and value-added products. Particularly, no study was reported on combined production of methyl ester and ɛ-polylysine. This work aimed to investigate the potential of lipid and carbohydrate containing biomass from Chlamydomonas sp. for the integrated production of methyl ester and ɛ-polylysine. Moreover, the objective of the work also extends to the determination of the flocculation condition for the Chlamydomonas sp. which added the benefit towards the biorefinery approach.
2.2. Harvesting efficiency Harvesting studies using flocculants were carried out in 20 ml cylindrical glass tubes. The algal suspension was mixed and swirled with flocculant and left to sediment. Various concentrations of flocculants were used in the range of 20–140 mg/L. The harvesting efficiency was calculated using the following equation,
Harvesting efficiency (%) =
(ODi−ODf ) × 100 ODi
ODi and ODf are initial and final optical density at 730 nm of non-sedimenting portion of sample respectively. 2.3. In-situ transesterification Lipid content in Chlamydomonas sp. was determined by solvent extraction method (Sivaramakrishnan and Incharoensakdi, 2017c). In a screw-capped glass vial, 1 g of dried microalgal biomass was mixed with an appropriate amount of dimethyl carbonate. The immobilized lipase was added to the mixture as a catalyst and appropriate amount of distilled water was added to start the reaction in a temperature controlled shaking incubator (180 rpm) at 45 °C. The reaction was run up to 36 h. The lipase (Novozyme – Lipozyme CAL-B from Candida antartica, Novozymes, Denmark) as a catalyst was immobilized on Celite material with 190 U/g enzyme activity. Lipase immobilization was performed as previously described by Sivaramakrishnan and Muthukumar (2012). Various factors affecting methyl ester yield were determined as follows: solvent algae ratio (2:1, 3:1, 4:1, 5:1, 6:1, 7:1 and 8:1), catalyst concentration (2, 4, 6, 8, 10 and 12 w/w %), water addition (0.2, 0.4, 0.6, 0.8, 1 and 1.2 w/w %), reaction temperature (35, 40, 45, 50, 55, 60 and 65 °C), reaction time (6, 12, 18, 24, 30 and 36 h) and the analysis of by-product (glycerol carbonate, glycerol dicarbonate and glycerol). After the reaction was stopped, the mixture was filtered to separate methyl ester and biomass, the traces of methyl ester present in the residues were collected by solvent (DMC) wash. The recovered samples were centrifuged at 20,000g for 15 min to remove the remaining contaminants. The obtained methyl ester content was determined, and its properties were analyzed. 2.4. Fermentative production of ɛ-polylysine Streptomyces albulus (MTCC 503) (Streptomyces sp.) obtained from microbial type culture collection, Chandigarh, India was used for the production of ɛ-polylysine utilizing microalgal hydrolysate as substrate. The hydrolysate was prepared by acid hydrolysis of the spent biomass (i.e. after in-situ transesterification). The hydrolysis was optimized, and the best conditions were found to be 0.3 N H2SO4 with autoclave (120 °C and 15 psi) for 20 min (data not shown). The fermentative medium contained 50 g/L sugars, 10 g/L peptone, 8 g/L (NH4)2SO4, 0.5 g/L MgSO4·7H20, 0.03 g/L FeSO4·7H2O, 0.04 g/L ZnSO4, 0.8 g/L K2HPO4, 1.36 g/L KH2PO4 and the pH was adjusted to 6.8 using 0.1 M phosphate buffer (pH 6.8) The fermentation was initiated by adding 2.5 ml of the culture (8.0 × 108 cells/ml) and grown for 110 h at 30 °C with shaking at 180 rpm. The analysis of ɛ-polylysine production was determined by harvesting the cells after the fermentation and centrifuging at 10,000g for 10 min. One ml of 1 mM methyl orange was
2. Materials and methods 2.1. Microorganism and culture conditions The Chlamydomonas sp. used in this study was isolated from the 282
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added to 1 ml of the supernatant followed by mixing for 60 min at 37 °C. The incubated samples were centrifuged again at 10,000g for 10 min and the supernatant was measured for its absorbance at 465 nm on UV–Vis spectrophotometer (Itzhaki, 1972). 2.5. Analytical methods The determination of methyl ester and glycerol carbonate contents was done by using gas chromatography according to Sivaramakrishnan and Incharoensakdi (2016). The methyl ester properties of saponification value (SV), iodine value (IV), cetane number (CN) and degree of unsaturation (DU) were determined as described by Sivaramakrishnan and Incharoensakdi (2017d). 2.6. Statistical analysis The results obtained are mean of triplicate values and the error bars are standard deviation (means ± SD, n = 3) values. The statistical significance (p < 0.05) was analyzed by t-test comparisons using graph pad software. 3. Results and discussion 3.1. Process overview The present study involved the production of methyl ester using Chlamydomonas sp. lipids and its carbohydrate content was utilized for the ɛ-polylysine production. The overall processing steps are divided into three different stages. 1) Concentration of microalgae by flocculation method using FeCl3 as a flocculant, 2) Production of methyl ester by in-situ transesterification of Chlamydomonas sp. using immobilized lipase as a catalyst and DMC as a solvent, and 3) Spent biomass after insitu transesterification was further hydrolyzed and the hydrolysate was used for the ɛ-polylysine production. The overall scheme of the present study is depicted in Fig. 1 showing the utilization of microalgal feedstock as a biorefinery model for the methyl ester and ɛ-polylysine production.
Fig. 2. (a) Effect of flocculants concentration on harvesting. Conditions: 0.25 g/ L of biomass and 1 h. (b) Effect of flocculant (FeCl3) on harvesting at different biomass densities. Conditions: 10 mg/L of flocculant, 0.25 g/L of biomass and 1 h. Data points were mean of triplicate values with the error bar showing standard deviations.
Al2(SO4)3 are considered the best flocculants for Chlamydomonas sp. and they were used for further experiments. The concentrations of FeCl3 and Al2(SO4)3 were varied to achieve the maximum harvesting efficiency. As shown in Fig. 2a, increasing the concentration of flocculants up to 100 mg/L increased the harvesting efficiency. Comparatively, FeCl3 showed higher harvesting efficiency than the Al2(SO4)3; however, both the flocculants showed their maximum harvesting efficiency at 100 mg/L. The obtained results agreed with the previously reported study, where 200 mg/L of FeCl3 was used as a coagulant at low pH to harvest Chlorella sp. (Seo et al., 2015). Different microalgae have different properties including shape, size, specific gravity, charge and motility, all of which can affect the harvesting efficiency (Udom et al., 2013). Moreover, harvesting efficiency of algae is also affected by the constituents of the culture medium, pH and ionic strength. The use of multivalent metal cations for the flocculation has been reported as a simple method with the minimum energy requirement. Such metal ions in the growth medium coagulate negatively charged microalgal cells leading to flocculation of cells. The changes in physical properties of the microalgae also affect the flocculation (Liu et al., 2013). The suspension of cells with flocculants results in charge neutralization and polysaccharide bridging ensures maximum aggregation and agglomeration of cells. Hence it is necessary to add a flocculating agent to achieve maximum harvesting efficiency to obtain larger flocs which settle down the bottom of the vessel and thereby leaving a clear supernatant. Different algal cell concentrations were varied to determine the suitable cell concentration for the harvesting (biomass concentration – 0.25–2 g/L) and the absence of FeCl3 is set as control. Increasing biomass concentration up to 1.5 g/L increased the harvesting efficiency (Fig. 2b). Using the FeCl3 (100 mg/L) with 1.5 g/L of biomass yielded
3.2. Harvesting of microalgae by flocculation In the present study, the four different flocculants were tested for their efficiency of harvesting Chlamydomonas sp. It was observed that the flocculants greatly influence the harvesting efficiency of Chlamydomonas sp. Among the four flocculants at 80 mg/L FeCl3 and Al2(SO4)3 showed maximum harvesting efficiency of 73 and 66% respectively. Polyacrylamide showed very low harvesting efficiency (35%) which is close to the control value (28%) whereas NaOH showed moderate harvesting efficiency of 47%. From the results, FeCl3 and
Fig. 1. Schematic representation of integrated production of methyl ester and ɛpolylysine. 283
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biomass ratio is considered as the optimum and used for further studies. Lipozyme is an important enzyme and highly preferable for the lipase mediated transesterification (Wang et al., 2017). The enzyme loading is a crucial factor for successful industrial application. To study the effect of enzyme content on in-situ transesterification, different enzyme contents ranging from 2 to 12% (w/w of microalgae) were studied. The increase in the catalyst content from 2 to 8% (w/w of microalgae) resulted in an increase of the methyl ester yield (Table 1). Increasing enzyme content above 8% (w/w of microalgae) decreased the methyl ester yield. The mass transfer resistance contributes to the decrease in the yield on higher enzyme content. It is economically necessary to determine the optimum enzyme content to cut the cost of enzyme wastage. Water content is another important parameter which can affect the activity and stability of the lipases. The methyl ester yield of in-situ transesterification was increased with the increase in water addition up to 0.8% (w/w of microalgae) (Table 1). A further increase in the water addition decreased the yield due to enzyme inactivation. To maintain an interfacial area of lipase it is necessary to add essential water. Excessive water also affects the enzyme and reduces the product yield. Excess water forms hydrogen bonds with the protein functional group (Sivaramakrishnan and Incharoensakdi, 2017a). The reaction temperature also affects the in-situ transesterification of Chlamydomonas sp. lipids. Increasing temperature up to 50 °C increased the methy ester yield, whereas the yield decreased considerably at higher than 50 °C (Table 1). The optimal temperature at which the enzyme operates is very crucial. The increase of temperature increases the rate of enzyme-catalyzed reaction; however, an increased temperature damages the enzyme structure leading to a decrease of reaction rate (Huang et al., 2015). Hence, the determination of optimal reaction temperature is necessary to achieve the maximal yield with maximum reaction rate. In this study, the enzyme requires 50 °C to perform transesterification of maximum lipids into methyl esters. The influence of reaction time on in-situ transesterification of Chlamydomonas sp. lipids is shown in Table 1. The methyl ester yield was increased from 22 to 92% when the reaction time was increased from 6 to 24 h. No significant change was observed in methyl ester yield after 24 h. These results were in agreement with those reported by Shuit et al. (2010). The reaction time of 24 h was considered as an optimal reaction time since longer reaction time would add extra energy cost. Glycerol carbonate (1.6 ± 0.21%), glycerol dicarbonate (5.1 ± 0.36%) and glycerol (1.2 ± 0.12%) all of which are by products during DMC mediated transesterification were obtained at the optimized conditions (Table 1). Glycerol molecule, detached from the triglyceride during transesterification, reacts with DMC to produce glycerol carbonate molecule. Glycerol carbonate has many applications, for example, in detergents, surfactants, paint and coatings, and gas separation membrane industries (Jung et al., 2017). The glycerol carbonate production is the promising by-product with regard to biorefinery approach. Moreover, the DMC mediated glycerol carbonate synthesis in this study is more environmentally advantageous when compared to chemically synthesized glycerol carbonate. At the optimized conditions of in-situ transesterification using DMC, the highest methyl ester yield of 92% was obtained from the Chlamydomonas sp. simultaneously with 1.6% glycerol carbonate production.
Table 1 Parameters influencing in-situ transesterification of Chlamydomonas sp. Parameters DMC/biomass 2 3 4 5 6 7 8
Yield (%) ratio (ml/g) 33 ± 1.52 45 ± 1.52 54 ± 0.57 63 ± 2.08 72 ± 2.08 67 ± 3.21 64 ± 2.08
Conditions
0.5 g Chlamydomonas sp., 10% catalyst (w/w of microalgae), 1% distilled water (w/w of microalgae), 45 °C and 36 h reaction time
Catalyst content (w/w of biomass) 2 38 ± 2.08 0.5 g Chlamydomonas sp., 6 ml DMC/g microalgae, 1% 4 51 ± 1.52 distilled water (w/w of microalgae), 45 °C and 36 h 6 64 ± 1.00 reaction time 8 78 ± 1.00 10 72 ± 2.51 12 66 ± 1.52 Water content (w/w of biomass) 0.2 56 ± 2.88 0.5 g Chlamydomonas sp., 6 ml DMC/g microalgae, 8% 0.4 62 ± 0.57 catalyst (w/w of microalgae), 45 °C and 36 h reaction 0.6 71 ± 0.57 time 0.8 85 ± 1.52 1 78 ± 1.32 1.2 69 ± 1.21 Temperature (°C) 35 57 ± 1.52 40 73 ± 1.52 45 85 ± 1.52 50 92 ± 2.0 55 81 ± 0.57 60 66 ± 3.6 65 43 ± 1.52 Time (min) 6 12 18 24 30 36
22 46 71 92 92 92
± ± ± ± ± ±
0.57 0.57 1.73 1.52 0.57 0.57
0.5 g Chlamydomonas sp., 6 ml DMC/g microalgae, 8% catalyst (w/w of microalgae), 0.8% water addition (w/w of microalgae) and 36 h reaction time
0.5 g Chlamydomonas sp., 6 ml DMC/g microalgae, 8% catalyst (w/w of microalgae), 0.8% water addition (w/w of microalgae), 50 °C
highest harvesting efficiency of 92% in 30 min. The sample without FeCl3 required 180 min to achieve its maximum of 80% harvesting efficiency. Above the 1.5 g/L of biomass the harvesting efficiency decreased. The results were in agreement with those reported by Zhao et al. (2014). Hence ferric chloride was the most suitable flocculant to harvest the Chlamydomonas sp. Moreover, addition of ferric chloride did not affect the algal growth and all cell components. The growth medium with low concentrations of ferric chloride can be reused although alum compounds can affect the microalgal growth significantly (Zhao et al., 2014). The FeCl3 at 100 mg/L flocculates Chlamydomonas sp. at 1.5 g/L of biomass efficiently in a short time with minimum energy. 3.3. In-situ transesterification Various process parameters involved in the in-situ transesterification of Chlamydomonas sp. were optimized and the results are shown in Table 1. The solvent DMC was used to simultaneously extract and transesterify the Chlamydomonas sp. lipids. The optimal DMC to biomass ratio not only increases the yield, but also avoids the excess use of solvent. The lipid transformation into methyl ester increased with the increase in the DMC to biomass ratio and the maximum methyl ester yield was achieved at a ratio of 6:1 (Table 1). On the other hand, a decrease in the yield was observed with the increase of DMC to biomass ratio from 6:1 to 8:1. Below the optimized level, the DMC cannot transform all the available lipids into methyl ester (Lee et al., 2010). Addition of DMC higher than the optimum level dilutes the lipid concentration resulting in inefficient transesterification. The 6:1 DMC to
3.4. Influence of nutrient composition on ɛ-polylysine production It is necessary to enhance the ɛ-polylysine production after the methyl ester production. The improved ɛ-polylysine production increases the chance of biorefinery commercialization. Hence, the critical medium compositions were optimized to improve the ɛ-polylysine production. It is very important to obtain the optimal concentration of sugar for the maximum ɛ-polylysine yield. The sugar concentrations in the hydrolysate of spent biomass were varied from 35 to 60 g/L and tested for their effect on ɛ-polylysine production. The results clearly 284
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because the addition of amino acid above the maximum extent inhibits the enzymes involved in the ɛ-polylysine synthesis pathway, such as aspartokinase or phosphoenol pyruvate carboxylase (Bankar and Singhal, 2011). The influence of citric acid and α-ketoglutaric acid on ɛ-polylysine production is shown in Fig. 3. At 4 mM citric acid and α-ketoglutaric acid showed the highest ɛ-polylysine yields. Among the various organic acids, citric acid is the important organic acid, which can greatly influence the ɛ-polylysine production (Bankar and Singhal, 2013). Addition of α-ketoglutaric acid also showed satisfactory results, but the yield was slightly lower than that of the citric acid at optimum concentration. Hence, citric acid is the critical TCA cycle component which greatly affects the ɛ-polylysine production. Overall, the concentration of sugar plays an important role in the ɛpolylysine production and showed the maximum yield at 50 g/L of sugar. The yield was further improved after MgSO4 optimization. The addition of key amino acids and TCA cycle intermediates considerably increases the ɛ-polylysine production. Particularly, citric acid (4 mM) showed highest improvement in the ɛ-polylysine production.
indicate that the sugar concentration in the fermentation plays a vital role in the ɛ-polylysine production. The 50 g/L sugar gave the highest ɛpolylysine yield. Shukla and Mishra (2012) reported that the glucose and glycerol in a 1:1 ratio increases the ɛ-polylysine production. The yield was further increased when molasses were used as the carbon source. The increased ɛ-polylysine production was reported in Streptomyces sp. when glucose was a carbon source and when added with the intermediates in the TCA cycle (Shih and Shen, 2006). In the present study 50 g/L sugar produced a maximum of 1.54 g/L ɛ-polylysine. When testing for the effect of glucose on the yield of ɛ-polylysine, it was found that the maximum ɛ-polylysine production of 1.77 g/L was obtained at 50 g/L glucose. Hence it is clear that the 50 g/L sugar in the hydrolysate of spent biomass contained fermentable sugar producing 1.54 g/L ɛ-polylysine, which accounted for about 90% of the yield produced by 50 g/L glucose. This indicated the presence of non-fermentable sugar in the hydrolysate of spent biomass. Another critical component in the media is magnesium, which is necessary for the cell division in bacteria. Apart from the cell division, magnesium is important for the activation of the ɛ-polylysine synthetase reaction (Chheda and Vernekar, 2015). The MgSO4 concentrations were varied from 0.15 to 0.6 g/L to determine their effect on ɛ-polylysine production. The results showed that 0.15 g/L MgSO4 yielded ɛpolylysine of 1.54 g/L. Increasing MgSO4 to 0.3 g/L yielded the maximum ɛ-polylysine of 1.78 g/L. The appropriate concentration of sugar (50 g/L) and MgSO4 (0.3 g/L) is required to produce maximum yield of ɛ-polylysine.
3.6. Time-course of ɛ-polylysine and biomass content The time-course of ɛ-polylysine yield and biomass content is shown in Fig. 4. Increasing time increased both biomass and ɛ-polylysine up to 110 h, at which time the maximum ɛ-polylysine production was obtained at about 2.24 g/L. As reported in the previous studies, maximum ɛ-polylysine production by Streptomyces sp. was achieved during 48–72 h and thereafter the production rate was reduced (Hirohara et al., 2006). For Streptomyces sp., the maximum ɛ-polylysine production was achieved in 144 h (Xu et al., 2015), which was similarly observed in the study with Streptomyces albulus PD-1 (Xia et al., 2014). The decrease in the ɛ-polylysine production after 120 h is due to the ɛpolylysine degrading enzyme released by the organism during late stationary phase which changes the medium pH to acidic condition (Yamanaka et al., 2010). In the present study, the maximum product yield of ɛ-polylysine was achieved at 110 h.
3.5. Effect of critical amino acids and TCA cycle intermediates on ɛpolylysine production In the ɛ-polylysine synthesis pathway, aspartate family amino acids are primarily involved, which are L-lysine, L-aspartate, L-methonine, and L-threonine. There is no significant change of ɛ-polylysine yield with the addition of methionine and threonine because these two amino acids are not participating in the biosynthesis of ɛ-polylysine (Hirohara et al., 2006). Various TCA cycle intermediates are involved in ɛ-polylysine production, but citric acid and α-ketoglutaric acid are the two important TCA cycle intermediates which can greatly influence the ɛpolylysine production (Bankar and Singhal, 2013). The important component for the synthesis ɛ-polylysine is lysine, which is the direct amino acid influencing the ɛ-polylysine production. In general, the synthesis of lysine occurs in the aspartate pathway. Addition of 2 mM lysine resulted in the maximum ɛ-polylysine production of 1.86 g/L (Fig. 3). The increase in ɛ-polylysine production after lysine addition is due to the utilization of lysine as a precursor for ɛ-polylysine production. Addition of 2 mM aspartate also increased the ɛ-polylysine production up to 1.91 g/L whereas further increase in the aspartate concentration decreased the ɛ-polylysine production. This is
3.7. Feasibility analysis with literature data Based on the process design, raw material and many other factors greatly influence the cost of biofuel ($1.65–$33.16 Gal−1). The metaanalysis methods reduce the cost considerably in the range of $11.68–$ 14.31 Gal−1 (Quinn and Davis, 2015). The cost of biodiesel production through open ponds and closed photobioreactors were found to be $9.84 Gal−1 and $20.53 Gal−1 respectively (Davis et al., 2011). The fuel cost can also be reduced by $1.59–3.67 Gal−1 if the techno-
Fig. 3. Effect of critical amino acids and precursors on ɛ-polylysine production. Conditions: 50 g/L sugar, 0.15 g/L MgSO4, 5% (v/v) inoculum, 90 h at 30 °C with 180 rpm. Data points were mean of triplicate values with the error bar showing standard deviations.
Fig. 4. Time-course of ɛ-polylysine production and biomass content. Conditions: 50 g/L sugar, 0.15 g/L MgSO4, 4 mM citric acid, 5% (v/v) inoculum, 30 °C with 180 rpm. Data points were mean of triplicate values with the error bar showing standard deviations. 285
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the limits when compared to ASTM and EN standards. The satisfactory methyl ester properties were obtained from the direct transesterification of Chlamydomonas sp.
Table 2 Properties of Chlamydomonas sp. methyl ester. Properties
Values
EN
ASTM
SV IV CN DU
209.65 54.16 60.15 51.44
NA 120 > 51 NA
NA NA > 47 NA
4. Conclusion The present study demonstrated the effective harvesting method of microalgae and integrated production of methyl ester and ɛ-polylysine as a biorefinery approach. The DMC mediated transesterification of Chlamydomonas sp. efficiently produces the methyl ester and valuable glycerol carbonate. The sugar and MgSO4 at optimal concentration play an important role in the ɛ-polylysine production by Streptomyces sp. The addition of TCA cycle intermediates in the fermentation medium considerably increases the ɛ-polylysine production. The promising path of the biorefinery concept in the present study will help to develop the economy based sustainable fuels and value-added compounds production in the near future.
Units for properties: SV (mg KOH g−1); IV (g I2 100 g−1); CN (> 47); DU (wt %). EN – European standards, ASTM – American society for testing and materials; NA – not available.
economic analysis was done based on mass balance studies (Leung and Guo, 2006). Novel findings can improve the economic feasibility of biodiesel as alternative for crude oil. Biorefinery approach can be adopted to cut the biofuel prices by producing more than one product. Silva et al. (2014) reported that 33% of the cost can be cut by utilizing both lipid and carbohydrate. In the present study, utilization of Chlamydomonas sp. as a biorefinery approach for the production of more than one product such as methyl ester, glycerol carbonate and ɛ-polylysine can be the promising biorefinery model. The overall mass balance of methyl ester and ɛ-polylysine production from Chlamydomonas sp. is shown in Fig. 1. One gram of biomass contains 0.25 g of transesterifiable lipids which can be used to produce 0.23 g of methyl ester, and 0.18 g of fermentable sugar was obtained from hydrolysate of spent biomass, which is used for the production of 0.0080 g of ɛ-polylysine. The remaining spent biomass residues can be further used as a substrate for biogas or as fertilizer.
Acknowledgements R. Sivaramakrishnan is thankful to the Graduate School and Faculty of Science, Chulalongkorn University (CU), for senior post-doctoral fellowship from Ratchadaphiseksomphot Endowment Fund. A.I. acknowledges the research grant from CU on the Frontier Research Energy Cluster (CU-59-048-EN) and from the Thailand Research Fund (IRG 5780008). Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.biortech.2018.10.026.
3.8. Fatty acid compositions of Chlamydomonas sp. and its fuel properties The fatty acid compositions of Chlamydomonas sp. were found to be palmitic acid: 54.12 ± 0.67%, palmitoleic acid: 1.38 ± 0.04%, stearic acid: 14. 86 ± 0.77%, oleic acid: 9.22 ± 0.62%, linoleic acid: 12.31 ± 0.52% and linolenic acid: 8.11 ± 0.43%. The overall saturated fatty acid content was 68.98% and mono and poly unsaturated fatty acids were 31.02%. The saturation index (SI) is defined as the stearic to oleic acid ratio, the SI of obtained methyl ester was found to be 1.6 ± 0.2. The palmitic acid is the major component of Chlamydomonas sp. Palmitic acid is the suitable composition for the biodiesel production and ensures good cold flow property (Gua et al., 2018). Transesterification performed with palm oil using DMC improves the fuel quality (Gua et al., 2018). The combination of both saturated and unsaturated fatty acids is important for the good fuel quality (Miao et al., 2009). The Chlamydomonas sp. used in this study showed 69% saturated and 31% unsaturated fatty acids which was in agreement with the previous study (Miao et al., 2009). The methyl ester properties were determined according to the calculations mentioned in the methods section. The results were compared with the values mentioned in American society for testing and materials (ASTM D6751) (Table 2). The cetane number (CN) is an important index of the methyl ester quality which relates to combustion properties and the ignition delay time. Hence CN value influences the engine performance and high CN value (> 47) showed good engine performance as well as low smoke emission. The obtained CN value in the present study was in compliance with the standard value and found to be satisfactory. Iodine value (IV) is a measure of total unsaturation of methyl ester and the value is often used to represent the oxidative stability of fuel. Hence high IV value reduces the oxidative stability. In the present study, the obtained IV value was satisfactory (< 120) and ensures the satisfactory oxidative stability. Degree of unsaturation (DU) is a measure of oxidative stability which is calculated from the amount of monounsaturated and polyunsaturated fatty acids. The DU value in this study agreed with the results reported by Sivaramakrishnan and Incharoensakdi (2018a,b,c). The important properties of methyl ester from the present study listed in Table 2 showed that all the values are in
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