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Prospective technology on bioethanol production from photofermentation
Bioresource Technology 181 (2015) 330–337
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Prospective technology on bioethanol production from photofermentation Rosangela Lucio Costa, Thamayne Valadares Oliveira, Juliana de Souza Ferreira, Vicelma Luiz Cardoso, Fabiana Regina Xavier Batista ⇑ School of Chemical Engineering, Federal University of Uberlandia. Av. Joao Naves de Avila 2121, Santa Monica 38408-144, Uberlandia, MG, Brazil
h i g h l i g h t s C. reinhardtii and R. capsulatus were utilized to the ethanol production. Ethanol was produced from sulfur deprivation and mixotrophic carbon source. Hybrid system and co-cultivation increase the ethanol production by photofermentation.
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Article history: Received 21 November 2014 Received in revised form 20 January 2015 Accepted 22 January 2015 Available online 30 January 2015 Keywords: Ethanol Chlamydomonas reinhardtii Rhodobacter capsulatus Hybrid system Co-culture
a b s t r a c t The most important global demand is the energy supply from alternative source. Ethanol may be considered an environmental friendly fuel that has been produced by feedstock. The production of ethanol by microalgae represent a process with reduced environmental impact with efficient CO2 fixation and requiring less arable land. This work studied the production of ethanol from green alga Chlamydomonas reinhardtii through the cellular metabolism in a light/dark cycle at 25 °C in a TAP medium with sulfur depletion. The parameters evaluated were inoculum concentration and the medium supplementation with mixotrophic carbon sources. The combination of C. reinhardtii and Rhodobacter capsulatus through a hybrid or co-culture systems was also investigated as well. C. reinhardtii maintained in TAP-S produced 19.25 ± 4.16 g/L (ethanol). In addition, in a hybrid system, with medium initially supplemented with milk whey permeated and the algal effluent used by R. capsulatus, the ethanol production achieved 19.94 ± 2.67 g/L. Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction The use of ethanol to replace oil is the most viable way to ensure a sustainable future. However, up to now more emphasis has been given on the yeast performance in reactors for bioethanol production (Andrietta et al., 2008). On the other hand, considering the advantages of microalgae culture such as rapid growth rate and productivity (Li et al., 2008) and their use to minimize contamination, since microalgae may to applied in wastewater treatment from inorganic salts (NH+4, NO3 , PO34 ) using them as nutrient materials (Mata et al., 2010), Chlamydomonas reinhardtii could be preferentially selected as a prospective biological system for bioethanol production instead of yeast. Concerning on the application of microalgae, recent research have shown that a diversity of strains may be cultivated to produce ⇑ Corresponding author. Tel.: +55 34 32309400. E-mail address: [email protected] (F.R.X. Batista). http://dx.doi.org/10.1016/j.biortech.2015.01.090 0960-8524/Ó 2015 Elsevier Ltd. All rights reserved.
biodiesel, hydrogen, methane and ethanol. Specifically to the ethanol production from microalgae, it can be directly synthesized by the cellular metabolism or from the fermentation of microalgae biomass, mainly cellulose and starch that are readily converted to fermentable products by enzymatic or acidic pretreatment technology (Oncel, 2013; Chen et al., 2013). Microalgae based ethanol may be considered as part of integrated process and a promising environmentally friendly alternative, since they could be present a potential for fixing CO2, high growth at high yields and low costs utilizing light as the energy source. Furthermore, they do not require fertile land and portable water as feedstock based biofuel (Chen et al., 2013; John et al., 2011; Hirano et al., 1997). The unicellular green alga C. reinhardtii uses light to grow photoautotrophically or mixotrophically in the presence of small organic substrates (Goff et al., 2009). C. reinhardtii is widely studied for hydrogen production by biophotolysis of water. This method uses the same processes found in plants photosynthesis. Photosynthesis involves the absorption of light by two distinct
R.L. Costa et al. / Bioresource Technology 181 (2015) 330–337
photosynthetic systems operating in series: a water splitting and O2 evolving system (‘‘photosystem II’’ or PSII) and a second photosystem (PSI), which generates the reductant used for CO2 reduction (Das and Veziroglu, 2001). It is important to note that green algae could produce hydrogen not only under light conditions, but under dark anaerobic conditions (Gaffron and Rubin, 1942). Nevertheless, if dark and anaerobic conditions are established on the microalgae cultures, hydrogen yield is quite low corresponding to about onesixth of direct biophotolysis production (Kosourov et al., 2002). Besides hydrogen, the oxidative reaction of starch become incomplete and depending on the type of the microalga, carbon dioxide, ethanol, lactic acid, formic acid, acetic acid, malic acid, glycerol and other compounds are produced in varying proportions (John et al., 2011; Gfeller and Gibbs, 1984). In addition, the medium composition may be altered to induce anaerobic condition. Hemschemeier and Happe (2004) reported that, under sulfur depletion, C. reinhardtii stops growing and accumulates starch. The absence of sulfur forces the algae to reorganize the whole metabolism. Anaerobiosis is established and hydrogen and ethanol could be produced. These authors discussed that the accumulation of ethanol already indicate the activity of pyruvate formate-lyase (PFL). PFL cleaves pyruvate into acetyl-CoA and it can further be reduced to acetaldehyde by acetaldehyde dehydrogenase. Furthermore, ethanol can be formed from cleavage of pyruvate by pyruvate decarboxylase (PDC) producing acetaldehyde as intermediate that is converted to ethanol by alcohol dehydrogenase (ADH). Previous work suggested it is possible to produce ethanol from metabolism of C. reinhardtii maintained in a basal medium supplemented with the mixotrophic carbon source (Costa et al., 2014). And, the purpose of this work was at first to verify the possibility of C. reinhardtii produce ethanol using a basal medium with sulfur depletion added mixotrophic carbon source such as milk whey permeate (rich in lactose) and sodium acetate. In the second step, the algae association with the purple non sulfur bacterium, a Rhodobacter capsulatus, to improve ethanol content into the medium was also evaluated by hybrid system (two stages) and co-culture. The hybrid systems and co-cultures approaches are strategies used in order to improve the yield. The purpose is to integrate microorganisms with distinct biological routes. Thus, the metabolites produced by one type of microorganism may be the substrate to the second type. 2. Methods 2.1. Algal biomass C. reinhardtii CC-124 was purchased from the Canadian Culture Collection, the Chlamydomonas Resource Center. The green alga was maintained in the basal medium Tris Acetate Phosphate (TAP) (Andersen, 2005) at initial pH of 7.0. In order to guarantee enough amounts of cells for the fermentation assays, the algal inoculum was subcultured with the addition of 250 mL of fresh TAP medium in 250 mL of growing culture. The alga was kept in Erlenmeyer (500 mL) at 25 °C under light cycle (night/day) of 12 h at 30 lE m 2 s 1. 2.2. Photosynthetic bacterial biomass R. capsulatus was purchased from DSMZ German Collection of Microorganisms and Cell Culture. The strain was cultivated anaerobically in Erlenmeyers (500 mL) maintained at 30 °C using RCV medium (Weaver et al., 1975), at initial pH of 6.8, under photosynthetic conditions of 30 lE m 2 s 1 (light-grown cells). Sodium glutamate was used as nitrogen source instead of (NH4)2SO4.
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2.3. Biological strategies to ethanol production by photofermentation For ethanol production by fermentation, anaerobic condition was used in all strategies: by C. reinhardtii cultures, hybrid system and co-culture approach. Thus, 50 mL bioreactors were flushed with Ar gas (99.999%) for 3 min. The inoculum was 10 days age for green algae culture and five days age for the purple non sulfur bacteria culture. Five days of time fermentation was used in all assays. The photoperiod of 12 h (12 h dark/12 h light) was used in all experiments with algae. In the hybrid system, in the second stage by R. capsulatus, the assays were carried out under light continuously. 2.3.1. C. reinhardtii under sulfur depletion In the first step of the current work, assays were carried out to investigate the ethanol production by C. reinhardtii in TAP-S (Tris– acetatephosphate-minus-sulfur) medium. The evaluated variables were the initial cell concentration (0.05, 0.10 and 0.20 g/L) of green alga and the type of carbon source in a mixotrophic pathway. In these latter trials, besides acetic acid, as usually present in the TAP medium, it was added more 0.1 or 1.0 g/L of sodium acetate and milk whey permeate (MWP), individually or simultaneously. As the manufacturer, Sooro Concentrado Industria de Produtos Lácteos Ltda from Brazil, milk whey permeate contain lactose (93%), proteins (1.2%), ashes (4.6%), among others traces compounds. 2.3.2. Hybrid system The hybrid system was evaluated as an alternative to increment the biofuel accumulation. In the first stage, C. reinhardtii was cultivated in TAP-S medium and the effluent, free of cell and rich in soluble metabolites, was used as substrate by R. capsulatus, since these photosynthetic bacteria can convert the organic acids and the non-consumed original carbon source for additional ethanol production. Initially the effects of supplementing R. capsulatus’s medium with sodium glutamate, malic acid, milk whey permeate and micronutrient solution, according Table 1a, were evaluated. Subsequently, similar assays were carried out to verify the influence of mixotrophic carbon source on the ethanol production by C. reinhardtii adding to the TAP-S medium different concentrations of sodium acetate and milk whey permeate. In these assays, only the crude effluent was used in the second stage of hybrid system (Table 1b). For all assays, the initial cell concentration was 0.1 g/L and they were performed in duplicate. 2.3.3. Co-culture of algae and bacteria Co-cultures of C. reinhardtii and R. capsulatus, being both cultured in TAP-S medium and RCV medium, were evaluated. The initial cell densities were 0.1 g/L, mixed together at variant ratios (from 0% to 100%). Co-culture grew in bioreactor of 50 mL (working volume of 37.5 mL) at 25 °C using 30 lE m 2 s 1 of light intensity. 2.4. Analytical methods The growth of cells was measured via spectrophotometry (UVmini-1240, Shimadzu) and biomass dry weight. One milliliter of sample was appropriately diluted with deionizer water and the absorbance of the sample was read at 665 nm (algae) and 660 nm (bacteria). The concentrations of metabolites were determined by HPLC (Shimadzu MODEL LC-20A Prominence, Supelcogel, column C-610H), equipped by ultra-violet and refractive detectors. The UV–VIS was used to determine organic acid concentrations at a wave length of 210 nm and the RID detector quantified the contents of lactose and ethanol. The column temperature was kept at 32 °C and an aqueous solution of 0.1% M H3PO4 was used for elution at 0.5 mL/min. The sample volume injected into the HPLC
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R.L. Costa et al. / Bioresource Technology 181 (2015) 330–337 Table 1 (a) Schedule of Hybrid System to ethanol production. (b) Evaluation of carbon source in the Hybrid System to ethanol production. A control was performed in TAP-S medium without mixotrophic carbon source addition. Experiment 1 Culture conditions
Experiment 2 Culture conditions
Experiment 3 Culture conditions
a
C. reinhardtii TAP-S medium
R. capsulatus (1) Crude Supernatant from C. reinhardtii; (2) Supernatant from C. reinhardtii supplemented with glutamate (2.54 g/L), malic acid (4.02 g/L), milk whey permeate (6 g/L) and micronutrient solutiona.
C. reinhardtii (1) TAP-S medium supplemented with 0.1 g/L of sodium acetate or/and milk whey permeate
R. capsulatus Crude supernatant from C. reinhardtii
C. reinhardtii (2) TAP-S medium supplemented with 1 g/L of sodium acetate and milk whey permeate
R. capsulatus Crude supernatant from C. reinhardtii
The micronutrient solution was composed according to RCV medium (Weaver et al., 1975).
was 20 lL. The produced gas was collected in graduated syringes, and the oxygen concentration was determined by gas chromatography using the chromatograph ShimadzuÒ model GC 17A, equipped with a thermal conductivity detector (TCD) and a capillary column CarboxenÒ 1010 (length 30 m, internal diameter 0.53 mm). The operating temperatures of the injection port, the oven, and the detector were 230, 32, and 230 °C, respectively. Argon was used as carrier gas.
3. Results and discussion Photobiological production of biofuel by green algae has great potential to be used for generating renewable fuel from clean resources. As reported by Oncel (2013), microalgae can also excrete ethanol directly through the cell walls by means of intracellular processes under dark. In fact, the degradation of intracellular starch, which is the main endogenous carbon source stored during aerobic phototrophic metabolism, into pyruvate is accomplished by the Embden–Meyerhof–Parnas and pentose phosphate pathways using pyruvate decarboxylase and alcohol dehydrogenase enzymes. The metabolism of C. reinhardtii is complex including a broad variety of end by-products resulting from the fermentation under different growth conditions in order to keep the cellular redox and energy balance in the anaerobic medium. The fermentation process occurs in dark and anaerobic environment or in the light after sulfur depletion. These by-products are carbon dioxide, hydrogen, acetate, formate, ethanol, lactate, glycerol, malate and succinate (Philipps et al., 2011; Mus et al., 2007). According to Gfeller and Gibbs (1984), acetate, formate and ethanol are the major anaerobic compounds produced by sulfur-replete cultures and, as reported by Kosourov et al. (2002), glycerol and lactate are produced only at low pH. Through glycolytic pathway, the starch degradation produces pyruvate that is cleaved by pyruvate formate lyase (PFL1) into formate and acetyl-CoA. Acetyl-CoA can further be converted to acetate by the successive action of phosphotransacetylase (PTA) and acetate kinase (ACK), (Philipps et al., 2011; Mus et al., 2007; Gfeller and Gibbs, 1984). Another pathway leads to the conversion of pyruvate into D-lactate catalyzed by D-lactate dehydrogenase (DLDH) (Philipps et al., 2011). Fig. 1b summarizes the metabolic route clarifying the ethanol production. The formation of glycerol, malate and succinate from starch by C. reinhardtii follows an alternative pathway for pyruvate metabolism (Catalanotti et al., 2012; Philipps et al., 2011; Dubini et al., 2009). In addition, Catalanotti et al. (2012) using a double mutant
lacking pyruvate formate lyase (PFL1) and alcohol dehydrogenase (ADH1) obtained glycerol and lactate in a higher concentration level. In respect to succinate formation that proceeds via malate synthesis, Dubini et al. (2009) succeeded to achieve elevated content of this metabolite applying a mutant strain with no hydrogenase activity. The following results present the ethanol formation by only C. reinhardtii or in hybrid system and co-culture with R. capsulatus. Besides the ethanol concentration, data of other metabolites are shown, since the comprehension of the relationship between different metabolic pathways, might lead to achieve the conditions that improve ethanol production by photobiological processes.
3.1. Ethanol production by C. reinhardtii 3.1.1. Effect of inoculum concentration In respect to the influence of initial cell concentration, the concentrations of 0.05, 0.10 and 0.20 g/L were applied to the assays of the ethanol production by C. reinhardtii, and the results of O2 evolution, final cell concentrations and metabolites concentrations are presented in Fig. 1. The analysis of Fig. 1 indicated that the ethanol production did not show a direct relation to the variation of cell concentration. The highest ethanol content 19.25 ± 4.16 g/L (0.16 ± 0.03 g/L h) was achieved by using 0.05 g/L as inoculum concentration and, in this case, it was observed an increase of 14% in cell concentration. At second ethanol production, 13.78 ± 3.90 g/L (0.12 ± 0.03 g/L h) was attained for inoculum concentration of 0.20 g/L, and in this assay, a 22.5% reduction of biomass concentration was measured. In the assay using 0.10 g/L of inoculum, the final cell concentration was 75% higher; however the production of ethanol was negligible. The ethanol production by green algae in mixotrophic conditions is not fully understood. The findings indicated that under low cell inoculum density (0.05 g/L) C. reinhardtii used available nutrients, under specific operational culture conditions, to ethanol production instead of cell growth. Probably, when higher density was used, growth factors could have been synthesized by cells and metabolic rotes favoring growth were preferential. In addition, Fig. 1a and b also showed clearly that the condition that generated the highest O2 evolution resulted in the lowest ethanol content. Concerning on the production of by-products (Fig. 1b), two organic acids were detected, that is, propionic and acetic acids. The propionic acid was produced in trace amounts from 0.74 ± 0.8 10 2 mg/L up to 6.67 ± 0.2 10 2 mg/L. The analysis of acetic acid concentration allows identify a trend between this metabolite and ethanol. For the assays with the highest ethanol
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(a)
(b)
(c)
hexose
CoA
PDC
Pyruvate CO2
(PFR1)
AcetylCoA NADPH
Acetaldehyde NAD(P)H
Acetaldehyde
NADP+CoA NAD(P)+
NADPH
NAD+ Ethanol
Ethanol Fig. 1. (a) O2 productivity and biomass evolution. C. reinhardtii culture was kept in anaerobiose under sulfur depletion. (b) Monitoring of ethanol and organic acid concentration. The fermentation time and inoculum age was 5 days and 10 days, respectively. (c) Metabolism pathways of Chlamydomonas during dark anaerobiosis to ethanol production (Adapted from Philipps et al., 2011). PFR1: pyruvate ferredoxin oxidoreductase.
contents, 19.25 ± 4.16 (0.16 ± 0.03 g/L h) and 13.78 ± 3.90 g/L (0.11 ± 0.03 g/L h), the production of acetic acid were the highest as well, 3.22 ± 0.28 g/L and 3.53 ± 0.81 g/L, respectively. The assay with negligible ethanol production presented only 1.81 ± 0.098 g/ L of acetic acid, which is around 54% lower than the other assays. Ethanol production from green algae has not been intensively studied. Hirano et al. (1997) performed the screening for microalgae with high starch content and high ability for intracellular ethanol production. The green alga Chlorella vulgaris (IAM C-534) showed the highest starch content (37%) and the ethanol-conversion rate was 65% compared to the theoretical rate from starch. In the study of the intracellular production by Hirano et al. (1997), a maximum ethanol concentration (1 w/w%) was obtained from C. reinhardtii (UTEX2247) and Sak-l isolated from seawater. Hirano et al. (1997) also evaluated ethanol production with intracellular starch fermentation. In this work, pH medium, cell concentration and different microalgae strains were investigated under dark and anaerobic conditions. Ethanol was observed in almost all of the tested strains and the highest ethanol productivity was attained by C. reinhardtii (UTEX2247). In contrast of the current work, Hirano et al. (1997) observed that the formation of ethanol increased almost proportionally to the concentration of the algal cells. The analysis of the organic metabolites in one of the anaerobic fermented slurries of Chlamydomonas showed that ethanol was the main metabolite, followed by lactate and acetate. The analysis of organic metabolites in hydrogen production by C. reinhardtii CC-124 was performed by Philipps et al. (2011). These authors determined the production of formate and ethanol
achieved 441.6 and 331.2 ng/lg chlorophyll, respectively and in smaller production of D-lactate (102.68 ng/lg chlorophyll). In respect to acetate, this compound was detected in amounts 10–20 times lower than the concentration in TAP medium and, in a medium without acetate; the synthesis of acetate by the alga was 258.17 ng/lmol chlorophyll. Mus et al. (2007) determined the final composition of the organic metabolites in their study of hydrogen production as well. In dark and anoxic fermentation, C. reinhardtii produced malate, formate, acetate and ethanol. The major components were formate, acetate and ethanol in a molar ratio of 1:1:0.5. In the current work, the ratio molar of ethanol and acetate varied from 0.4:0.05 to 0.3:0.06 for those testes that showed significant amount of ethanol. Gfeller and Gibbs (1984) investigated the anaerobic degradation of starch into end products by C. reinhardtii F-60 in the dark and light. Their study resulted in the production of formate, acetate and ethanol in the ratios of 2.07:1.07:0.91, with roughly 100% of starch conversion. Specifically to ethanol, the authors concluded that ethanol synthesis was inhibited by the light in all conditions tested and on the other hand, formate production was least affected by light. Furthermore, the other organic compounds, glycerol and lactate, were detected in minor concentrations. In the current work, besides ethanol, only propionate and acetate were detected in the trials carried out in the evaluation of the effect of initial cell concentration. Lactate and formate were not identified as the works aforementioned described. In this work, formic acid was produced by C. reinhardtii only in medium
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supplemented with milk whey permeated and lactic acid was produced by the green alga only in RCV medium as it will be presented in the following sections. 3.1.2. Effect of carbon source In order to evaluate the influence of increasing the amount and the type of the carbon source on the ethanol production by C. reinhardtii, a set of trials were performed adding sodium acetate and/or milk whey permeate (MWP) to the TAP-S medium (Table 2). Heterotrophic cultivation is suitable for a restrict number of microalgae. According to Perez-Garcia et al. (2011), sucrose, lactate, lactose and ethanol have been tested under heterotrophic microalgae cultures with negative results in growth and metabolite production. Nevertheless, the results of Table 2 proved that C. reinhardtii CC-124 cultivated in mixotrophic conditions has the ability to produce ethanol under anaerobic fermentation with day/light cycles of 12 h supplemented by sodium acetate and, especially, with lactose from milk whey permeate. Table 2 indicated that the TAP-S medium without supplementation of carbon source was not adequate to produce ethanol. This result is in agreement with the aforementioned data. Nevertheless, the additions of 0.1 g/L of sodium acetate or milk whey permeate and the addition of both simultaneously promotes the production of the biofuel. The concentration of ethanol attained was 9.64 ± 1.84 g/L (0.08 ± 0.016 g/L h), 13.11 ± 2.57 g/L (0.11 ± 0.02 g/ L h), 14.94 ± 6.95 g/L (0.12 ± 0.02 g/L h) in the presence of sodium acetate, milk whey permeate and in the presence of both, respectively. Therefore, the medium supplementation with milk whey permeate resulted in increases of the target-product. The data shown in Table 2 also indicate that increasing the supplementation to 1.0 g/L of the same mixotrophic carbon sources do not improve the synthesis of ethanol, indicating that a substrate inhibition could have occurred. Besides ethanol, further metabolites were detected such as acetic acid, propionic acid and formic acid. Propionic acid and acetic acid are present in all compositions of medium. The concentration of the propionic acid is in the order of 1.4 ± 0.08 mg/L to 8.8 ± 1.75 10 3 mg/L. The content of acetic acid was superior to 1.8 g/L and achieved 3.0–3.5 g/L at the highest production of ethanol, similarly to the previous trials (Fig. 1b). Formic acid was synthesized only when milk whey permeate was added to TAP-S medium. By taking into account the supplementation of the mixotrophic carbon source of 0.1 g/L, its concentration range varied from 0.9 g/L for TAP-S medium with MWP to 1.54 g/ L for the medium with TAP-S medium with MWP and Na-acetate. For the assay with addition of 1.0 g/L of MWP to TAP-S medium, the final concentration of formic acid was 1.11 ± 0.45 g/L. The number of developed studies concerning on the mixotrophic cultivation of algae are rare. The aim of these works was to evaluate the effect of the different carbon source in growth rate
and bioproducts like biofuel and pigments (Perez-Garcia et al., 2011; Sahu and Adhikary, 1981). Nevertheless, the influence of mixotrophic carbon source in the ethanol production by C. reinhardtii has not found in the literature. The effect of lactose, fructose, mannose, xylose and sodium acetate on the growth rate and pigment composition by Anabaena sp. under light and dark conditions was investigated by Sahu and Adhikary (1981). They did not describe the ethanol formation, but reported that lactose was the most appropriated carbon source for growth of the alga. The present work indicated that industrial wastes containing this disaccharide are promising resource that can be mixed with water and be used as substrate to C. reinhardtii in the production of ethanol. Besides discussing the effect of carbon source as substrate to C. reinhardtii, it is worth to emphasize that the ethanol amount attained in the current work was considerable higher than certain strains of yeast. For instance, Khattak et al. (2014) investigated the production of ethanol by yeast varying the initial concentration of glucose from 10 to 50 g/L. They found out that the substrate conversion was complete only at 10 g/L of glucose; and the conversion decreased 33% at 50 g/L. The production of ethanol increased from 3.83 to 4.56 g/L corresponded to the initial glucose concentration of 10 and 50 g/L, respectively. The ethanol concentration achieved in Khattak et al. (2014) was approximately of 30% (4.56 g/L) of the ethanol production in this paper (14.94 g/L). Nevertheless, low cost feedstocks that are agroindustrial residues, such as whey cheese, enables to combine the wastewater treatment effluent with the production of value-added products and the addition of small amounts of organic carbon sources leads to higher growth rate without bacterial contamination (Perez-Garcia et al., 2011). 3.2. Ethanol from hybrid system: C. reinhardtii and R. capsulatus An alternative biological route to obtain ethanol is to use the effluent produced in the process by green algae as substrate to photosynthetic bacteria, particularly, the purple non sulfur bacteria. The integration of these photobiological processes, named hybrid system, may lead to higher degradation efficiency, since non purple bacteria may utilize the organic acids and non-converted carbon source present in the effluent by C. reinhardtii into additional ethanol (Batista et al., 2014; Srikanth et al., 2009). Hybrid systems have been widely studied for hydrogen formation, especially those based in the integration of dark fermentation by anaerobic agents and photofermentation by sulfur non-purple bacteria (Tawfik et al., 2014; Urbaniec et al., 2014). Nevertheless, they were not explored for ethanol production by microalgae and photosynthetic bacteria as it was proposed in the present work. In Experiment 1, TAP-S medium was used in the first stage of the hybrid system metabolized by C. reinhardtii. The potential of
Table 2 Effect of carbon source (0.1 and 1.0 g/L) on the production of ethanol. Medium
Cacetic
Concentration of C source (0.10 g/L) TAP-Sb TAP-S + Na-acetate TAP-S + MWPc TAP-S + Na-acetate + MWP
1.79 ± 0.07 2.21 ± 0.64 3.04 ± 0.75 3.49 ± 0.83
1.48 10 5.1 10 4.4 10 8.8 10
3
± 0.002 ± 4.3 10 3 3 ± 0.001 3 ± 1.75 10 3
NDa NDa 0.90 ± 0.37 1.54 ± 0.07
NDa 9.64 ± 1.84 13.11 ± 2.57 14.94 ± 6.95
Concentration of C source (1.0 g/L) TAP-Sb TAP-S + Na-acetate TAP-S + MWPc
1.88 ± 0.05 2.08 ± 0.05 1.89 ± 0.10
1.4 10 3.7 10 2.96 10
3
NDa NDa 1.11 ± 0.45
NDa NDa NDa
acid
(g/L)
The error estimates are derived from replicated (2) runs. a ND – not detected. b Concentration of acetic acid in TAP-S medium: 1.04 g/L. c MWP, milk whey permeate.
Cpropionic
acid
(g/L)
3
± 0.008 ± 0.37 10 2 3 ± 0.045 10 2 3
Cformic
acid
(g/L)
Cethanol (g/L)
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R.L. Costa et al. / Bioresource Technology 181 (2015) 330–337 Table 3 Effect of the composition medium for R. capsulatus on the production of ethanol in a hybrid system by C. reinhardtii and R. capsulatus. Triala
Composition of metabolites (g/L)c C. reinhardtii
1 2
R. capsulatus
Cacet
Cprop
1.79 ± 0.07 1.84 ± 0.05
1.5 10 0.7 10
3
± 0.002 3 ± 0.000
Ceth
Clact
Cform
NDb NDb
0.16 ± 0.11 5.95 ± 4.02
0.01 ± 0.04 10 0.21 ± 0.025
4
Cacet
Cprop
Cbut
Ceth
2.58 ± 0.41 4.38 ± 0.97
0.80 ± 0.56 5.52 ± 3.89
NDb 2.38 ± 1.60
12.61 ± 2.01 18.22 ± 1.89
The error estimates are derived from replicated (2) runs. a Trial 1: First stage, C. reinhardtii – TAP-S medium, Second stage R. capsulatus – crude effluent; Trial 2: First stage, C. reinhardtii – TAP-S medium, Second stage R. capsulatus – effluent with supplements. b ND – not detected. c Metabolites: Acet: acetic acid; prop: propionic acid; lact: lactic acid; form: formic acid; but: butyric acid; eth: ethanol.
Table 4 Effect of the enrichment with 0.1 g/L of extra heterotrophic carbon source in the medium for C. reinhardtii on the production of ethanol in a hybrid system by C. reinhardtii and R. capsulatus. Medium in the 1st stage of hybrid systema
TAP-S TAP-S + Na-acetate TAP-S + MWPc TAP-S + Na-acetate + MWP
Composition of metabolites (g/L)c C. reinhardtii
R. capsulatus
Cacet
Cprop
1.79 ± 0.07 2.21 ± 0.64 3.04 ± 0.75 3.50 ± 0.83
1.48 10 5.1 10 4.4 10 8.8 10
3
± 0.002 3 ± 4.3 10 3 3 ± 0.001 3 ± 1.75 10 3
Cform
Ceth
Cacet
Cprop
Cform
Ceth
NDb NDb 0.90 ± 0.37 1.54 ± 0.07
NDb 9.64 ± 1.84 13.11 ± 2.57 14.94 ± 6.95
2.32 ± 0.41 3.20 ± 0.22 4.08 ± 0.24 2.90 ± 0.28
0.36 ± 0.056 0.01 ± 0.003 0.05 ± 0.56 10 0.03 ± 0.02
NDb NDb 0.05 ± 0.01 NDb
10.26 ± 2.01 16.13 ± 2.69 19.94 ± 2.67 17.17 ± 2.63
2
The error estimates are derived from replicated (2) runs. a Second stage: R. capsulatus – crude effluent; b ND – not detected. c Metabolites: Acet: acetic acid; prop: propionic acid; lact: lactic acid; form: formic acid; but: butyric acid; eth: ethanol; MWP: milk whey permeate.
R. capsulatus of producing ethanol from the metabolites synthesized by the green alga was evaluated comparing the biofuel production using the crude fermentative broth and the effluent supplemented with sodium glutamate, malic acid, milk whey permeate and micronutrients. In both cases, the effluent was free of C. reinhardtii and fermentation time was 5 days in the two stages of the hybrid system. The results are presented in Table 3. The results shown in Table 3 proved that R. capsulatus synthesis ethanol even though the macro and micronutrients, usually found in RCV medium, were not present. Table 3 also indicated that ethanol production by R. capsulatus cultivated in effluent supplemented with malic acid, milk whey permeate and sodium glutamate was 44.5% superior (18.22 g/L) than when bacteria were cultivated in the crude effluent (12.61 g/L). In respect to the metabolites, the amount of organic acids was higher as well. In the crude effluent there were around 1.79 ± 0.07 g/L to 1.84 ± 0.55 g/L of acetic acid and at the end, the effluent produced by R. capsulatus in the medium without supplementation was composed by 0.16 ± 0.11 g/L of lactic acid, 2.58 ± 0.41 g/L of acetic acid, 0.01 ± 0.04 10 4 g/L of formic acid and 0.80 ± 0.56 g/L of propionic acid. The composition of the effluent by R. capsulatus in the supplemented medium was 5.95 ± 4.02 g/L of lactic acid, 4.38 ± 0.97 g/L of acetic acid, 5.52 ± 3.89 g/L of propionic acid, 0.21 ± 0.025 g/L of formic acid and 2.38 ± 1.60 g/L of butyric acid. In the following Experiments 2 and 3, an effluent produced by C. reinhardtii cultivated in a medium enriched with a carbon source was used by R. capsulatus. In the Experiment 2, the medium used by C. reinhardtii was supplemented with 0.1 g/L of sodium acetate or milk whey permeate or both of the components and the composition of the effluent is shown in Tables 2 and 4. In the Experiment 3, the supplementation of the medium was made by the addition of 1.0 g/L the same components and the composition of the effluent is shown in Tables 2 and 5.
As it was discussed aforementioned, the medium enriched with 0.1 g/L of milk whey permeate increased the ethanol production by C. reinhardtii. The effluent produced from this medium led to a higher final production of the biofuel, 19.94 ± 2.67 g/L (0.17 ± 0.02 g/L h) by R. capsulatus, as Table 4 demonstrated. Although the effluent utilized by the bacteria was not supplemented with malic acid and MWP, the acetic acid and formic acid may have provided carbon source enough to R. capsulatus metabolism. Concerning on the organic acids, except to TAP-S medium with sodium acetate and milk whey permeate, acetic acid and propionic acid were synthesized in both stages of the hybrid system. In the trials with TAP-S medium containing MWP, the formic acid produced in the first stage was consumed by the photosynthetic bacteria. Acetic acid produced by C. reinhardtii was consumed by R. capsulatus in the assay with TAP-S medium supplemented with sodium acetate and MWP. The analysis of Tables 2 and 5 showed that no production of ethanol was verified at higher concentration of carbon source (1.0 g/L), independently whether sodium acetate or milk whey permeate were added. Besides the ethanol, acetic acid was synthesized by R. capsulatus only in the trial of TAP-S medium without supplementation. In further trials, the addition of 1.0 g/L of sodium acetate or milk whey permeate led to a metabolic pathway in which consume of acetic acid and no production of ethanol by the bacterium were observed. Complementarily, to produce biofuels as hydrogen and ethanol, purple non-sulfur bacteria commonly synthesize polyhydroxyalkanoates. In the current work, the consumption of the organic acids without production of ethanol by R. capsulatus suggested that further metabolites could be synthesized, as it was demonstrated by Sawayama (2001) and Maeda et al. (1998). Hybrid systems incorporating two stages of algae and photosynthetic bacteria are hardly to find published, since the focus of
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Table 5 Effect of the enrichment with 1.0 g/L of extra heterotrophic carbon source in the medium for C. reinhardtii on the production of ethanol in a hybrid system by C. reinhardtii and R. capsulatus. Medium in the 1st stage of hybrid systema
Composition of metabolites (g/L)c Chlamydomonas reinhardtii
TAP-S TAP-S + Na-acetate TAP-S + MWPc
Cacet
Cprop
1.88 ± 0.16 2.08 ± 0.30 1.89 ± 0.35
1.4 10 3.7 10 2.96 10
Rhodobacter capsulatus
3
± 0.17 3 ± 0.001 3 ± 0.001
Ceth
Cacet
Cprop
Ceth
NDb NDb NDb
4.09 ± 0.15 1.08 ± 0.37 10 0.95 ± 0.28
NDb 0.03 ± 0.003 0.02 ± 0.002
12.57 ± 0.01 NDb NDb
2
The error estimates are derived from replicated (2) runs. a Second stage: R. capsulatus – crude effluent; b ND – not detected. c Metabolites: Acet: acetic acid; prop: propionic acid; lact: lactic acid; form: formic acid; but: butyric acid; eth: ethanol. MWP: milk whey permeate.
Table 6 Composition of metabolites produced by co-culture system based on different C reinhardtii (A) to R. capsulatus (B) ratios. Composition of metabolites (g/L)b Strain ratio
Clact
Basal medium
RCV
TAP-S
RCV
TAP-S
RCV
TAP-S
RCV
TAP-S
A A A A A A A
0.57 ± 0.18 0.15 ± 0.07 0.07 ± 0.02 0.31 ± 0.22 0.41 ± 0.28 0.78 ± 0.18 0.27 ± 0.07
NDa 0.01 ± 0.65 10 NDa 0.27 ± 0.19 NDa 0.26 ± 0.02 NDa
0.58 ± 0.33 1.47 ± 0.09 0.46 ± 0.10 0.32 ± 0.067 0.37 ± 0.069 0.93 ± 0.15 2.58 ± 1.00
3.45 ± 0.29 3.42 ± 0.33 4.94 ± 1.60 3.73 ± 1.82 3.64 ± 0.73 2.37 ± 2.20 3.09 ± 0.15
0.54 ± 0.15 0.20 ± 0.14 NDa NDa NDa 0.33 ± 0.20 0.91 ± 0.08
NDa 1.1 10 2 ± 0.83 10 4.4 10 2 ± 0.03 10 0.91 ± 0.64 2.4 10 2 ± 0.11 10 6.7 10 3 ± 0.53 10 1.5 10 3 ± 0.02
9.00 ± 6.36 NDa 0.79 ± 0.06 NDa NDa NDa 12.61 ± 8.91
NDa 0.08 ± 0.06 0.25 ± 0.12 0.22 ± 0.16 NDa 0.12 ± 0.02 0.09 ± 0.02
(0%): B (100%) (10%): B (90%) (30%): B (70%) (50%): B (50%) (70%): B (30%) (90%): B (10%) (100%): B (0%)
Cacet
2
Cprop
Ceth
2 2
2 2
The error estimates are derived from replicated (2) runs. a ND – not detected. b Metabolites: Acet: acetic acid; prop: propionic acid; lact: lactic acid; but: butyric acid; eth: ethanol.
the works developed by Maeda et al. (1998), Miura et al. (1997) and Miura et al. (1992) was the hydrogen production. In order to investigate hydrogen production, Maeda et al. (1998) proposed an integrated process where the effluent produced by Chlamydomonas sp. MGA161, cultivated under dark and anaerobic conditions, was used by Rhodovulum sulfidophilum W-1S. In the first stage, acetic acid, ethanol and glycerol were the main products excreted into the medium during Chlamydomonas sp. MGA161. In the second stage, there was consumption by R. sulfidophilum W1S of all these organic acids immediately after hydrogen evolution started. The amounts of the metabolites were not discussed by the authors. Miura et al. (1997) scaled up photobiological hydrogen production system to a pilot plant scale in the photosynthetic starch accumulation, followed by the fermentative production of metabolites from starch by Chlamydomonas MGA 161 and the hydrogen photoproduction by R. sulfidophilum W-1S from fermentative broth produced by Chlamydomonas MGA 161 in the natural day/ night cycle. The yield of acetic acid, ethanol and glycerol from starch of Chlamydomonas MGA 161 was 80–100% of the theoretical yield. Maeda et al. (1998) and Miura et al. (1997) also described that the decrease in the conversion of substrate into hydrogen production was due to the accumulation of polyhydroxybutyrate (PHB). Similar conclusion was withdrawn by Sawayama (2001) that studied the consumption of acetate by photosynthetic bacteria. The author reported the production of polyhydroxybutyrate (PHB) by phototrophic bacteria using as substrate an effluent from lighted upflow anaerobic sludge blanket (LUASB) reactor with sodium acetate under anaerobic light condition. The concentration of PHB enhanced in a secondary incubation of this effluent with Na-acetate.
3.3. Ethanol from co-culture: C. reinhardtii and R. capsulatus In this current work, strategies of co-cultivation of C. reinhardtii (green alga) and R. capsulatus (PNS Bacteria) to ethanol production were also attempted. In this coupled system, R. capsulatus could use the organic acids excreted by C. reinhardtii cells during anaerobic photofermentation process. Furthermore, the integration of these two biological systems may enhance the ethanol production with a reduction in energy cost, due to better solar irradiance utilization (visible and infrared) and integrated use of the nutrients (Melis and Melnicki, 2006). Table 6 summarizes the results of cocultivation. Two fermentation media were tested in the co-cultivation, that is, RCV and TAP-S. In the RCV medium used in the co-cultivation, lactic and acetic acids were produced in all algae to bacteria ratios. The production of propionic acid and ethanol did not present a trend by increasing the alga to bacteria ratio. Surprisingly, isolated green alga [Algae (100%): Bacteria (0%)] cultured in RCV medium (a basal medium to PNS bacteria) was fully capable to synthesize ethanol resulting in 12.6 ± 8.91 g/L (0.11 ± 0.07 g/L h). Similar procedure was also performed to co-culture maintained in TAP-S medium. However, R. capsulatus has not presented a good performance in relation to the ethanol production in this medium formulation, probably due the lack of essential nutrients. In TAP-S medium, our findings showed that C. reinhardtii and R. capsulatus contributed equally to acetic acid production, since at isolated bacterial culture [Algae (0%): Bacteria (100%)] resulted in 3.45 ± 0.29 g/L and isolated algal culture [Algae (100%): Bacteria (0%)] produced up to 3.09 ± 0.15 g/L. In addition, propionic acid was produced when the variant ration of 1:1 of cells was used in the co-cultivation. On the other hand, the final ethanol concentration [Algae (30%): Bacteria (70%)] was at least 3.2-fold lower than
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the minimum ethanol content observed in the co-culture maintained in RCV medium (0.79 ± 0.06 g/L) at the same variant ratio. The co-culture approach of microalgae and photosynthetic bacteria has not been explored and the influence of lots of variables is still unknown. However the results of ethanol content obtained through the co-cultivation strategy were lower than those from mixotrophic photofermentation by C. reinhardtii isolated or in hybrid system with R. capsulatus. It is necessary to enlarge the conditions of this technique by evaluating the composition of the medium, including the supplementation of organic carbon source, nutrients and light intensity as Melis and Melnicki (2006) did. These authors investigated the hydrogen production by coupling Rhodospirillum rubrum and C. reinhardtii, in a bioreactor containing all the nutrients of both Ormerod and TAP that are media used to the maintenance of bacteria and algae cultures, respectively. Moreover, other parameter evaluated was the light intensity (30–150 lmol photons m 2 s 1), since the level of irradiance could regulate the relative growth and production of metabolites of the two types of cells. Melis and Melnicki (2006) did not present data related to the composition of organic acids and ethanol. In general, for each system approached in this work, it is important to emphasis that a full study must be performed in order to enhance the comprehension of the effect of the medium composition and operational conditions on the ethanol production by C. reinhardtii and R. capsulatus. For instance, the light intensity, residual sulfur content, photoperiod and bioreactor configurations may influence the biofuel synthesis. The comprehension of metabolism of different organic compounds by C. reinhardtii and R. capsulatus requires more detailed studies. Further researches may lead to significant progress in improving the biomass accumulation, starch production and co-production of ethanol. The results of this work may be used in future design of ethanol production in integrated systems weather by hybrid or co-culture systems. 4. Conclusion In this study the effect of inoculum concentration and carbon source to C. reinhardtii, as well as the influence of hybrid system and co-culture (C. reinhardtii and R. capsulatus) on the photofermentative ethanol production were investigated. Maximum ethanol content 19.94 ± 2.67 g/L (0.17 ± 0.02 g/L h) was achieved by hybrid system in which the effluent of C. reinhardtii containing organic acids was used as substrate to R. capsulatus. The results from this work are beneficial to comprehend the potentiality of green alga and PNS photosynthetic bacteria to synthesize ethanol concerning several strategies such as media composition and different culture systems (hybrid and co-cultivation). Acknowledgements The authors wish to thank Brazilian agencies CNPq, CAPES and FAPEMIG (Grant No. APQ-01020-12) for financial support. References Andersen, R.A. (Ed.), 2005. Algal Culturing Techniques. London Elsevier Academic Press (p. 578). Andrietta, S.R., Steckelberg, C., Andrietta, M.G.S., 2008. Study of flocculent yeast performance in tower reactors for bioethanol production in a continuous fermentation process with no cell recycling. Bioresour. Technol. 99, 3002–3008. Batista, A.P., Moura, P., Marques, P.A., Ortigueira, J., Alves, L., Gouveia, L., 2014. Scenedesmus obliquus as feedstock for biohydrogen production by Enterobacter aerogenes and Clostridium butyricum. Fuel 117, 537–543. Catalanotti, C., Dubini, A., Subramanian, V., Yang, W., Magneschi, L., Mus, F., Seibert, M., Posewitz, M.C., Grossman, A.R., 2012. Altered fermentative metabolism in Chlamydomonas reinhardtii mutants lacking pyruvate formate lyase and both pyruvate formate lyase and alcohol dehydrogenase. Plant Cell 24, 692–707.
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Prospective technology on bioethanol production from photofermentation Rosangela Lucio Costa, Thamayne Valadares Oliveira, Juliana de Souza Ferreira, Vicelma Luiz Cardoso, Fabiana Regina Xavier Batista ⇑ School of Chemical Engineering, Federal University of Uberlandia. Av. Joao Naves de Avila 2121, Santa Monica 38408-144, Uberlandia, MG, Brazil
h i g h l i g h t s C. reinhardtii and R. capsulatus were utilized to the ethanol production. Ethanol was produced from sulfur deprivation and mixotrophic carbon source. Hybrid system and co-cultivation increase the ethanol production by photofermentation.
a r t i c l e
i n f o
Article history: Received 21 November 2014 Received in revised form 20 January 2015 Accepted 22 January 2015 Available online 30 January 2015 Keywords: Ethanol Chlamydomonas reinhardtii Rhodobacter capsulatus Hybrid system Co-culture
a b s t r a c t The most important global demand is the energy supply from alternative source. Ethanol may be considered an environmental friendly fuel that has been produced by feedstock. The production of ethanol by microalgae represent a process with reduced environmental impact with efficient CO2 fixation and requiring less arable land. This work studied the production of ethanol from green alga Chlamydomonas reinhardtii through the cellular metabolism in a light/dark cycle at 25 °C in a TAP medium with sulfur depletion. The parameters evaluated were inoculum concentration and the medium supplementation with mixotrophic carbon sources. The combination of C. reinhardtii and Rhodobacter capsulatus through a hybrid or co-culture systems was also investigated as well. C. reinhardtii maintained in TAP-S produced 19.25 ± 4.16 g/L (ethanol). In addition, in a hybrid system, with medium initially supplemented with milk whey permeated and the algal effluent used by R. capsulatus, the ethanol production achieved 19.94 ± 2.67 g/L. Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction The use of ethanol to replace oil is the most viable way to ensure a sustainable future. However, up to now more emphasis has been given on the yeast performance in reactors for bioethanol production (Andrietta et al., 2008). On the other hand, considering the advantages of microalgae culture such as rapid growth rate and productivity (Li et al., 2008) and their use to minimize contamination, since microalgae may to applied in wastewater treatment from inorganic salts (NH+4, NO3 , PO34 ) using them as nutrient materials (Mata et al., 2010), Chlamydomonas reinhardtii could be preferentially selected as a prospective biological system for bioethanol production instead of yeast. Concerning on the application of microalgae, recent research have shown that a diversity of strains may be cultivated to produce ⇑ Corresponding author. Tel.: +55 34 32309400. E-mail address: [email protected] (F.R.X. Batista). http://dx.doi.org/10.1016/j.biortech.2015.01.090 0960-8524/Ó 2015 Elsevier Ltd. All rights reserved.
biodiesel, hydrogen, methane and ethanol. Specifically to the ethanol production from microalgae, it can be directly synthesized by the cellular metabolism or from the fermentation of microalgae biomass, mainly cellulose and starch that are readily converted to fermentable products by enzymatic or acidic pretreatment technology (Oncel, 2013; Chen et al., 2013). Microalgae based ethanol may be considered as part of integrated process and a promising environmentally friendly alternative, since they could be present a potential for fixing CO2, high growth at high yields and low costs utilizing light as the energy source. Furthermore, they do not require fertile land and portable water as feedstock based biofuel (Chen et al., 2013; John et al., 2011; Hirano et al., 1997). The unicellular green alga C. reinhardtii uses light to grow photoautotrophically or mixotrophically in the presence of small organic substrates (Goff et al., 2009). C. reinhardtii is widely studied for hydrogen production by biophotolysis of water. This method uses the same processes found in plants photosynthesis. Photosynthesis involves the absorption of light by two distinct
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photosynthetic systems operating in series: a water splitting and O2 evolving system (‘‘photosystem II’’ or PSII) and a second photosystem (PSI), which generates the reductant used for CO2 reduction (Das and Veziroglu, 2001). It is important to note that green algae could produce hydrogen not only under light conditions, but under dark anaerobic conditions (Gaffron and Rubin, 1942). Nevertheless, if dark and anaerobic conditions are established on the microalgae cultures, hydrogen yield is quite low corresponding to about onesixth of direct biophotolysis production (Kosourov et al., 2002). Besides hydrogen, the oxidative reaction of starch become incomplete and depending on the type of the microalga, carbon dioxide, ethanol, lactic acid, formic acid, acetic acid, malic acid, glycerol and other compounds are produced in varying proportions (John et al., 2011; Gfeller and Gibbs, 1984). In addition, the medium composition may be altered to induce anaerobic condition. Hemschemeier and Happe (2004) reported that, under sulfur depletion, C. reinhardtii stops growing and accumulates starch. The absence of sulfur forces the algae to reorganize the whole metabolism. Anaerobiosis is established and hydrogen and ethanol could be produced. These authors discussed that the accumulation of ethanol already indicate the activity of pyruvate formate-lyase (PFL). PFL cleaves pyruvate into acetyl-CoA and it can further be reduced to acetaldehyde by acetaldehyde dehydrogenase. Furthermore, ethanol can be formed from cleavage of pyruvate by pyruvate decarboxylase (PDC) producing acetaldehyde as intermediate that is converted to ethanol by alcohol dehydrogenase (ADH). Previous work suggested it is possible to produce ethanol from metabolism of C. reinhardtii maintained in a basal medium supplemented with the mixotrophic carbon source (Costa et al., 2014). And, the purpose of this work was at first to verify the possibility of C. reinhardtii produce ethanol using a basal medium with sulfur depletion added mixotrophic carbon source such as milk whey permeate (rich in lactose) and sodium acetate. In the second step, the algae association with the purple non sulfur bacterium, a Rhodobacter capsulatus, to improve ethanol content into the medium was also evaluated by hybrid system (two stages) and co-culture. The hybrid systems and co-cultures approaches are strategies used in order to improve the yield. The purpose is to integrate microorganisms with distinct biological routes. Thus, the metabolites produced by one type of microorganism may be the substrate to the second type. 2. Methods 2.1. Algal biomass C. reinhardtii CC-124 was purchased from the Canadian Culture Collection, the Chlamydomonas Resource Center. The green alga was maintained in the basal medium Tris Acetate Phosphate (TAP) (Andersen, 2005) at initial pH of 7.0. In order to guarantee enough amounts of cells for the fermentation assays, the algal inoculum was subcultured with the addition of 250 mL of fresh TAP medium in 250 mL of growing culture. The alga was kept in Erlenmeyer (500 mL) at 25 °C under light cycle (night/day) of 12 h at 30 lE m 2 s 1. 2.2. Photosynthetic bacterial biomass R. capsulatus was purchased from DSMZ German Collection of Microorganisms and Cell Culture. The strain was cultivated anaerobically in Erlenmeyers (500 mL) maintained at 30 °C using RCV medium (Weaver et al., 1975), at initial pH of 6.8, under photosynthetic conditions of 30 lE m 2 s 1 (light-grown cells). Sodium glutamate was used as nitrogen source instead of (NH4)2SO4.
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2.3. Biological strategies to ethanol production by photofermentation For ethanol production by fermentation, anaerobic condition was used in all strategies: by C. reinhardtii cultures, hybrid system and co-culture approach. Thus, 50 mL bioreactors were flushed with Ar gas (99.999%) for 3 min. The inoculum was 10 days age for green algae culture and five days age for the purple non sulfur bacteria culture. Five days of time fermentation was used in all assays. The photoperiod of 12 h (12 h dark/12 h light) was used in all experiments with algae. In the hybrid system, in the second stage by R. capsulatus, the assays were carried out under light continuously. 2.3.1. C. reinhardtii under sulfur depletion In the first step of the current work, assays were carried out to investigate the ethanol production by C. reinhardtii in TAP-S (Tris– acetatephosphate-minus-sulfur) medium. The evaluated variables were the initial cell concentration (0.05, 0.10 and 0.20 g/L) of green alga and the type of carbon source in a mixotrophic pathway. In these latter trials, besides acetic acid, as usually present in the TAP medium, it was added more 0.1 or 1.0 g/L of sodium acetate and milk whey permeate (MWP), individually or simultaneously. As the manufacturer, Sooro Concentrado Industria de Produtos Lácteos Ltda from Brazil, milk whey permeate contain lactose (93%), proteins (1.2%), ashes (4.6%), among others traces compounds. 2.3.2. Hybrid system The hybrid system was evaluated as an alternative to increment the biofuel accumulation. In the first stage, C. reinhardtii was cultivated in TAP-S medium and the effluent, free of cell and rich in soluble metabolites, was used as substrate by R. capsulatus, since these photosynthetic bacteria can convert the organic acids and the non-consumed original carbon source for additional ethanol production. Initially the effects of supplementing R. capsulatus’s medium with sodium glutamate, malic acid, milk whey permeate and micronutrient solution, according Table 1a, were evaluated. Subsequently, similar assays were carried out to verify the influence of mixotrophic carbon source on the ethanol production by C. reinhardtii adding to the TAP-S medium different concentrations of sodium acetate and milk whey permeate. In these assays, only the crude effluent was used in the second stage of hybrid system (Table 1b). For all assays, the initial cell concentration was 0.1 g/L and they were performed in duplicate. 2.3.3. Co-culture of algae and bacteria Co-cultures of C. reinhardtii and R. capsulatus, being both cultured in TAP-S medium and RCV medium, were evaluated. The initial cell densities were 0.1 g/L, mixed together at variant ratios (from 0% to 100%). Co-culture grew in bioreactor of 50 mL (working volume of 37.5 mL) at 25 °C using 30 lE m 2 s 1 of light intensity. 2.4. Analytical methods The growth of cells was measured via spectrophotometry (UVmini-1240, Shimadzu) and biomass dry weight. One milliliter of sample was appropriately diluted with deionizer water and the absorbance of the sample was read at 665 nm (algae) and 660 nm (bacteria). The concentrations of metabolites were determined by HPLC (Shimadzu MODEL LC-20A Prominence, Supelcogel, column C-610H), equipped by ultra-violet and refractive detectors. The UV–VIS was used to determine organic acid concentrations at a wave length of 210 nm and the RID detector quantified the contents of lactose and ethanol. The column temperature was kept at 32 °C and an aqueous solution of 0.1% M H3PO4 was used for elution at 0.5 mL/min. The sample volume injected into the HPLC
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R.L. Costa et al. / Bioresource Technology 181 (2015) 330–337 Table 1 (a) Schedule of Hybrid System to ethanol production. (b) Evaluation of carbon source in the Hybrid System to ethanol production. A control was performed in TAP-S medium without mixotrophic carbon source addition. Experiment 1 Culture conditions
Experiment 2 Culture conditions
Experiment 3 Culture conditions
a
C. reinhardtii TAP-S medium
R. capsulatus (1) Crude Supernatant from C. reinhardtii; (2) Supernatant from C. reinhardtii supplemented with glutamate (2.54 g/L), malic acid (4.02 g/L), milk whey permeate (6 g/L) and micronutrient solutiona.
C. reinhardtii (1) TAP-S medium supplemented with 0.1 g/L of sodium acetate or/and milk whey permeate
R. capsulatus Crude supernatant from C. reinhardtii
C. reinhardtii (2) TAP-S medium supplemented with 1 g/L of sodium acetate and milk whey permeate
R. capsulatus Crude supernatant from C. reinhardtii
The micronutrient solution was composed according to RCV medium (Weaver et al., 1975).
was 20 lL. The produced gas was collected in graduated syringes, and the oxygen concentration was determined by gas chromatography using the chromatograph ShimadzuÒ model GC 17A, equipped with a thermal conductivity detector (TCD) and a capillary column CarboxenÒ 1010 (length 30 m, internal diameter 0.53 mm). The operating temperatures of the injection port, the oven, and the detector were 230, 32, and 230 °C, respectively. Argon was used as carrier gas.
3. Results and discussion Photobiological production of biofuel by green algae has great potential to be used for generating renewable fuel from clean resources. As reported by Oncel (2013), microalgae can also excrete ethanol directly through the cell walls by means of intracellular processes under dark. In fact, the degradation of intracellular starch, which is the main endogenous carbon source stored during aerobic phototrophic metabolism, into pyruvate is accomplished by the Embden–Meyerhof–Parnas and pentose phosphate pathways using pyruvate decarboxylase and alcohol dehydrogenase enzymes. The metabolism of C. reinhardtii is complex including a broad variety of end by-products resulting from the fermentation under different growth conditions in order to keep the cellular redox and energy balance in the anaerobic medium. The fermentation process occurs in dark and anaerobic environment or in the light after sulfur depletion. These by-products are carbon dioxide, hydrogen, acetate, formate, ethanol, lactate, glycerol, malate and succinate (Philipps et al., 2011; Mus et al., 2007). According to Gfeller and Gibbs (1984), acetate, formate and ethanol are the major anaerobic compounds produced by sulfur-replete cultures and, as reported by Kosourov et al. (2002), glycerol and lactate are produced only at low pH. Through glycolytic pathway, the starch degradation produces pyruvate that is cleaved by pyruvate formate lyase (PFL1) into formate and acetyl-CoA. Acetyl-CoA can further be converted to acetate by the successive action of phosphotransacetylase (PTA) and acetate kinase (ACK), (Philipps et al., 2011; Mus et al., 2007; Gfeller and Gibbs, 1984). Another pathway leads to the conversion of pyruvate into D-lactate catalyzed by D-lactate dehydrogenase (DLDH) (Philipps et al., 2011). Fig. 1b summarizes the metabolic route clarifying the ethanol production. The formation of glycerol, malate and succinate from starch by C. reinhardtii follows an alternative pathway for pyruvate metabolism (Catalanotti et al., 2012; Philipps et al., 2011; Dubini et al., 2009). In addition, Catalanotti et al. (2012) using a double mutant
lacking pyruvate formate lyase (PFL1) and alcohol dehydrogenase (ADH1) obtained glycerol and lactate in a higher concentration level. In respect to succinate formation that proceeds via malate synthesis, Dubini et al. (2009) succeeded to achieve elevated content of this metabolite applying a mutant strain with no hydrogenase activity. The following results present the ethanol formation by only C. reinhardtii or in hybrid system and co-culture with R. capsulatus. Besides the ethanol concentration, data of other metabolites are shown, since the comprehension of the relationship between different metabolic pathways, might lead to achieve the conditions that improve ethanol production by photobiological processes.
3.1. Ethanol production by C. reinhardtii 3.1.1. Effect of inoculum concentration In respect to the influence of initial cell concentration, the concentrations of 0.05, 0.10 and 0.20 g/L were applied to the assays of the ethanol production by C. reinhardtii, and the results of O2 evolution, final cell concentrations and metabolites concentrations are presented in Fig. 1. The analysis of Fig. 1 indicated that the ethanol production did not show a direct relation to the variation of cell concentration. The highest ethanol content 19.25 ± 4.16 g/L (0.16 ± 0.03 g/L h) was achieved by using 0.05 g/L as inoculum concentration and, in this case, it was observed an increase of 14% in cell concentration. At second ethanol production, 13.78 ± 3.90 g/L (0.12 ± 0.03 g/L h) was attained for inoculum concentration of 0.20 g/L, and in this assay, a 22.5% reduction of biomass concentration was measured. In the assay using 0.10 g/L of inoculum, the final cell concentration was 75% higher; however the production of ethanol was negligible. The ethanol production by green algae in mixotrophic conditions is not fully understood. The findings indicated that under low cell inoculum density (0.05 g/L) C. reinhardtii used available nutrients, under specific operational culture conditions, to ethanol production instead of cell growth. Probably, when higher density was used, growth factors could have been synthesized by cells and metabolic rotes favoring growth were preferential. In addition, Fig. 1a and b also showed clearly that the condition that generated the highest O2 evolution resulted in the lowest ethanol content. Concerning on the production of by-products (Fig. 1b), two organic acids were detected, that is, propionic and acetic acids. The propionic acid was produced in trace amounts from 0.74 ± 0.8 10 2 mg/L up to 6.67 ± 0.2 10 2 mg/L. The analysis of acetic acid concentration allows identify a trend between this metabolite and ethanol. For the assays with the highest ethanol
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(a)
(b)
(c)
hexose
CoA
PDC
Pyruvate CO2
(PFR1)
AcetylCoA NADPH
Acetaldehyde NAD(P)H
Acetaldehyde
NADP+CoA NAD(P)+
NADPH
NAD+ Ethanol
Ethanol Fig. 1. (a) O2 productivity and biomass evolution. C. reinhardtii culture was kept in anaerobiose under sulfur depletion. (b) Monitoring of ethanol and organic acid concentration. The fermentation time and inoculum age was 5 days and 10 days, respectively. (c) Metabolism pathways of Chlamydomonas during dark anaerobiosis to ethanol production (Adapted from Philipps et al., 2011). PFR1: pyruvate ferredoxin oxidoreductase.
contents, 19.25 ± 4.16 (0.16 ± 0.03 g/L h) and 13.78 ± 3.90 g/L (0.11 ± 0.03 g/L h), the production of acetic acid were the highest as well, 3.22 ± 0.28 g/L and 3.53 ± 0.81 g/L, respectively. The assay with negligible ethanol production presented only 1.81 ± 0.098 g/ L of acetic acid, which is around 54% lower than the other assays. Ethanol production from green algae has not been intensively studied. Hirano et al. (1997) performed the screening for microalgae with high starch content and high ability for intracellular ethanol production. The green alga Chlorella vulgaris (IAM C-534) showed the highest starch content (37%) and the ethanol-conversion rate was 65% compared to the theoretical rate from starch. In the study of the intracellular production by Hirano et al. (1997), a maximum ethanol concentration (1 w/w%) was obtained from C. reinhardtii (UTEX2247) and Sak-l isolated from seawater. Hirano et al. (1997) also evaluated ethanol production with intracellular starch fermentation. In this work, pH medium, cell concentration and different microalgae strains were investigated under dark and anaerobic conditions. Ethanol was observed in almost all of the tested strains and the highest ethanol productivity was attained by C. reinhardtii (UTEX2247). In contrast of the current work, Hirano et al. (1997) observed that the formation of ethanol increased almost proportionally to the concentration of the algal cells. The analysis of the organic metabolites in one of the anaerobic fermented slurries of Chlamydomonas showed that ethanol was the main metabolite, followed by lactate and acetate. The analysis of organic metabolites in hydrogen production by C. reinhardtii CC-124 was performed by Philipps et al. (2011). These authors determined the production of formate and ethanol
achieved 441.6 and 331.2 ng/lg chlorophyll, respectively and in smaller production of D-lactate (102.68 ng/lg chlorophyll). In respect to acetate, this compound was detected in amounts 10–20 times lower than the concentration in TAP medium and, in a medium without acetate; the synthesis of acetate by the alga was 258.17 ng/lmol chlorophyll. Mus et al. (2007) determined the final composition of the organic metabolites in their study of hydrogen production as well. In dark and anoxic fermentation, C. reinhardtii produced malate, formate, acetate and ethanol. The major components were formate, acetate and ethanol in a molar ratio of 1:1:0.5. In the current work, the ratio molar of ethanol and acetate varied from 0.4:0.05 to 0.3:0.06 for those testes that showed significant amount of ethanol. Gfeller and Gibbs (1984) investigated the anaerobic degradation of starch into end products by C. reinhardtii F-60 in the dark and light. Their study resulted in the production of formate, acetate and ethanol in the ratios of 2.07:1.07:0.91, with roughly 100% of starch conversion. Specifically to ethanol, the authors concluded that ethanol synthesis was inhibited by the light in all conditions tested and on the other hand, formate production was least affected by light. Furthermore, the other organic compounds, glycerol and lactate, were detected in minor concentrations. In the current work, besides ethanol, only propionate and acetate were detected in the trials carried out in the evaluation of the effect of initial cell concentration. Lactate and formate were not identified as the works aforementioned described. In this work, formic acid was produced by C. reinhardtii only in medium
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supplemented with milk whey permeated and lactic acid was produced by the green alga only in RCV medium as it will be presented in the following sections. 3.1.2. Effect of carbon source In order to evaluate the influence of increasing the amount and the type of the carbon source on the ethanol production by C. reinhardtii, a set of trials were performed adding sodium acetate and/or milk whey permeate (MWP) to the TAP-S medium (Table 2). Heterotrophic cultivation is suitable for a restrict number of microalgae. According to Perez-Garcia et al. (2011), sucrose, lactate, lactose and ethanol have been tested under heterotrophic microalgae cultures with negative results in growth and metabolite production. Nevertheless, the results of Table 2 proved that C. reinhardtii CC-124 cultivated in mixotrophic conditions has the ability to produce ethanol under anaerobic fermentation with day/light cycles of 12 h supplemented by sodium acetate and, especially, with lactose from milk whey permeate. Table 2 indicated that the TAP-S medium without supplementation of carbon source was not adequate to produce ethanol. This result is in agreement with the aforementioned data. Nevertheless, the additions of 0.1 g/L of sodium acetate or milk whey permeate and the addition of both simultaneously promotes the production of the biofuel. The concentration of ethanol attained was 9.64 ± 1.84 g/L (0.08 ± 0.016 g/L h), 13.11 ± 2.57 g/L (0.11 ± 0.02 g/ L h), 14.94 ± 6.95 g/L (0.12 ± 0.02 g/L h) in the presence of sodium acetate, milk whey permeate and in the presence of both, respectively. Therefore, the medium supplementation with milk whey permeate resulted in increases of the target-product. The data shown in Table 2 also indicate that increasing the supplementation to 1.0 g/L of the same mixotrophic carbon sources do not improve the synthesis of ethanol, indicating that a substrate inhibition could have occurred. Besides ethanol, further metabolites were detected such as acetic acid, propionic acid and formic acid. Propionic acid and acetic acid are present in all compositions of medium. The concentration of the propionic acid is in the order of 1.4 ± 0.08 mg/L to 8.8 ± 1.75 10 3 mg/L. The content of acetic acid was superior to 1.8 g/L and achieved 3.0–3.5 g/L at the highest production of ethanol, similarly to the previous trials (Fig. 1b). Formic acid was synthesized only when milk whey permeate was added to TAP-S medium. By taking into account the supplementation of the mixotrophic carbon source of 0.1 g/L, its concentration range varied from 0.9 g/L for TAP-S medium with MWP to 1.54 g/ L for the medium with TAP-S medium with MWP and Na-acetate. For the assay with addition of 1.0 g/L of MWP to TAP-S medium, the final concentration of formic acid was 1.11 ± 0.45 g/L. The number of developed studies concerning on the mixotrophic cultivation of algae are rare. The aim of these works was to evaluate the effect of the different carbon source in growth rate
and bioproducts like biofuel and pigments (Perez-Garcia et al., 2011; Sahu and Adhikary, 1981). Nevertheless, the influence of mixotrophic carbon source in the ethanol production by C. reinhardtii has not found in the literature. The effect of lactose, fructose, mannose, xylose and sodium acetate on the growth rate and pigment composition by Anabaena sp. under light and dark conditions was investigated by Sahu and Adhikary (1981). They did not describe the ethanol formation, but reported that lactose was the most appropriated carbon source for growth of the alga. The present work indicated that industrial wastes containing this disaccharide are promising resource that can be mixed with water and be used as substrate to C. reinhardtii in the production of ethanol. Besides discussing the effect of carbon source as substrate to C. reinhardtii, it is worth to emphasize that the ethanol amount attained in the current work was considerable higher than certain strains of yeast. For instance, Khattak et al. (2014) investigated the production of ethanol by yeast varying the initial concentration of glucose from 10 to 50 g/L. They found out that the substrate conversion was complete only at 10 g/L of glucose; and the conversion decreased 33% at 50 g/L. The production of ethanol increased from 3.83 to 4.56 g/L corresponded to the initial glucose concentration of 10 and 50 g/L, respectively. The ethanol concentration achieved in Khattak et al. (2014) was approximately of 30% (4.56 g/L) of the ethanol production in this paper (14.94 g/L). Nevertheless, low cost feedstocks that are agroindustrial residues, such as whey cheese, enables to combine the wastewater treatment effluent with the production of value-added products and the addition of small amounts of organic carbon sources leads to higher growth rate without bacterial contamination (Perez-Garcia et al., 2011). 3.2. Ethanol from hybrid system: C. reinhardtii and R. capsulatus An alternative biological route to obtain ethanol is to use the effluent produced in the process by green algae as substrate to photosynthetic bacteria, particularly, the purple non sulfur bacteria. The integration of these photobiological processes, named hybrid system, may lead to higher degradation efficiency, since non purple bacteria may utilize the organic acids and non-converted carbon source present in the effluent by C. reinhardtii into additional ethanol (Batista et al., 2014; Srikanth et al., 2009). Hybrid systems have been widely studied for hydrogen formation, especially those based in the integration of dark fermentation by anaerobic agents and photofermentation by sulfur non-purple bacteria (Tawfik et al., 2014; Urbaniec et al., 2014). Nevertheless, they were not explored for ethanol production by microalgae and photosynthetic bacteria as it was proposed in the present work. In Experiment 1, TAP-S medium was used in the first stage of the hybrid system metabolized by C. reinhardtii. The potential of
Table 2 Effect of carbon source (0.1 and 1.0 g/L) on the production of ethanol. Medium
Cacetic
Concentration of C source (0.10 g/L) TAP-Sb TAP-S + Na-acetate TAP-S + MWPc TAP-S + Na-acetate + MWP
1.79 ± 0.07 2.21 ± 0.64 3.04 ± 0.75 3.49 ± 0.83
1.48 10 5.1 10 4.4 10 8.8 10
3
± 0.002 ± 4.3 10 3 3 ± 0.001 3 ± 1.75 10 3
NDa NDa 0.90 ± 0.37 1.54 ± 0.07
NDa 9.64 ± 1.84 13.11 ± 2.57 14.94 ± 6.95
Concentration of C source (1.0 g/L) TAP-Sb TAP-S + Na-acetate TAP-S + MWPc
1.88 ± 0.05 2.08 ± 0.05 1.89 ± 0.10
1.4 10 3.7 10 2.96 10
3
NDa NDa 1.11 ± 0.45
NDa NDa NDa
acid
(g/L)
The error estimates are derived from replicated (2) runs. a ND – not detected. b Concentration of acetic acid in TAP-S medium: 1.04 g/L. c MWP, milk whey permeate.
Cpropionic
acid
(g/L)
3
± 0.008 ± 0.37 10 2 3 ± 0.045 10 2 3
Cformic
acid
(g/L)
Cethanol (g/L)
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R.L. Costa et al. / Bioresource Technology 181 (2015) 330–337 Table 3 Effect of the composition medium for R. capsulatus on the production of ethanol in a hybrid system by C. reinhardtii and R. capsulatus. Triala
Composition of metabolites (g/L)c C. reinhardtii
1 2
R. capsulatus
Cacet
Cprop
1.79 ± 0.07 1.84 ± 0.05
1.5 10 0.7 10
3
± 0.002 3 ± 0.000
Ceth
Clact
Cform
NDb NDb
0.16 ± 0.11 5.95 ± 4.02
0.01 ± 0.04 10 0.21 ± 0.025
4
Cacet
Cprop
Cbut
Ceth
2.58 ± 0.41 4.38 ± 0.97
0.80 ± 0.56 5.52 ± 3.89
NDb 2.38 ± 1.60
12.61 ± 2.01 18.22 ± 1.89
The error estimates are derived from replicated (2) runs. a Trial 1: First stage, C. reinhardtii – TAP-S medium, Second stage R. capsulatus – crude effluent; Trial 2: First stage, C. reinhardtii – TAP-S medium, Second stage R. capsulatus – effluent with supplements. b ND – not detected. c Metabolites: Acet: acetic acid; prop: propionic acid; lact: lactic acid; form: formic acid; but: butyric acid; eth: ethanol.
Table 4 Effect of the enrichment with 0.1 g/L of extra heterotrophic carbon source in the medium for C. reinhardtii on the production of ethanol in a hybrid system by C. reinhardtii and R. capsulatus. Medium in the 1st stage of hybrid systema
TAP-S TAP-S + Na-acetate TAP-S + MWPc TAP-S + Na-acetate + MWP
Composition of metabolites (g/L)c C. reinhardtii
R. capsulatus
Cacet
Cprop
1.79 ± 0.07 2.21 ± 0.64 3.04 ± 0.75 3.50 ± 0.83
1.48 10 5.1 10 4.4 10 8.8 10
3
± 0.002 3 ± 4.3 10 3 3 ± 0.001 3 ± 1.75 10 3
Cform
Ceth
Cacet
Cprop
Cform
Ceth
NDb NDb 0.90 ± 0.37 1.54 ± 0.07
NDb 9.64 ± 1.84 13.11 ± 2.57 14.94 ± 6.95
2.32 ± 0.41 3.20 ± 0.22 4.08 ± 0.24 2.90 ± 0.28
0.36 ± 0.056 0.01 ± 0.003 0.05 ± 0.56 10 0.03 ± 0.02
NDb NDb 0.05 ± 0.01 NDb
10.26 ± 2.01 16.13 ± 2.69 19.94 ± 2.67 17.17 ± 2.63
2
The error estimates are derived from replicated (2) runs. a Second stage: R. capsulatus – crude effluent; b ND – not detected. c Metabolites: Acet: acetic acid; prop: propionic acid; lact: lactic acid; form: formic acid; but: butyric acid; eth: ethanol; MWP: milk whey permeate.
R. capsulatus of producing ethanol from the metabolites synthesized by the green alga was evaluated comparing the biofuel production using the crude fermentative broth and the effluent supplemented with sodium glutamate, malic acid, milk whey permeate and micronutrients. In both cases, the effluent was free of C. reinhardtii and fermentation time was 5 days in the two stages of the hybrid system. The results are presented in Table 3. The results shown in Table 3 proved that R. capsulatus synthesis ethanol even though the macro and micronutrients, usually found in RCV medium, were not present. Table 3 also indicated that ethanol production by R. capsulatus cultivated in effluent supplemented with malic acid, milk whey permeate and sodium glutamate was 44.5% superior (18.22 g/L) than when bacteria were cultivated in the crude effluent (12.61 g/L). In respect to the metabolites, the amount of organic acids was higher as well. In the crude effluent there were around 1.79 ± 0.07 g/L to 1.84 ± 0.55 g/L of acetic acid and at the end, the effluent produced by R. capsulatus in the medium without supplementation was composed by 0.16 ± 0.11 g/L of lactic acid, 2.58 ± 0.41 g/L of acetic acid, 0.01 ± 0.04 10 4 g/L of formic acid and 0.80 ± 0.56 g/L of propionic acid. The composition of the effluent by R. capsulatus in the supplemented medium was 5.95 ± 4.02 g/L of lactic acid, 4.38 ± 0.97 g/L of acetic acid, 5.52 ± 3.89 g/L of propionic acid, 0.21 ± 0.025 g/L of formic acid and 2.38 ± 1.60 g/L of butyric acid. In the following Experiments 2 and 3, an effluent produced by C. reinhardtii cultivated in a medium enriched with a carbon source was used by R. capsulatus. In the Experiment 2, the medium used by C. reinhardtii was supplemented with 0.1 g/L of sodium acetate or milk whey permeate or both of the components and the composition of the effluent is shown in Tables 2 and 4. In the Experiment 3, the supplementation of the medium was made by the addition of 1.0 g/L the same components and the composition of the effluent is shown in Tables 2 and 5.
As it was discussed aforementioned, the medium enriched with 0.1 g/L of milk whey permeate increased the ethanol production by C. reinhardtii. The effluent produced from this medium led to a higher final production of the biofuel, 19.94 ± 2.67 g/L (0.17 ± 0.02 g/L h) by R. capsulatus, as Table 4 demonstrated. Although the effluent utilized by the bacteria was not supplemented with malic acid and MWP, the acetic acid and formic acid may have provided carbon source enough to R. capsulatus metabolism. Concerning on the organic acids, except to TAP-S medium with sodium acetate and milk whey permeate, acetic acid and propionic acid were synthesized in both stages of the hybrid system. In the trials with TAP-S medium containing MWP, the formic acid produced in the first stage was consumed by the photosynthetic bacteria. Acetic acid produced by C. reinhardtii was consumed by R. capsulatus in the assay with TAP-S medium supplemented with sodium acetate and MWP. The analysis of Tables 2 and 5 showed that no production of ethanol was verified at higher concentration of carbon source (1.0 g/L), independently whether sodium acetate or milk whey permeate were added. Besides the ethanol, acetic acid was synthesized by R. capsulatus only in the trial of TAP-S medium without supplementation. In further trials, the addition of 1.0 g/L of sodium acetate or milk whey permeate led to a metabolic pathway in which consume of acetic acid and no production of ethanol by the bacterium were observed. Complementarily, to produce biofuels as hydrogen and ethanol, purple non-sulfur bacteria commonly synthesize polyhydroxyalkanoates. In the current work, the consumption of the organic acids without production of ethanol by R. capsulatus suggested that further metabolites could be synthesized, as it was demonstrated by Sawayama (2001) and Maeda et al. (1998). Hybrid systems incorporating two stages of algae and photosynthetic bacteria are hardly to find published, since the focus of
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Table 5 Effect of the enrichment with 1.0 g/L of extra heterotrophic carbon source in the medium for C. reinhardtii on the production of ethanol in a hybrid system by C. reinhardtii and R. capsulatus. Medium in the 1st stage of hybrid systema
Composition of metabolites (g/L)c Chlamydomonas reinhardtii
TAP-S TAP-S + Na-acetate TAP-S + MWPc
Cacet
Cprop
1.88 ± 0.16 2.08 ± 0.30 1.89 ± 0.35
1.4 10 3.7 10 2.96 10
Rhodobacter capsulatus
3
± 0.17 3 ± 0.001 3 ± 0.001
Ceth
Cacet
Cprop
Ceth
NDb NDb NDb
4.09 ± 0.15 1.08 ± 0.37 10 0.95 ± 0.28
NDb 0.03 ± 0.003 0.02 ± 0.002
12.57 ± 0.01 NDb NDb
2
The error estimates are derived from replicated (2) runs. a Second stage: R. capsulatus – crude effluent; b ND – not detected. c Metabolites: Acet: acetic acid; prop: propionic acid; lact: lactic acid; form: formic acid; but: butyric acid; eth: ethanol. MWP: milk whey permeate.
Table 6 Composition of metabolites produced by co-culture system based on different C reinhardtii (A) to R. capsulatus (B) ratios. Composition of metabolites (g/L)b Strain ratio
Clact
Basal medium
RCV
TAP-S
RCV
TAP-S
RCV
TAP-S
RCV
TAP-S
A A A A A A A
0.57 ± 0.18 0.15 ± 0.07 0.07 ± 0.02 0.31 ± 0.22 0.41 ± 0.28 0.78 ± 0.18 0.27 ± 0.07
NDa 0.01 ± 0.65 10 NDa 0.27 ± 0.19 NDa 0.26 ± 0.02 NDa
0.58 ± 0.33 1.47 ± 0.09 0.46 ± 0.10 0.32 ± 0.067 0.37 ± 0.069 0.93 ± 0.15 2.58 ± 1.00
3.45 ± 0.29 3.42 ± 0.33 4.94 ± 1.60 3.73 ± 1.82 3.64 ± 0.73 2.37 ± 2.20 3.09 ± 0.15
0.54 ± 0.15 0.20 ± 0.14 NDa NDa NDa 0.33 ± 0.20 0.91 ± 0.08
NDa 1.1 10 2 ± 0.83 10 4.4 10 2 ± 0.03 10 0.91 ± 0.64 2.4 10 2 ± 0.11 10 6.7 10 3 ± 0.53 10 1.5 10 3 ± 0.02
9.00 ± 6.36 NDa 0.79 ± 0.06 NDa NDa NDa 12.61 ± 8.91
NDa 0.08 ± 0.06 0.25 ± 0.12 0.22 ± 0.16 NDa 0.12 ± 0.02 0.09 ± 0.02
(0%): B (100%) (10%): B (90%) (30%): B (70%) (50%): B (50%) (70%): B (30%) (90%): B (10%) (100%): B (0%)
Cacet
2
Cprop
Ceth
2 2
2 2
The error estimates are derived from replicated (2) runs. a ND – not detected. b Metabolites: Acet: acetic acid; prop: propionic acid; lact: lactic acid; but: butyric acid; eth: ethanol.
the works developed by Maeda et al. (1998), Miura et al. (1997) and Miura et al. (1992) was the hydrogen production. In order to investigate hydrogen production, Maeda et al. (1998) proposed an integrated process where the effluent produced by Chlamydomonas sp. MGA161, cultivated under dark and anaerobic conditions, was used by Rhodovulum sulfidophilum W-1S. In the first stage, acetic acid, ethanol and glycerol were the main products excreted into the medium during Chlamydomonas sp. MGA161. In the second stage, there was consumption by R. sulfidophilum W1S of all these organic acids immediately after hydrogen evolution started. The amounts of the metabolites were not discussed by the authors. Miura et al. (1997) scaled up photobiological hydrogen production system to a pilot plant scale in the photosynthetic starch accumulation, followed by the fermentative production of metabolites from starch by Chlamydomonas MGA 161 and the hydrogen photoproduction by R. sulfidophilum W-1S from fermentative broth produced by Chlamydomonas MGA 161 in the natural day/ night cycle. The yield of acetic acid, ethanol and glycerol from starch of Chlamydomonas MGA 161 was 80–100% of the theoretical yield. Maeda et al. (1998) and Miura et al. (1997) also described that the decrease in the conversion of substrate into hydrogen production was due to the accumulation of polyhydroxybutyrate (PHB). Similar conclusion was withdrawn by Sawayama (2001) that studied the consumption of acetate by photosynthetic bacteria. The author reported the production of polyhydroxybutyrate (PHB) by phototrophic bacteria using as substrate an effluent from lighted upflow anaerobic sludge blanket (LUASB) reactor with sodium acetate under anaerobic light condition. The concentration of PHB enhanced in a secondary incubation of this effluent with Na-acetate.
3.3. Ethanol from co-culture: C. reinhardtii and R. capsulatus In this current work, strategies of co-cultivation of C. reinhardtii (green alga) and R. capsulatus (PNS Bacteria) to ethanol production were also attempted. In this coupled system, R. capsulatus could use the organic acids excreted by C. reinhardtii cells during anaerobic photofermentation process. Furthermore, the integration of these two biological systems may enhance the ethanol production with a reduction in energy cost, due to better solar irradiance utilization (visible and infrared) and integrated use of the nutrients (Melis and Melnicki, 2006). Table 6 summarizes the results of cocultivation. Two fermentation media were tested in the co-cultivation, that is, RCV and TAP-S. In the RCV medium used in the co-cultivation, lactic and acetic acids were produced in all algae to bacteria ratios. The production of propionic acid and ethanol did not present a trend by increasing the alga to bacteria ratio. Surprisingly, isolated green alga [Algae (100%): Bacteria (0%)] cultured in RCV medium (a basal medium to PNS bacteria) was fully capable to synthesize ethanol resulting in 12.6 ± 8.91 g/L (0.11 ± 0.07 g/L h). Similar procedure was also performed to co-culture maintained in TAP-S medium. However, R. capsulatus has not presented a good performance in relation to the ethanol production in this medium formulation, probably due the lack of essential nutrients. In TAP-S medium, our findings showed that C. reinhardtii and R. capsulatus contributed equally to acetic acid production, since at isolated bacterial culture [Algae (0%): Bacteria (100%)] resulted in 3.45 ± 0.29 g/L and isolated algal culture [Algae (100%): Bacteria (0%)] produced up to 3.09 ± 0.15 g/L. In addition, propionic acid was produced when the variant ration of 1:1 of cells was used in the co-cultivation. On the other hand, the final ethanol concentration [Algae (30%): Bacteria (70%)] was at least 3.2-fold lower than
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the minimum ethanol content observed in the co-culture maintained in RCV medium (0.79 ± 0.06 g/L) at the same variant ratio. The co-culture approach of microalgae and photosynthetic bacteria has not been explored and the influence of lots of variables is still unknown. However the results of ethanol content obtained through the co-cultivation strategy were lower than those from mixotrophic photofermentation by C. reinhardtii isolated or in hybrid system with R. capsulatus. It is necessary to enlarge the conditions of this technique by evaluating the composition of the medium, including the supplementation of organic carbon source, nutrients and light intensity as Melis and Melnicki (2006) did. These authors investigated the hydrogen production by coupling Rhodospirillum rubrum and C. reinhardtii, in a bioreactor containing all the nutrients of both Ormerod and TAP that are media used to the maintenance of bacteria and algae cultures, respectively. Moreover, other parameter evaluated was the light intensity (30–150 lmol photons m 2 s 1), since the level of irradiance could regulate the relative growth and production of metabolites of the two types of cells. Melis and Melnicki (2006) did not present data related to the composition of organic acids and ethanol. In general, for each system approached in this work, it is important to emphasis that a full study must be performed in order to enhance the comprehension of the effect of the medium composition and operational conditions on the ethanol production by C. reinhardtii and R. capsulatus. For instance, the light intensity, residual sulfur content, photoperiod and bioreactor configurations may influence the biofuel synthesis. The comprehension of metabolism of different organic compounds by C. reinhardtii and R. capsulatus requires more detailed studies. Further researches may lead to significant progress in improving the biomass accumulation, starch production and co-production of ethanol. The results of this work may be used in future design of ethanol production in integrated systems weather by hybrid or co-culture systems. 4. Conclusion In this study the effect of inoculum concentration and carbon source to C. reinhardtii, as well as the influence of hybrid system and co-culture (C. reinhardtii and R. capsulatus) on the photofermentative ethanol production were investigated. Maximum ethanol content 19.94 ± 2.67 g/L (0.17 ± 0.02 g/L h) was achieved by hybrid system in which the effluent of C. reinhardtii containing organic acids was used as substrate to R. capsulatus. The results from this work are beneficial to comprehend the potentiality of green alga and PNS photosynthetic bacteria to synthesize ethanol concerning several strategies such as media composition and different culture systems (hybrid and co-cultivation). Acknowledgements The authors wish to thank Brazilian agencies CNPq, CAPES and FAPEMIG (Grant No. APQ-01020-12) for financial support. References Andersen, R.A. (Ed.), 2005. Algal Culturing Techniques. London Elsevier Academic Press (p. 578). Andrietta, S.R., Steckelberg, C., Andrietta, M.G.S., 2008. Study of flocculent yeast performance in tower reactors for bioethanol production in a continuous fermentation process with no cell recycling. Bioresour. Technol. 99, 3002–3008. Batista, A.P., Moura, P., Marques, P.A., Ortigueira, J., Alves, L., Gouveia, L., 2014. Scenedesmus obliquus as feedstock for biohydrogen production by Enterobacter aerogenes and Clostridium butyricum. Fuel 117, 537–543. Catalanotti, C., Dubini, A., Subramanian, V., Yang, W., Magneschi, L., Mus, F., Seibert, M., Posewitz, M.C., Grossman, A.R., 2012. Altered fermentative metabolism in Chlamydomonas reinhardtii mutants lacking pyruvate formate lyase and both pyruvate formate lyase and alcohol dehydrogenase. Plant Cell 24, 692–707.
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