Biohydrogen and methane production via a two-step process using an acid pretreated native microalgae consortium

Bioresource Technology 221 (2016) 324–330

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Biohydrogen and methane production via a two-step process using an acid pretreated native microalgae consortium Julian Carrillo-Reyes, Germán Buitrón ⇑ Laboratory for Research on Advanced Processes for Water Treatment, Unidad Académica Juriquilla, Instituto de Ingeniería, Universidad Nacional Autónoma de México, Blvd. Juriquilla 3001, Querétaro 76230, Mexico

h i g h l i g h t s
A native microalgae consortium was pretreated using thermal-acidic hydrolysis.
Hydrogen and methane were produced sequentially with the acidic hydrolysates.
The lower acid concentration gave the highest H2 and CH4 production. 1


H2 and CH4 yields were up to 45.4 and 432 mL g VS

a r t i c l e

i n f o

Article history: Received 21 July 2016 Received in revised form 7 September 2016 Accepted 11 September 2016 Available online 13 September 2016 Keywords: Dark fermentation Hydrogen Methane Acidic hydrolysis Microalgae Consortium

, respectively.

a b s t r a c t A native microalgae consortium treated under thermal-acidic hydrolysis was used to produce hydrogen and methane in a two-step sequential process. Different acid concentrations were tested, generating hydrogen and methane yields of up to 45 mL H2 g VS1 and 432 mL CH4 g VS1, respectively. The hydrogen production step solubilized the particulate COD (chemical oxygen demand) up to 30%, creating considerable amounts of volatile fatty acids (up to 10 g COD L1). It was observed that lower acid concentration presented higher hydrogen and methane production potential. The results revealed that thermal acid hydrolysis of a native microalgae consortium is a simple but effective strategy for producing hydrogen and methane in the sequential process. In addition to COD removal (50–70%), this method resulted in an energy recovery of up to 15.9 kJ per g of volatile solids of microalgae biomass, one of the highest reported. Ó 2016 Elsevier Ltd. All rights reserved.

1. Introduction The energy crisis has driven a search for renewable fuels that can be produced using substrates such as photosynthetic biomass or wastes. The term microalga often generalizes all photosynthetic unicellular or simple multi-cellular prokaryotic and eukaryotic microorganisms, such as cyanobacteria, green and red algae, and diatoms. Microalgae are a potential biomass for biofuel production because of their fast growth rate, their lipid and carbohydrate content, and their cultivation in wastewater, which is coupled to their effective role in nutrient removal. When compared to terrestrial crops, microalgae cultures consume less water, reaching higher productivities per culture area, and do not compromise the production of food (Brennan and Owende, 2010). In this sense, a sustainable biofuel production based on microalgae is only

⇑ Corresponding author. E-mail address: [email protected] (G. Buitrón). http://dx.doi.org/10.1016/j.biortech.2016.09.050 0960-8524/Ó 2016 Elsevier Ltd. All rights reserved.

possible under a biorefinery approach, producing gaseous biofuels and other value-added products from microalgae biomass (Sarkar et al., 2015). Microalgae cultivation in wastewater will promote the development of a consortium, contrasting with the mono-algal cultures evaluated in most studies of fuel production. The importance of evaluating a native microalgae consortium lies in the wide diversity of cell wall composition among microalgae species (Domozych et al., 2012), implying different grades of resistance between species. The high carbohydrate content in microalgae makes them a suitable substrate for fermentative fuel production, producing fuels such as biohydrogen, bioethanol, and methane. However, carbohydrates are difficult to extract from microalgae because they are part of the microfibrillar polysaccharides embedded in matrix of polysaccharides and proteoglycans, making necessary a pretreatment step to liberate them (Domozych et al., 2012; Günerken et al., 2015). Different pretreatment technologies have been suggested to break down complex biopolymers in microalgae cells, among them biological, mechanical or chemical.

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Biological pretreatments involve the cell degradation by purified enzymes or by microorganisms with enzymatic activity capable of hydrolyzing the microalgae cell wall (Carrillo-Reyes et al., 2016). In this sense, significant differences were observed in the fermentative step when microalgae biomass (Chlorella vulgaris) was used directly or received an enzymatic pretreatment. Specifically, the former resulted in a yield of 11.3 mL H2 g(volatile solids) VS1 (Lakaniemi et al., 2011), whereas enzymatic pretreatment produced 135 mL H2 g VS1 (Wieczorek et al., 2014). Methane production from microalgal biomass has been improved by applying pretreatments to solubilize the microalgae and digest their organic content. For instance, applying a thermal pretreatment there was an increase of 50% in the methane production from Chlorella vulgaris (Mendez et al., 2014). Chemical pretreatment has an economic advantage over enzymatic pretreatment or thermal pretreatments; however, its application to native microalgae biomass for hydrogen and methane production is still limited (Passos et al., 2014). To the best of our knowledge, the scarce works applying chemical pretreatments are combined with harsh physical disruption strategies, such as ultrasonic or high pressure (Cheng et al., 2014; Liu et al., 2012; Yun et al., 2013). Among chemical pretreatments for microalgae, acidic hydrolysis has been successful in carbohydrate recovery for bioethanol production. For instance, thermal-acidic hydrolysis, under optimized conditions, achieved 95.6% sugar extraction from Scenedesmus obliquus (Miranda et al., 2012), and 97% from Chlorella vulgaris (Ho et al., 2013). Regarding biohydrogen production, acidic hydrolysis recovered almost 100% of the carbohydrate concentrations as reducing sugars; however, this procedure has only been optimized for pure microalgae strains (Liu et al., 2012), which is different from the microalgae consortium that could be recovered from wastewater treatment. Moreover, the acidic hydrolysate concentration is a key parameter to evaluate for increasing the specific hydrogen-producing potential from microalgae biomass, since it has been observed the generation of inhibitors such as furans and 5-hydroxymethyl furfural (HMF) (Yun et al., 2013). Two-step processes have been proposed to improve the energetic gain from microalgal biomass (Yang et al., 2011; Lü et al., 2013; Wieczorek et al., 2014). In such processes, the carbohydrates are first fermented producing hydrogen and volatile fatty acids (VFA). Then, in a second step, the VFA are easily digested under methanogenic conditions to generate methane. This two-step strategy has been applied in lipid-extracted microalgal biomass residues increasing the methane yield by 22% (Yang et al., 2011), and up to 67% compared to methanogenesis using a single step (Wieczorek et al., 2014). Lü et al. (2013) found a 9.4% increase in the energy yield in a two-step process when compared with the one-step process, using bacterial bioaugmentation. Despite the advances on microalgal pretreatments, most of the previously cited works used mono-algal cultures as feedstock, leaving unresolved the potential barriers of hydrolyzing mixed cultures, such as the developed in wastewater. Therefore, the aim of the present work was to evaluate the energy recovery through the hydrogen and methane production using a two-step process. A thermal acidic pretreated native microalgae consortium was evaluated under different hydrolysate conditions.

2. Materials and methods 2.1. Microalgae biomass A native microalgae consortium enriched from a local lake in Queretaro, Mexico (20°420 07.000 N 100°270 36.700 W) was used as

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the biomass source. The microalgae culture was enriched in Bold’s medium in tubular plastic bags (8 L) as reactors illuminated by 12 h light-dark cycles supplied via a 54 W daylight neon lamp with an intensity of 100 lmol m2 s1 (LT 300, Extech Instruments, Nashua, NH, USA) and aeration flow rate of 1 L min1 (Cea-Barcia et al., 2014). The culture composition was determined by optical microscopy (Leica DM500, Japan), and direct counting was performed with a 0.1 mm Neubauer chamber (Wehr and Sheath, 2003). The main genera identified was Scenedesmus (79%), with the remaining microalgae belonging to Keratococcus (19%), Oscillatoria and undetermined species (<2%). The microalgal culture was concentrated by centrifugation (4500 rpm, 10 min). Biochemical fractioning of the microalgae consortium revealed that biomass was composed of 20% of carbohydrates, 19% of lipids and 50% of proteins. 2.2. Thermal-acidic pretreatment The microalgae biomass was hydrolyzed in 2% HCl solution (v/w) at 40 g TS (total solids) L1, heated at 90 °C for 2 h with constant mixing at 300 rpm in a total volume of 300 mL. After the pretreatment, the hydrolysate was neutralized with 10 N NaOH. The pretreatment protocol was carried out in triplicate to evaluate its reproducibility. After evaluating different dilutions of 2% HCl hydrolysates, microalgae biomass was hydrolyzed with 1% HCl using 20 and 10 g TS L1, at the same temperature and mixing conditions. Hydrolysis conditions were based on a previous work that optimized the saccharification using dilute acid hydrolysis for microalgae biomass (Castro et al., 2015). 2.3. Hydrogen- and methane-producing inoculum The inoculum for hydrogen and methane tests was a granular anaerobic sludge from a digester treating wastewater from the brewery industry; the sludge had a solids content of 27 g TS L1 and 19 g volatile solids (VS) L1. Prior to its utilization for hydrogen tests, a thermal pretreatment was applied to the inoculum (105 °C, 24 h) to select those hydrogen-producing bacteria capable of sporulating. Then, the dried sludge was ground with a mortar to homogenize, and the resulting powder was used as inoculum (Buitrón and Carvajal, 2010). For methane tests, the granular anaerobic sludge was kept under endogenous conditions for 2 weeks to reduce the remaining substrate and the exogenous biogas generation. No further treatment was applied to that sludge. 2.4. Hydrogen production batch tests Hydrogen production batch tests were performed in triplicate in sealed 120 mL serum bottles in a 60 mL working volume and 6.7 g VS L1 of inoculum. The specific hydrogen production and rate were evaluated using two different acid concentrations for the hydrolysate. In a first round, three dilutions of 2% HCl hydrolysates were tested and named according to the final solid concentrations obtained as 40, 20 and 10 g TS L1. Then, a second set of experiments using 1% HCl hydrolysates was evaluated. Here, the solids concentrations of 20 and 10 g TS L1 were selected. The mineral medium composition was described previously (Mizuno et al., 2000). The initial pH was adjusted to 6.5 with 5 N HCl or 5 N NaOH. The head space was purged with N2 for 1 min to ensure anaerobic conditions, incubating the bottles at 36 °C with 150 rpm of horizontal shaking (WiseCube, Daihan Scientific Co., Korea) until the gas production stopped. Blank tests containing only inoculum and mineral medium were carried out to determine the endogenous hydrogen production from the inoculum. Gas production was measured daily by the liquid displacement method (an acidic

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water-filled inverted burette was used). To evaluate the effect of hydrolysis on the hydrogen production from the microalgae, control tests using microalgae without treatment were performed at 20 and 10 g TS L1 with the inoculum and mineral medium under the conditions already described. 2.5. Methane production batch tests

2.7. Data analysis 2.7.1. COD solubilization and consumption The percentage of COD solubilization (CODSOL) during the hydrolysis step and the hydrogen production tests was evaluated according to Eq. (1) (Alzate et al., 2012):

CODSOL ¼

The methane production potential was evaluated using the remaining material from hydrogen production tests. Methane production batch tests were performed in triplicate in sealed 120 mL serum bottles with a 45 mL working volume. Only anaerobic sludge as the inoculum was added to the residual material from the hydrogen tests, using a ratio of 0.5 g VS inoculum (g VS microalgae)1 (Alzate et al., 2012). The initial pH was adjusted to 7.5 with 5 N NaOH. The head space was purged with N2 for 1 min to ensure anaerobic conditions, and the bottles were incubated at 36 °C with 150 rpm of horizontal shaking until gas production stopped. Blank tests containing only inoculum and mineral medium were carried out to measure the endogenous methane production from the inoculum. Gas production was monitored three times a week using a pressure transducer, releasing the gas to equal atmospheric pressure. To evaluate the effect of hydrolysis on the methane production from the microalgae, control tests using microalgae without treatment were performed at 40, 20 and 10 g TS L1 with the inoculum and mineral medium under the same conditions as described above. 2.6. Analytical methods The hydrogen, carbon dioxide and methane content of the gas produced was analyzed using a gas chromatograph (8610C, SRI Instruments, Torrance, CA, USA) equipped with a thermal conductivity detector (TCD) and two packed columns (a 60  1/800 silica gel packed column and a 60  1/800 molecular sieve 13 packed column). The injector and detector temperatures were 90 °C and 150 °C, respectively. The initial column temperature was 40 °C, which was held for 4 min and then gradually increased to 110 °C at a rate of 20 °C min1. The final column temperature was held for 3 min. Nitrogen was used as a carrier gas at a flow rate of 20 mL min1. From the soluble phase, volatile fatty acids (VFA) and ethanol were determined at the end of batch runs and from the hydrolysate using a gas chromatograph (7890 B, Agilent Technologies, Santa Clara, CA, USA) fitted with a FID detector and a 15 m long (530 lm  1 lm) DB-FFAP column. Injector and detector temperatures were maintained at 190 and 210 °C, respectively. The initial temperature of the column was 60 °C; then, it was increased to 90 °C at a rate of 15 °C min1, subsequently to 170 °C at a rate of 25 °C min1 and hold during 3.5 min. The carrier gas was nitrogen at 25 mL min1. COD and TS and VS were measured according to standard methods (APHA/AWWA/WFE, 2005). Total sugars were measured using the phenol-sulphuric acid method (Dubois et al., 1956), and reducing sugars using the DNS (Dinitrosalicylic Acid) method (Miller, 1959).

  ½ðCODs Þf  ðCODs Þi  100 CODt  ðCODs Þi

ð1Þ

where subscript letters i and f are the initial and final values, respectively, and s and t refer to the soluble and total fractions. COD consumption was determined at the end of the methane production tests. 2.7.2. Kinetics of hydrogen and methane production The kinetics of hydrogen and methane from batch tests (expressed at 273 K and 1 atm) were fitted to the modified Gompertz equation (Eq. (2)) using KaleidaGraph 4.0 (Synergy software):

   2:71828Rmax HðtÞ ¼ Hmax exp  exp ðk  tÞ þ 1 Hmax

ð2Þ

where H(t) (mL g VS1) is the accumulated hydrogen or methane production at time t (d); Hmax (mL g VS1) is the maximum gas produced; Rmax is the maximum production rate [mL (g VS d)1] and k is the lag-phase time before the exponential hydrogen or methane production (d). Fitting results are presented in the Supplementary data (Tables S1 and S2). The performance of the hydrogen and methane tests were evaluated by calculating the Rmax and Hmax values, and the statistical significance of differences among them was determined by analysis of variance (ANOVA F-test, p = 0.05). 3. Results and discussion 3.1. Thermal acidic microalgal pretreatment The percentage of COD solubilization (CODSOL) during pretreatment was 37.7%, 39.8% and 50.5% at different hydrolysate conditions of 40, 20 and 10 g TS L1, respectively. A release of volatile fatty acids (Table 1) was observed and explained by cellulose and hemicellulose hydrolysis (Hendriks and Zeeman, 2009). During pretreatment, the sugars solubilization reached 85% and 100% of the total sugar content using 2% HCl and 1% HCl (Table 1), respectively. A higher recovery of reducing sugars (close to 90%, with respect to total sugars) was obtained with the 1% HCl concentration, than the value obtained with the 2% HCl concentration (49%). The reducing sugars concentration is crucial because of the role carbohydrate complexity plays in determining the hydrogenproduction efficiency and yield (Quéméneur et al., 2011). An improvement on the hydrogen production is expected at higher concentrations of reducing sugars. In a previous study, a native consortium of microalgae primarily dominated by Scenedesmus, was treated with 1 M H2SO4 and the optimized thermal acidic

Table 1 Thermal-acid hydrolysates characterization. TS content (g L1)

40 20 10

COD (g L1)

Sugars (g L1) a

VFA (mg L1)

Total

Soluble

Total

Soluble

67.4 29.2 13.6

25.4 11.6 6.9

5.5 2.8 1.3

4.7 2.8 1.3

a

Soluble 2.3 2.5 1.2

b

Acetate

Propionate

Butyrate

1106 206 85

356 0 0

619 249 130

TS, total solids; COD, chemical oxygen demand; VFA, volatile fatty acids. aDetermined by acid-sulfuric method; breducing sugars determined by DNS method. 40 g ST L1 HCl at 2%; 20 and 10 g ST L1 HCl at 1%.

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hydrolysis achieved a yield of 166 gsugars kg1 alga (Castro et al., 2015). Although the acid concentration was significant lower in our study (1% HCl or 0.33 M), a similar sugar recovery yield of 120 gsugars kg-alga 1 was observed. The lower yield is attributed to the lower sugars content in the microalgae culture (Table 1). Castro et al. (2015) evaluated the hydrolysis with 0.35 M H2SO4, similar to our 1% HCl condition (0.33 M), achieving a lower sugar recovery (71%) than in our best result. This comparison shows how the microalgae composition may affect the efficiency of the pretreatment. 3.2. Hydrogen production No significant effects on the specific maximum hydrogen production (Hmax) nor in the production rates were observed for the 2% HCl hydrolysate among the evaluated dilutions (40, 20 and

Fig. 1. Specific hydrogen production of microalgae biomass by dark fermentation, pretreated and without pretreatment at different microalgae concentrations. Symbols indicate the hydrogen molar yield in terms of soluble sugars consumed.

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10 g TS L1) as well as for both solids concentration evaluated with the 1% HCl (Fig. 1). However, comparing both acid concentrations, the biomass hydrolyzed with 1% HCl presented a significant higher hydrogen production than the hydrolyzed with 2% HCl, achieving the maximal production within 96 h (Fig. 1). In terms of production rate, the maximum value using the 1% HCl hydrolysate was 54.6 ± 17.4 mL H2 (g VS d)1, increasing up to 5.8-fold and 10fold with respect to the best results obtained with the 2% HCl condition and the untreated biomass, respectively. The higher hydrogen production from biomass hydrolyzed with 1% HCl is not only explained by the higher sugar solubilization, but also by the higher molar yield achieved from the consumed sugars (Fig. 1). In this sense, a probable inhibition occurred with the 2% HCl hydrolysates, fostering the formation of hydrogen inhibitors such as furfurals. It has been suggested that 1.6% HCl concentration and a pretreatment time longer than 60 min produce at least 1.5 g L1 of HMF from a C. vulgaris biomass (Yun et al., 2013). Regarding the hydrogen metabolic pathway, acetate has a direct relationship with hydrogen production (Yang et al., 2010). In our study, even though acetate was produced in higher concentration at 2% HCl than 1% HCl (Fig. 2a), the average hydrogen molar yield in the latter was two times higher (Fig. 1). Homoacetogenesis can explain the higher acetate concentration observed at 2% HCl, a hydrogen consuming metabolic pathway that has been already suggested in microalgae biomass fermentation (Yang et al., 2010). An additional factor for the lower hydrogen production at 2% HCl was the propionate production, another hydrogenconsuming pathway, which is present in a higher proportion than the observed for 1% HCl hydrolysate (Fig. 2a, Supplementary data Table S3). The specific hydrogen production with the 1% HCl hydrolysates was similar to those obtained in most of the hydrogen production tests (Table 2); however, in the present research, the proposed strategy was milder compared with others strategies combining acid hydrolysis with ultrasound or microwave or higher acid concentrations, for example. Higher hydrogen production rates were observed only in the case where a single fermentative bacteria was used (Batista et al., 2014). Results indicated that another important factor is the carbohydrate content in the microalgae biomass. The more carbohydrate content in the microalgae culture, the more hydrogen is produced. Liu et al. (2012) using a C. vulgaris culture with a carbohydrate content of 57% (dry weight basis)

Fig. 2. Electron balance for the first and second steps. (A) Percentage of the initial COD invested to the production of metabolites (ethanol, volatile fatty acids and remaining soluble COD), hydrogen and biomass during the fermentative step; (B) Percentage of COD converted to methane during the second stage, either with respect to the initial COD as well as in respect to the metabolites produced during the first step.

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Table 2 Relevant results of hydrogen production from microalgae biomass by dark fermentation. Microalgae biomass Mesophilic fermentation C. vulgaris C. vulgaris S. obliquus Wastewater consortium Chlorella sp. Algae bloom in lake Native consortium Native consortium

Pretreatment

Inoculum

Ta (°C)

mL H2 g VS1

mmol H2 g VS1

1.5% HCl, 121 °C, 20 min 0.79% HCl, 49600 kJ kg1 algae (ultrasound) 121 °C, 15 min 121 °C, 15 min None 1% H2SO4, 140 °C, 15 min (microwave) 1% HCl, 90 °C, 2 h None

Clostridium butyricum Treated anaerobic sludge

37 35

81 42.1c

3.19 1.67

Liu et al. (2012) Yun et al. (2013)

Clostridium butyricum Enterobacter aerogenes Treated anaerobic sludge Mixed hydrogen-producing bacteria

37 30 35 35

113.1 46.8 7.1 47.1

4.44 1.89 0.28 1.86

Batista et al. (2014) Batista et al. (2015) Sun et al. (2011) Cheng et al. (2014)

Treated anaerobic sludge Treated anaerobic sludge

36 36

45.4 15.0

2.02 0.67

This study This study

Cellulolytic thermophilic culture

60

N.R.

Carver et al. (2011)

Treated anaerobic sludge

60

135.0

4.2 2.1 4.94

Thermophilic fermentation C. vulgaris None D. tertiolecta C. vulgaris Enzymes mixture (Cellulase and pectinase activity) a b c

b

Reference

Wieczorek et al. (2014)

Temperature; ca. Calculated assuming pressure = 1 atm. mL H2/g dried algae.

using 1% HCl hydrolysate was four times higher than at 2% HCl (Fig. 2a). The total soluble products shown the solubilization role of the fermentation stage, transforming the initial COD fed as microalgae biomass from 52% to 74%. Other works have already found CODSOL during hydrogen production from microalgae, reporting values ranging from 20 to 80% (Lakaniemi et al., 2011; Yang et al., 2010), as well as 60% protein solubilization (Lakaniemi et al., 2011). The total metabolites concentration at the end of hydrogen batch tests (2.98–14.5 g COD L1) and the partial CODSOL made these residual materials a suitable substrate for methane production. 3.3. Methane production

Fig. 3. Specific methane production of residual biomass from fermentation of hydrolyzed and untreated microalgae. Symbols indicate the methane yield in terms of COD fed.

produced 80% more hydrogen than our study (Table 2). However, the molar yield in our study, using a native consortium, doubled the best reported in the former work, showing the efficient use of reducing sugars by fermenters with the 1% HCl condition. In this sense, using a native consortium as feedstock is a more suitable application when wastewater is used to growth microalgae than pure cultures. High hydrogen production was also reported (Table 2) using an enzymatic thermophilic pretreatment (Wieczorek et al., 2014); however, the price of enzymes should be considered (Günerken et al., 2015). Despite the clear different performance in terms of hydrogen production promoted by the acid concentration, the metabolites produced during the fermentation had a similar distribution (Fig. 2). The average production of organic matter was 535 ± 49 mg CODequivalent g VS1, being the major metabolites ethanol and isovalerate, followed by isobutyrate. The fermentative stage favored the formation of soluble products in both hydrolysis conditions and the untreated biomass; in this sense, the COD conversion to ethanol and VFAs had similar proportions ranging from 30% to 41% (Fig. 2a). However, the COD directed to H2 production

The whole of the residual material from hydrogen tests was treated by anaerobic digestion for methane production. The average maximum methane production (Hmax) increased by 46% using the 1% HCl hydrolysate compared with the 2% HCl condition, both with 20 g TS L1 (Fig. 3). Following the same trend as seen in the hydrogen tests, batch tests pretreated with the same acid concentration were statistically similar. It was observed that digesting the pretreated residual biomass resulted in a significant higher methane production in comparison to the untreated biomass (Fig. 3). Examples of two-step processes for hydrogen and methane production from microalgae are scarce, and some relevant works are cited in Table 3, where the molar yield achieved in our study is shown to be greater than 35% of the molar yield obtained by others. Yields from hydrolysates at 1% HCl are also significantly higher than those from one-step methanogenic processes from microalgae, which range from 140 to 400 mL CH4 g VS1 according to Uggetti et al. (2014). However, other results using thermal pretreated (120 °C, 40 min) microalgae C. vulgaris strains reached yields up to 225 mL CH4 g COD1. Comparisons between studies are complicated because of the complex microalgae cell wall composition and its intrinsic diversity among species (Domozych et al., 2012), in addition to the variety of pretreatment methods tested. However, further studies using native or wastewater microalgae consortium are needed because this type of fuel production is a suitable economic growth strategy. Following the same trend that methane production, total COD consumption increased from 68 to 83% using hydrolysates at 2% HCl and 1% HCl, respectively. The COD balance showed that during anaerobic digestion in the second step, up to 60% of the metabolites produced using 1% HCl hydroly-

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J. Carrillo-Reyes, G. Buitrón / Bioresource Technology 221 (2016) 324–330 Table 3 Relevant results of methane production form microalgae biomass from two and single step processes. Pretreatment

Previous step

Ta (°C)

mL CH4 g VS1

mmol CH4 g VS1

None

Dark fermentation

37

394

15.5

Yang et al. (2011)

Dark fermentation Dark fermentation + photofermentation Dark fermentation

55 37

321 254

11.9 10.0

C. vulgaris

None 1% H2SO4, 140 °C, 15 min (autoclave) Enzymes mixture

35

362

14.3

Native consortium

1% HCl, 90 °C, 2 h

Dark fermentation

36

431

21.8

Lü et al. (2013) Cheng et al. (2014) Wieczorek et al. (2014) This study

Single stage process Scenedesmus sp.

Freeze-thaw cycling

–

35

410

16.2

Microalgae biomass Two stages process Lipid-extracted microalgae residues C. vulgaris Algae bloom in lake

a b c

b

C. vulgaris

120 °C, 40 min

–

35

458

18.1

Native consortium

None

–

36

245

10.9

c

Reference

Frigon et al. (2013) Mendez et al. (2014) This study

Temperature. Calculated from data provided. Calculated assuming pressure = 1 atm.

sates were converted to methane. However, using 2% HCl hydrolysates only 28% of the metabolites produced during fermentation were converted to methane (Fig. 2b). This lower methane conversion using 2% HCl hydrolysates suggests the inhibition of the methanogenic microorganisms.

using the heating value is not the most suitable method regarding hydrogen production, considering that the driving force for hydrogen production is related to its use in fuel cells for direct electricity production and its portability. 4. Conclusions

3.4. Energy conversion In the present work, hydrogen and methane production was inversely dependent on acid concentration during the hydrolysis step. Lakaniemi et al. (2013) reviewed the energy recovery from microalgae using different downstream processes, with the best recovery ranging from 14.4 to 14.8 kJ g1, which was produced by a direct methanogenic digestion, acid hydrolysis and ethanol fermentation, as well as by a two-step process (H2-CH4) from microalgal biomass residues. Using the same method, according to the lower heating values (10.78 and 35.9 MJ m3 for hydrogen and methane, respectively) the total energy recovery from algae hydrolysate under 2% HCl was, in average, 9.9 kJ g VS1 (20 and 10 g TS L1); whereas the hydrolysis at 1% of acid generated 15.9 kJ g VS1 with 20 g TS L1. Considering the energy density of the biochemical fractions in microalgae (17, 37 and 17 kJ g1 for carbohydrates, lipids and proteins; respectively) the energy content of the feedstock was 19.3 kJ g VS1; therefore, the balance of the two stages process resulted in a recovery up to 82% of total energy available. Our best condition exceed by 10% the best results for energy recovery already reported (Harun and Danquah, 2011) and using a Chlorococcum humicola culture for bioethanol production. Sarkar et al. (2015) presented the integration of thermo-chemical process with microalgae cultivation. They found that the thermal decomposition of a mixed microalgae consortia by pyrolysis produced 111 mL H2 g biomass1 and 48 mL CH4 g biomass1, equivalent to 2.9 kJ g biomass1. Those values represent only a fraction of the values obtained in the present study. That comparison highlights the relevance of the development of biological process to produce microalgae based biofuels. The main contribution for the energy recovery in our study was due to the methane yield (P97% of total energy recovery), which can be explained by the lower energy density of hydrogen. A relevant comparison regarding energy recovery is provided by Cheng et al. (2014), where despite the high hydrogen yield (257– 283 mL H2 g SV1) obtained by two-step processes (dark and photo-fermentation), the energy recovery after a sequential methane production (167–254 mL CH4 g SV1) was between 9.03–11.84 kJ g SV1. In this sense, calculating the energy recovery

Thermal-acidic microalgal hydrolysis increases the hydrogen and methane production potential of a native consortium compared to those that were not pretreated. The acid concentration was a crucial parameter. In that sense, it was observed that lower acid concentration presented higher hydrogen and methane production potential due to a high recovery of reducing sugars. Results from diluted hydrolysate showed a high energy conversion and methane yield of 15.9 kJ g SV1 and 432 CH4 g SV1, respectively, resulting in a total COD consumption of 82.7%. Acknowledgements This research was supported through the Dirección General de Asuntos del Personal Académico, Universidad Nacional Autónoma de México – Mexico (PAPIIT IN 101716) and CONACYT – Mexico (249590) projects. The authors are grateful to Jaime Perez and Gloria Moreno for the technical support and fruitful discussions. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.biortech.2016.09. 050. References Alzate, M.E., Muñoz, R., Rogalla, F., Fdz-Polanco, F., Pérez-Elvira, S.I., 2012. Biochemical methane potential of microalgae: influence of substrate to inoculum ratio, biomass concentration and pretreatment. Bioresour. Technol. 123, 488–494. http://dx.doi.org/10.1016/j.biortech.2012.06.113. APHA/AWWA/WFE, 2005. Standard Methods for the Examination of Water and Wastewater, 21th ed. Washington DC, USA. Batista, A.P., Ambrosano, L., Graça, S., Sousa, C., Marques, P.A.S.S., Ribeiro, B., Botrel, E.P., Castro Neto, P., Gouveia, L., 2015. Combining urban wastewater treatment with biohydrogen production – an integrated microalgae-based approach. Bioresour. Technol. 184, 230–235. http://dx.doi.org/10.1016/j. biortech.2014.10.064. Advances in biofuels and chemicals from algae. Batista, A.P., Moura, P., Marques, P.A.S.S., Ortigueira, J., Alves, L., Gouveia, L., 2014. Scenedesmus obliquus as feedstock for biohydrogen production by Enterobacter aerogenes and Clostridium butyricum. Fuel 117 (Part A), 537–543. http://dx.doi. org/10.1016/j.fuel.2013.09.077.

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