Bioresource Technology 157 (2014) 60–67

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Methane production from marine microalgae Isochrysis galbana Nathalia O. Santos a, Suzana M. Oliveira a, Larissa C. Alves b, Magali C. Cammarota a,⇑ a Department of Biochemical Engineering, School of Chemistry, Federal University of Rio de Janeiro, Cidade Universitária, Centro de Tecnologia, Bl. E, Sl. 203, Ilha do Fundão, 21941-909 Rio de Janeiro, Brazil b Petroleum Engineering, Estácio de Sá University, Av. Presidente Vargas, 2560, Centro, 20210-030 Rio de Janeiro, Brazil

h i g h l i g h t s

g r a p h i c a l a b s t r a c t

 Methane production from marine

microalgae Isochrysis galbana was assessed.  Mechanical pretreatment resulted in a 61.7% increase in soluble COD.  Best hydrolysis condition was 40 °C/ 0.2% (v/v) acid/16 h (9.27 L CH4/ kg VS).  Low methane yields were attributed to inhibitory sodium concentrations.  Biomass prewashing eliminated inhibitory sodium and increased methane yield in 71.5%.

a r t i c l e

i n f o

Article history: Received 8 December 2013 Received in revised form 17 January 2014 Accepted 21 January 2014 Available online 30 January 2014 Keywords: Anaerobic digestion Biogas Isochrysis galbana Marine microalgae Thermal pre-treatment

a b s t r a c t Methane production from marine microalgae Isochrysis galbana was assessed before and after mechanical and chemical pretreatments. Mechanical pretreatment resulted in a 61.7% increase in soluble Chemical Oxygen Demand. Different hydrolysis conditions were evaluated by varying temperature – T, sulfuric acid concentration – AC and biomass suspension concentration (measured as particulate COD – CODp) using an experimental design. The most significant interaction occurred between AC and T and the hydrolysis condition that showed the best result in the anaerobic digestion step was the condition at 40 °C with addition of 0.2% (v/v) acid for 16 h (9.27 L CH4/kg VS). The low methane yields were attributed to inhibitory sodium concentrations for anaerobic digestion. Eliminating inhibitory sodium in the anaerobic digestion by biomass prewashing, there was a 71.5% increase in methane yield for biomass after acid hydrolysis, demonstrating the need for pretreatment and reduction in sodium concentration in the anaerobic digestion. Ó 2014 Elsevier Ltd. All rights reserved.

1. Introduction Recently, several studies have investigated the potential of microalgae for CO2 capture and storage and generation of biofuels. Many microalgae species such as Pyrrosia laevis, Dunaliella sp, Chlorella vulgaris, Neochloris oleoabundans and Isochrysis galbana accumulate lipids (Deng et al., 2009), which can be extracted and used for biodiesel production, allowing the generation of third⇑ Corresponding author. Tel.: +55 21 2562 7568; fax: +55 21 2562 7567. E-mail addresses: [email protected] (L.C. Alves), [email protected] (M.C. Cammarota). http://dx.doi.org/10.1016/j.biortech.2014.01.091 0960-8524/Ó 2014 Elsevier Ltd. All rights reserved.

generation biofuel with reduced environmental impact (Scott et al., 2010; Sánchez et al., 2013). However, cultivating microalgae for CO2 capture and biodiesel production generates residual algae biomass that must be managed to prevent that its improper disposal into the environment contaminates soil and surface water and accelerates the eutrophication process (Sialve et al., 2009). In this context, anaerobic treatment emerges as a promising technology for the management of this waste, seeking adequate treatment and final disposal and at the same time to obtain energy in the form of methane, which can contribute to the sustainability of biodiesel production from

N.O. Santos et al. / Bioresource Technology 157 (2014) 60–67

microalgae (Ehimen et al., 2009; Yang et al., 2011; Schwede et al., 2013). The anaerobic digestion of microalgae can eliminate the steps of biomass collection and drying, reducing costs (Vonshak, 1997; Costa and Morais, 2011). The treatment/utilization of this biomass through anaerobic digestion is an alternative that deserves attention because it promotes the formation of biogas containing methane, a product of great commercial interest as fuel. However, the combined application of biomass production with anaerobic digestion of residual biomass has few studies in literature. The management of this residual biomass linked to energy production, to control biological processes driven by microorganisms present in the medium, and the use of nutrients such as nitrogen and phosphorus, are important factors for the application of this methodology on an industrial scale (Sialve et al., 2009). However, some studies have shown that during the anaerobic digestion process, the intracellular material remains intact, i.e., there is no disruption of the cell membrane (Sialve et al., 2009; Debowski et al., 2013). In order to release the material retained within the cell, some studies have been aimed at the disruption of the cell membrane (Yen and Brune, 2007). In addition, some microalgae have a cellulose coating or mucilage, which requires a pretreatment step in order to improve the access of microorganisms to the organic matter (Habig, 1985). Chen and Oswald (1998) reported that only 40% of the microalgae constituents are readily available for methane production, the remaining 60% require pretreatment steps to make the intracellular content available. Some pretreatment steps are well known to increase methane production from activated sludge and other waste, among which mechanical and chemical (acid, alkalis, ozone), thermal, ultrasonic and enzymatic treatments stand out (Bonmati et al., 2001; Bougrier et al., 2006; Schwede et al., 2013). Mechanical and chemical pretreatments have been applied in algal biomass in order to increase biodegradability (De Schamphelaire and Verstraete, 2009). Physical methods such as applying homogenizers or glass beads are more favorable for cell disruption due to economic limitations of other methods (Saboya et al., 2003). According to Harun et al. (2011), acid and alkaline pretreatments are those that require lower operating cost when compared to enzymatic treatments. Acid treatments have been widely studied and predominantly used in raw materials with high carbohydrate content, while alkaline treatments are best applied to raw materials with high concentration of proteins, and have been recently applied to microalgae. Most current studies on methane production from microalgae biomass are conducted on bench scale and batch regime. Such studies do not allow concluding on the technical and economic feasibility of the process as well as the necessity of a pretreatment step before anaerobic digestion of the biomass. Thus, studies on the anaerobic degradation of microalgae biomass should be carried out, particularly due to the different results obtained with different species and operating conditions, both in the cultivation of microalgae as in the anaerobic digestion step. The use of marine microalgae in studies of CO2 capture and biomass production for biofuel production is aimed at the use of seawater as culture medium at a greatly reduced cost. Although biomass is collected by centrifugation for biomass concentration and reuse of the culture medium, it is possible that a residual salt concentration remains in the biomass, contributing to low biodegradability and methane production (McCarty and McKinney, 1961; Chernicharo et al., 2007; Schwede et al., 2013). Thus, this study assessed the effectiveness of thermal and chemical (acid) pretreatment methods for cellular content availability and the effect of salinity of marine microalgae I. galbana biomass on methane production.

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2. Methods 2.1. Collection and characterization of algal biomass and anaerobic sludge In this study, experiments were conducted using microalgae I. galbana biomass (Laboratory for Applied Research in Photosynthesis/Institute of Chemistry/Federal University of Rio de Janeiro – UFRJ). The maintenance and cultivation of microalgae were performed at the Laboratory of Hydrorefining, Process Engineering and Applied Thermodynamics/School of Chemistry/UFRJ. Cultivations were carried out in modified f/2 medium (Guillard, 1975), in batch-fed system and with nitrogen and phosphorus adjustments as required (Picardo et al., 2013). Upon reaching the stationary phase, the culture was stopped and the algal biomass was concentrated in refrigerated centrifuge (CIENTEC, model CT6000R) at 4000 rpm/20 °C for 10 min. The cell pellet was resuspended in distilled water and characterized in terms of pH, solids, COD, BOD5, Carbon, Nitrogen, Phosphorus, Oil and Grease, carbohydrates and proteins. The remainder was kept at 4 °C until use. The sludge used as inoculum was collected from an upflow anaerobic sludge blanket (UASB) – type anaerobic reactor operating in a poultry processing industry, characterized in terms of total volatile solids (11,000 mg/L). 2.2. Mechanical pretreatment About 5 mL of algal biomass suspension were placed in glass tubes with screw cap (8.4  1.5 cm) with glass beads (mean diameter of 3 mm) and submitted to vortexing. Assays were performed by varying the mass of glass beads (1 and 5 g) and the stirring time (1–5 min). After shaking, the glass beads were removed, the suspension was filtered through membrane with pore size of 0.45 lm and the soluble Chemical Oxygen Demand (CODs) was determined in the filtrate. 2.3. Thermal and acid hydrolysis tests of algal biomass Different hydrolysis conditions, based on results of Yawson et al. (2011) and Ho et al. (2013), were evaluated by varying temperature (40, 50, 60 °C), sulfuric acid concentration (0, 0.1, and 0.2% v/v) and biomass suspension concentration (measured as particulate COD CODp, 1500, 3000 and 4500 mg/L). Glass tubes (10 mL) with screw cap (8.4  1.5 cm) were filled with 5 mL of algae biomass suspension after mechanical pretreatment and certain volumes of sulfuric acid pa, then immersed in a thermostated bath (Memmert) at the desired temperature for 16 h, which was the time that yielded the best results in milder hydrolysis conditions (results not shown). To better evaluate the interaction between variables involved, a 23 complete factorial design (with 3 central points, totaling 11 experiments) was performed with 3 variables (temperature, sulfuric acid concentration and biomass suspension concentration) and 2 levels ( 1 and +1). The coded and current values of variables are shown in Table 2. The results of the analysis of variance, using the increase in CODs as response variable, were analyzed with the Statistica 7 software (StatSoft) for a significance level of 95% (p level < 0.05). 2.4. Anaerobic biodegradability tests of algal biomass The tests were conducted under two control conditions (biomass with no hydrolysis, CODp 4500 mg/L): Condition 1 – biomass with no mechanical pretreatment, and Condition 2 – biomass after mechanical pretreatment. In addition to these conditions, three

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conditions were selected, based on the analysis of results of the experimental design: Condition 3 (CODp 4500 mg/L, 40 °C, 0.2% acid, 16 h); Condition 4 (CODp 4500 mg/L, 60 °C, 16 h) and Condition 5 (CODp 4500 mg/L, 40 °C, 16 h). After hydrolysis assays, the biomass suspension pH was adjusted to 7.0 ± 0.2 with NaHCO3 and was inoculated with sludge in penicillin flasks of 100 mL, with usable volume of 90 mL. The inoculum was introduced into flasks so as to maintain a Total Volatile Solids: biomass COD ratio of 1:1. The biomass and sludge mixture was supplemented with solutions of macro and micronutrients (Chernicharo et al., 2007) for a COD:N:P ratio of 350:5:1. After introduction of inoculum, the flasks were sealed with rubber stoppers and aluminum seals and incubated at 30 ± 2 °C. Biogas production over time was measured by the displacement of the plunger of one or more 20 mL plastic syringes attached to the rubber seal, and the biogas volume was reported at 30 °C and 1 atm. After biogas production stabilization, the gas present in the syringes was transferred to gas vials and submitted to gas chromatography to quantify the methane. In the supernatant, pH and CODs were analyzed. In tests with unwashed biomass, the algal suspension from the culture was centrifuged, the supernatant (culture medium) was discarded and the biomass pellet was resuspended in distilled water. Before or after hydrolysis, the suspension pH was adjusted with sodium bicarbonate. In tests with prewashed biomass, the algae suspension from the culture was centrifuged and the supernatant was discarded 3 times. In the fourth washing, the biomass resuspended in distilled water was used in tests, and after hydrolysis, its pH was adjusted with calcium hydroxide to minimize the sodium concentration in the sample.

2.5. Analytical methods The characterization of suspended biomass and sludge, as well as the parameters used in the monitoring of tests were performed with standard methods (Greenberg et al., 2005). Carbohydrates and proteins were determined using the methods of Dubois et al. (1956) and Lowry et al. (1951). Biogas composition was measured in gas chromatograph Micro CG VARIAN (CP-4900), using a 10 m  0.32 mm PPQ column, column temperature of 50 °C, thermal conductivity detector (TCD) at 250 °C, injector temperature of 80 °C and helium as carrier gas.

3. Results and discussion 3.1. Characterization of suspended microalgae biomass Table 1 shows the characterization of suspended microalgae biomass. The suspension had pH near neutrality, which is favorable for anaerobic digestion without adjustments. The low proportion of total volatile solids in relation to total solids (about 25%) indicates the presence of salts used in the culture medium, even after centrifugation and discard of the culture medium. Part of these salts is in the form of carbonates and bicarbonates, as shown by the inorganic carbon values. The much lower total COD values, compared to total solids, also show residual salinity. This salinity can inhibit the activity of anaerobic microorganisms, requiring gradual adaptation (Chen et al., 2008). The biomass suspension showed low soluble COD (only 17.4% of total COD) and low biodegradability, as shown in the COD/BOD5 ratio greater than 5, indicating the need for a physicochemical treatment step prior to biological treatment. The CODtotal:N:P ratio (1370:79:1) obtained shows, based on the optimal ratio of 350:5:1 (Chernicharo et al., 2007), sufficient amounts of nitrogen, but low

Table 1 Characterization of microalgae I. galbana suspended biomass.

*

Parameter*

Value

pH Total solids Total volatile solids Total COD Soluble COD BOD5 COD/BOD5 Total carbon Inorganic carbon Total organic carbon Total nitrogen soluble phosphorus Oils and greases Carbohydrates (Glucose eq.) Proteins

7.4 45,133 11,112 3564 620 686 5.2 1037 147 890 205 2.6 2533 718 1700

All parameters in mg/L, except pH e BOD5/COD.

Table 2 Coded and current levels (in parentheses) of independent variables and results of the experimental design to assess the different conditions of acid and thermal pretreatment of algal biomass. Test 1 2 3 4 5 6 7 8 9 10 11

CODp (mg/L) 1 1 1 1 1 1 1 1 0 0 0

(1500) (4500) (1500) (4500) (1500) (4500) (1500) (4500) (3000) (3000) (3000)

Temperature (°C) 1 1 1 1 1 1 1 1 0 0 0

(40) (40) (60) (60) (40) (40) (60) (60) (50) (50) (50)

Acid (% v/v)

D CODs (16 h)

Solubilization of COD (%)

1 (0) 1 (0) 1 (0) 1 (0) 1 (0.2) 1 (0.2) 1 (0.2) 1 (0.2) 0 (0.1) 0 (0.1) 0 (0.1)

252 434 202 418 95 351 50 389 116 100 115

11.8 6.6 10.3 12.0 12.4 8.4 12.2 8.2 1.8 1.5 2.0

concentrations of soluble phosphorus, which is supplemented to achieve satisfactory levels in anaerobic biodegradability tests. There was a high concentration of oil and grease (O&G), which may impair anaerobic digestion (Chen et al., 2008). This high value is due to the fact that biomass was cultivated under conditions that favor the accumulation of lipids and it was used fresh, before the lipid extraction step for biodiesel production. According to Mata et al. (2010), the lipid concentration in I. galbana would be 7–40% (dry weight). The data obtained indicate percentage of 22.8% (considering that VS quantify suspended biomass). The percentage values of carbohydrates (6.5%) and protein (15.3%) are much lower than those reported in literature for I. galbana biomass. Tokusoglu and Unal (2003), for example, reported 16.98% of carbohydrates and 26.99% of proteins. Schwede et al. (2013) determined the organic composition of Nannochloropsis salina biomass and verified that the organic fraction consisted mainly of lipids (36.1% of the total solids) and nitrogen-free extractive (34.7%). The high protein content led to low C/N ratio (12.2). In the present study, C/N ratio of 5.1 was obtained for I. galbana suspension. Low C/N ratios due to high protein contents can result in inhibition of anaerobic digestion by ammonia nitrogen (Sanchez and Travieso, 1993; Yen and Brune, 2007). The knowledge on the cellular composition in terms of proteins, carbohydrates and lipids is important for anaerobic digestion, considering that these constituents have different potentials for methane production. Mata et al. (2010) reported that proteins,

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carbohydrates and lipids have increasing potential for methane production in this order. However, Habig (1985) evaluated several conditions in which combined intracellular proteins and carbohydrates did not play a significant role in biogas production; however, when evaluating each cell constituent separately, they observed that carbohydrates and proteins are the components that provide the greatest positive contribution to biogas production. They also found that soluble carbohydrates are the most important components in determining biogas yield and experiments conducted with algae after protein extraction showed lower digestion time and increased biogas production when compared to the digestion process with untreated biomass. 3.2. Mechanical pretreatment Stirring of algal biomass with 1 g of glass beads for 1 min resulted in an increase of COD compared to initial COD of 61.7%. Keeping the amount of glass beads in 1 g and increasing the stirring time to 5 min resulted in a 63% increase in COD. The increased mass of glass beads and stirring time to 5 min and 5 g led to an increase in COD of only 24.7%, probably due to the difficult stirring with larger mass of beads in the tube. Thus, considering the small difference between the 2 times with 1 g of glass beads (or 20% w/v), time of 1 min was adopted in the mechanical pretreatment of suspended biomass used in subsequent experiments. 3.3. Thermal and acid hydrolysis tests of algal biomass At this step, 11 trials were done to a 23 complete factorial design. The results showed a maximum COD solubilization of only 12.4% and in many trials, small solubilization was observed. Table 2 shows the results of hydrolysis tests conducted for time of 16 h. The software used generated a linear regression curve with 0.995 correlation coefficient for 95% confidence interval. To check which effects were significant or not on COD solubilization, the Student’s t test was used. The tabulated Student’s t value is

obtained through the number of degrees of freedom for the trials provided by the software (degree of freedom in the 23 complete factorial design was 6). Assuming a confidence interval of 95% and 6 degrees of freedom, t = 2.447 was obtained from tables in statistical books. Based on this value, the effects were analyzed for their significance on COD solubilization. For an effect or interaction to be considered important, its value should be greater than the tabulated value (2.447). These analyses are best visualized by using Pareto’s graph, where the black vertical line (p = 0.05, the tabulated Student’s t value) indicates the minimum magnitude of the statistically significant effects for 95% confidence level for the system under analysis. The effects of factors and interactions surpassing that line indicate that they are important to the analysis in question. Fig. 1 on the manuscript shows the effect analysis of the 11 trials using Pareto’s graph. The values shown in the horizontal columns of Pareto’s graph correspond to calculated Student’s t test of each factor or interaction (t = coefficient of each factor/standard error provided by the software). The effects of these factors can be classified as main or interaction effects. The main positive effects indicate that the factors should be used at their highest level so as to provide the best response to the system; the negative effects indicate that they should be used at their lowest level. Interactions can also be positive or negative. Thus, Pareto’s graph shows that the main effect for COD solubilization that was found in the trials was ‘‘CODp concentration’’ (positive) followed by ‘‘acid percentage’’ (negative) and CODp concentration  acid percentage (positive) and CODp concentration  temperature (positive) interactions. The effect of temperature, analyzed separately, was not significant to a 95% confidence level in the Student’s t distribution. This suggests that, the temperature range evaluated (40–60 °C) was not enough for COD solubilization to take place in an efficient fashion. Fig. 2 shows the surface charts of interactions between variables CODp concentration, acid percentage and temperature. For the interaction between CODp and acid percentage, it is possible to observe that better results were obtained from intermediate to high CODp concentration values associated with low acid percentages. High response values are obtained when high CODp

Pareto Chart of Standardized Effects; Variable: CDOs (16h) 2**(3-0) design; MS Pure Error=80,33333 DV: CDOs (16h)

(1)CODp (mg/L)

39.17025

(3)Acid (%)

-16.6069

1by3

1by2

(2)Temperature (ºC)

7.770936

4.615226

-2.87959

p=,05 Standardized Effect Estimate (Absolute Value) Fig. 1. Pareto’s graph of the experimental design to evaluate different acid and thermal hydrolysis conditions of algal biomass. Numbers 1, 2 and 3 represent factors and interactions (1 = CODp concentration – mg/L; 2 = temperature – °C; 3 = acid percentage). The values appearing the horizontal columns of the graph correspond to the Student’s t value generated for each factor and the black vertical line indicates the tabulated Student’s t test value (2.447). Factors and interactions surpassing the vertical line indicate that they are important to the analysis in question.

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A

B

5 00 400

500 400 300

CODs

CODs

300 20 0 1 00

0,0 4 -0 , , 8 -0

8 0, 4 0, 0 0, 4 , -0

400 400 300 200 100

,8 -0 2 , -1

> < < <
400 < 340 < 240 < 140 < 40

C 5 00 400

CODs

300 200 100

0,

4 0,

8

d

) (%

e tur er a ) p em ( º C

0 0, 4 , -0 ,8 -0 2 , -1

i Ac

0 ,4 0, 0 4 - 0,

2 1,

1 ,2 8 0,

8 - 0 , ,2 -1

T

Fig. 2. Surface charts for CODp  acid, temperature  CODp and temperature  acid interactions in the experimental design with 16 h of hydrolysis. For charts A, B and C, the third variable has been set at 1, namely, 60 °C, 0.2% v/v acid and 4500 mg/L CODp, respectively.

concentrations are combined with higher or lower temperatures, confirming the importance of CODp and demonstrating that in this association, temperature does not significantly interfere with the result. It was observed through the slope of the chart that there is an interaction between these factors. For all factors considered, it was possible to observe a characteristic pattern for COD solubilization, in which in all cases, the maximum COD variation value was close. Comparatively analyzing the three graphs of Fig. 2, it was observed that the greatest response was obtained in the association between CODp and temperature, CODp and acid and followed by acid and temperature, a fact that shows that CODp is even more important. This result is very satisfactory, since CODp represents a factor of low operational costs compared to other factors. Although the analysis of graphs does not allow a more detailed definition of the relevance of interactions, the analysis of coefficients indicates a greater contribution of CODp and acid interaction in relation to CODp and temperature. Again, regardless of interaction, the importance of CODp in the process was observed. 3.4. Anaerobic biodegradability tests of algal biomass Fig. 3 and Table 3 show the biogas production (30 °C, 1 atm) over time and biogas composition and production data, respectively, for each hydrolysis conditions based on the experimental results along with controls with untreated biomass, with and without mechanical pretreatment. The mechanical treatment resulted in low biogas production, indicating that membrane disruption alone is not sufficient to promote anaerobic digestion. It was found that Condition 3 (40 °C/0.2% v/v acid/16 h) showed biogas volume (26.0 mL) slightly higher than control without mechanical pretreatment (22.0 mL). The initial biogas production rate was higher

Fig. 3. Biogas volume (30 °C, 1 atm) produced over time in the anaerobic biodegradation test with algal biomass (CODp 4500 mg/L) under conditions Control 1 (no mechanical pretreatment), Control 2 (with mechanical pretreatment) and with hydrolysed biomass under conditions 3 (40 °C/0.2% v/v acid/16 h), 4 (60 °C/ 16 h) and 5 (40 °C/16 h).

in experiment with hydrolyzed biomass (11 mL/d) than in Controls 1 (2.5 mL/d) and 2 (2.0 mL/d), indicating that hydrolysis released a higher amount of substrate for assimilation by microorganisms. In fact, a higher CODs value (436 mg/L) was observed in Condition 3, compared to Condition 1 (343 mg/L) and 2 (363 mg/L). After digestion of the solubilized material, the biogas production stabilized,

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Table 3 Results of anaerobic biodegradability tests: volume and biogas composition on conditions Control 1 (without mechanical pretreatment), Control 2 (with mechanical pretreatment) and with biomass hydrolyzed under conditions 3 (40 °C/0.2% v/v acid/16 h), 4 (60 °C/16 h) and 5 (40 °C/16 h). All conditions with CODp of 4500 mg/L. Test

Initial pH

Initial CODs (mg/L)

Final pH

Final CODs (mg/L)

COD Removal (%)

Biogas volume at 30 °C (mL)

% CH4

1 2 3 4 5

6.9 ± 0.1 6.9 ± 0.1 6.8 ± 0.1 6.9 ± 0.1 7.0 ± 0.1

343 ± 10 363 ± 23 436 ± 14 249 ± 14 236 ± 10

6.6 ± 0.1 6.6 ± 0.1 7.2 ± 0.1 6.6 ± 0.1 7.0 ± 0.1

98 ± 1 74 ± 3 227 ± 14 22 ± 3 44 ± 6

71.4 ± 0.6 79.6 ± 2.0 47.8 ± 4.5 91.3 ± 1.2 81.3 ± 2.7

22.0 ± 1.0 12.7 ± 0.6 26.0 ± 1.0 3.7 ± 0.6 3.0 ± 0.0

79.4 ± 0.1 79.0 ± 0.1 63.1 ± 0.7 77.6 ± 2.6 76.3 ± 0.2

Note: All values are averages and standard deviations for triplicates.

probably due to substrate limitation. Thermal hydrolysis conditions without addition of acid (Conditions 4 and 5) showed the lowest biogas and methane production values, probably due to little changes in the cell structure under these conditions (low temperature and without addition of acid). Other authors have reported improved results in the anaerobic digestion after pretreatment under higher temperatures. Chen and Oswald (1998) studied different pretreatments for an algal biomass produced in sewage treatment ponds. The temperature appeared to have the most important effect, and optimal pretreatment consisted of heating at 100 °C during 8 h resulting in a 33% methane production improvement. Samson and LeDuy (1983) obtained the best performance for Spirulina maxima with thermal pretreatment at 150 °C and a pH = 11. Based on the methane percentage obtained in the biogas (Table 3) and in the suspended biomass (CODp of 4500 mg/L), the specific methane production was evaluated, obtaining values of 43.1, 24.8, 40.5, 7.1 and 5.7 mL CH4/g CODp (30 °C) for conditions 1–5, respectively. These values confirm that Conditions 1 and 3 showed better methane production compared to the other conditions. To investigate the relationship between hydrolysis products and anaerobic digestion results, analyses of carbohydrates and proteins were carried out in the soluble fraction of algal biomass before (Control) and after hydrolysis. Habig (1985) reports that increased levels of carbohydrates in the soluble phase positively influence biogas production. Briand and Morand (1997) reported that high concentrations of carbohydrates are favorable to methane production because their fractionation forms methanogenesis precursors. The hydrolysis condition that showed the highest biogas production (Condition 3) also showed higher concentration of available carbohydrate in the soluble phase (180 mg/L) versus 70–85 mg/L in the Control conditions and 32–36 mg/L in the other two conditions. Protein concentrations in hydrolysates were much lower, ranging from 24 to 92 mg/L. As the culture medium of the microalgae contains 35 g NaCl/L, it is possible that a residual salt concentration remains in the biomass after centrifugation. Therefore, the low specific methane production values can also be associated with residual salinity in the biomass. Chen et al. (2008) reported that moderate amounts of sodium (100–350 mg/L) can stimulate the growth of microorganisms; however, excessive amounts (3500–8000 mg/L) can inhibit their growth. Seeking a higher methane production, the effect of Na+ on the anaerobic digestion of algal biomass was evaluated. For this experiment, the best hydrolysis condition found (Condition 3–40 °C/0.2% v/v acid/16 h) was compared, using biomass with and without intensive prewash step using distilled water, as shown in Fig. 4. The condition without prewash was used as a control, with pH being adjusted with the aid of sodium bicarbonate, while the condition with prewash had its pH adjusted with calcium hydroxide in order to minimize the sodium concentration in the medium. Contrary to expectations, similar biogas production profiles were

Fig. 4. Biogas production over time with suspended biomass with and without prewashing step to reduce salinity.

Table 4 Sodium concentration and its effect on methane and carbon dioxide production with biomass washed and not washed subjected to hydrolysis condition 40 °C/0.2% v/v acid/16 h. Pre-washed

Na+ (mg/L) Residual Added

Washed 64.8 Not washed 1101.4 *

Total

Biogas volume % CH4* at 30 °C (mL)*

– 64.8 15.4 ± 0.7 2464.3 3565.7 17.7 ± 0.8

% CO2*

86.5 ± 0.1 12.0 ± 0.1 29.8 ± 0.1 69.0 ± 0.1

Average and standard deviations for triplicates.

found for both conditions and the final biogas volume was smaller than that obtained in the previous test (Fig. 3). This variability may be due to different batches of samples used in the experiments, since cultivation was performed in batch-fed system. Habig (1985) reported that such fluctuations in biogas production are related to different batches of the same substrate, which occurred in this study. In order to confirm the effect of sodium concentration on the result previously obtained, sodium concentration was estimated in the washed and unwashed biomass suspension, and the result is shown in Table 4. In the test with unwashed biomass, besides residual sodium, sodium in the form of sodium bicarbonate was also added for pH adjustment. Finally, sodium concentration of 3565.7 mg/L was obtained for unwashed biomass and only 64.8 mg/L (55 times lower) for washed biomass. Although biogas production results have been similar for unwashed and washed samples, the methane volume produced was much higher (86.5%, 13 mL) with washed biomass than with unwashed biomass (30%, 5 mL), suggesting that there is no total inhibition of the anaerobic digestion, but an impairment in the methanogenic step. The sodium concentration results indicate the occurrence of moderate inhibition. Chernicharo et al. (2007)

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reported that sodium concentrations from 3500 to 5500 mg/L are moderately inhibitory in the anaerobic digestion process. McCarty and McKinney (1961) showed that chlorides of monovalent ions such as sodium cause higher toxicity to the sludge than chlorides of bivalent ions such as calcium. They also reported that NaCl concentration of 2370 mg/L exerts inhibitory effect on anaerobic digestion. In the present study, the residual NaCl concentration was 2801.4 mg/L, corroborating data from literature. Schwede et al. (2013) also found that the biodegradability of N. saline microalgae suspension was significantly improved using thermally pretreated algal biomass. Besides the rate limiting hydrolysis of complex protein and lipid structures accumulation of sodium and ammonium influenced the biodegradability of the feedstock in semi-continuous digestion. The results obtained in this work lead to a series of reflections on methane production from microalgae I. galbana biomass, namely: (1) the anaerobic digestion of fresh biomass is too slow (biogas production stabilizes after 10 days) and shows very low methane yield: 9.87 L CH4/kg VS (Control 1 – Table 3). In order to increase the use of organic matter contained in the biomass and consequently the methane yield, mechanical, chemical, thermal or enzymatic pretreatment methods should be used, which provide larger amounts of organic matter to be assimilated by microorganisms in the anaerobic digestion step; (2) among these pretreatment methods, chemical treatment would be one of the treatments with the best cost-benefit ratio. However, the use of acid hydrolysis as a pretreatment requires the addition of acid and then alkali to adjust the pH to levels appropriate for microbial activity. This pH adjustment not only increases the consumption of chemicals but also increases sodium concentration in the reaction medium, whereas traditionally, it is done with sodium bicarbonate, which can lead to inhibition of the activity of anaerobic microorganisms; (3) the use of marine species also leads to high sodium concentrations in the biomass being digested. To overcome this problem, an alternative is washing the biomass to reduce the sodium concentration, leading to the consumption of clean water; (4) after washing the biomass, methane production reaches higher levels. In the present study, after acid hydrolysis, biomass presents yield of 15.9 L CH4/ kg VS (prewash biomass – Table 4) as compared to 6.14 L CH4/ kg VS obtained with unwashed biomass (Table 4), reinforcing the idea of inhibition by the addition of sodium to the culture medium and pH adjustment after hydrolysis. Thus, under Condition 3, there is a 71.5% increase in methane production compared to 9.27 L CH4/ kg VS for unwashed biomass (Condition 3, Table 3), confirming the importance of reducing sodium concentration in the anaerobic digestion step. Future studies should investigate the inhibitory sodium concentrations and their effect on successive biomass contacts with the same sludge and determine new techniques for adjusting pH after acid hydrolysis. Other digestion methods such as batch-fed can be tested in order to increase the efficiency of the process.

4. Conclusion Mechanical pretreatment increased COD solubilization, allowing the reduction of temperature and the percentage of acid applied in the hydrolysis step. All the variables studied in the experimental design exerted influence on the acid hydrolysis of microalgae biomass; however, the most significant interaction occurred between acid concentration and temperature. The hydrolysis condition that showed the best result in the anaerobic digestion was: CODp of 4500 mg/L, 40 °C, 0.2% (v/v) acid, and 16 h. The unwashed biomass suspension produced 6.14 L CH4/kg VS, while the washed suspension produced 15.9 L CH4/kg VS, confirming the efficiency of the washing method.

Acknowledgements This work was supported by project funds from the Brazilian National Council for Research and Development (CNPq), Carlos Chagas Filho Foundation for Research Support in the State of Rio de Janeiro (FAPERJ), and Petrobras. The authors thank the Laboratory of Hydrorefining, Process Engineering and Applied Thermodynamics, School of Chemistry, UFRJ, for providing algal biomass.

References Bonmati, A., Flotats, X., Mateu, I., Campos, E., 2001. Study of thermal hydrolysis as a pretreatment to mesophilic anaerobic digestion of pig slurry. Water Sci. Technol. 44, 109–116. Bougrier, C., Albasi, C., Delgenès, J.P., Carrère, H., 2006. Effect of ultrasonic thermal and ozone pretreatments on waste activated sludge solubilisation and anaerobic biodegradability. Chem. Eng. Proc. 45, 711–718. Briand, X., Morand, P., 1997. Anaerobic digestion of Ulva sp. 1. Relationship between Ulva composition and methanisation. J. Appl. Phycol. 9, 511–524. Chen, P.H., Oswald, W.J., 1998. Thermochemical treatment for algal fermentation. Environ. Int. 24, 889–897. Chen, Y., Cheng, J.J., Creamer, K.S., 2008. Inhibition of anaerobic digestion process: a review. Bioresour. Technol. 99, 4044–4064. Chernicharo, C.A.L., 2007. Biological Wastewater Treatment, Vol. 4, Anaerobic Reactors, first ed. IWA Publishing, London. Costa, J.A.V., Morais, M.G., 2011. The role of biochemical engineering in the production of biofuels from microalgae. Bioresour. Technol. 102, 2–9. De Schamphelaire, L., Verstraete, W., 2009. Revival of the biological sunlight-to biogas energy conversion system. Biotechnol. Bioeng. 103, 296–304. Debowski, M., Zielinski, M., Grala, A., Dudek, M., 2013. Algae biomass as an alternative substrate in biogas production technologies-review. Renew. Sust. Energy Rev. 27, 596–604. Deng, X., Li, Y., Fei, X., 2009. Microalgae: a promising feedstock for biodiesel. Afr. J. Microbiol. Res. 3, 1008–1014. Dubois, M., Gilles, K.A., Hamilton, J.K., Rebers, P.A., Smith, F., 1956. Colorimetric method for determination of sugars and related compounds. Anal. Chem. 28, 350–356. Ehimen, E.A., Connaughton, S., Sun, Z., Carrington, C., 2009. Energy recovery from lipid extracted, transesterified and glycerol co-digested microalgae biomass. GCB Bioenergy 1, 371–881. Greenberg, A.E., Clesceri, L.S., Eaton, A.D., 2005. Standard Methods for the Examination of Water and Wastewater, 21st ed. American Public Health Association, American Water Works Association, Water Pollution Control Federation, Washington, DC. Guillard, R.R.L., 1975. Culture of phytoplankton for feeding marine invertebrates. In: Smith, W.L., Chanley, M.H. (Eds.), Culture of Marine Invertebrate Animals. Plenum, New York, pp. 29–60. Habig, C., 1985. Influence of substrate composition on biogas yields of methanogenic digesters. Biomass 8, 245–253. Harun, R., Jason, W.S.Y., Cherrington, T., Danquah, M.K., 2011. Exploring pretreatment of microalgal biomass for bioethanol production. Appl. Energy 88, 3464–3467. Ho, S.H., Huang, S.W., Chen, C.Y., Hasunuma, T., Kondo, A., Chang, J.S., 2013. Bioethanol production using carbohydrate-rich microalgae biomass as feedstock. Bioresour. Technol. 135, 191–198. Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J., 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265–276. Mata, T.M., Martins, A.A., Caetano, N.S., 2010. Microalgae for biodiesel production and other applications: a review. Renew. Sust. Energy Rev. 14, 217–232. McCarty, P.L., McKinney, R.E., 1961. Salt Toxicity in anaerobic digestion. J. Water Pollut. Control Fed. 33, 399–415. Picardo, M.C., Medeiros, J.L., Araujo, O.Q.F., Chaloub, R.M., 2013. Effects of CO2 enrichment and nutrients supply intermittency on batch cultures of Isochrysis galbana. Bioresour. Technol. 143, 242–250. Saboya, L.V., Maillard, M.B., Lortal, S., 2003. Efficient mechanical disruption of Lactobacillus heiveticus, Lactococcus lactis and Propionibacterium freudenreichii by a new high-pressure homogenizer and recovery of intracellular aminotransferase activity. J. Ind. Microbiol. Biotechnol. 30, 1–5. Samson, R., LeDuy, A., 1983. Influence of mechanical and thermochemical pretreatments on anaerobic digestion of Spirulina maxima algal biomass. Biotechnol. Lett. 5, 671–676. Sanchez, E.P., Travieso, L., 1993. Anaerobic digestion of Chlorella vulgaris for energy production. Resour. Conserv. Recy. 9, 127–132. Sánchez, A., Maceiras, R., Cancela, A., Pérez, A., 2013. Culture aspects of Isochrysis galbana for biodiesel production. Appl. Energy 101, 192–197. Schwede, S., Rehman, Z.-U., Gerber, M., Theiss, C., Span, R., 2013. Effects of thermal pretreatment on anaerobic digestion of Nannochloropsis salina biomass. Bioresour. Technol. 143, 505–511. Scott, S.A., Davey, M.P., Dennis, J.S., Horst, I., Howe, C.J., Lea-Smith, D.J., Smith, A.G., 2010. Biodiesel from algae: challenges and prospects. Biotechnology 21, 277– 286.

N.O. Santos et al. / Bioresource Technology 157 (2014) 60–67 Sialve, B., Bernet, N., Bernard, O., 2009. Anaerobic digestion of microalgae as a necessary step to make microalgal biodiesel sustainable. Biotechnol. Adv. 27, 409–416. Tokusoglu, Ö., Ünal, M.K., 2003. Biomass nutrient profile of three microalgae: Spirulina platensis, Chlorella vulgaris, and Isochrisis galbana. J. Food Sci. 68, 1144– 1148. Vonshak, A., 1997. Spirulina platensis (Arthrospira): Physiology, Cell-biology and Biotechnology. Taylor & Francis, London.

67

Yang, Z., Guo, R., Xu, X., Fan, X., Luo, S., 2011. Hydrogen and methane production from lipid-extracted microalgal biomass residues. Int. J. Hydrogen Energy 36, 3465–3470. Yawson, S.K., Liao, P.H., Lo, K.V., 2011. Two-stage dilute acid hydrolysis of dairy manure for nutrient release, solid reduction and reducing sugar production. Nat. Resour. 2, 224–233. Yen, H.W., Brune, D.E., 2007. Anaerobic co-digestion of algal sludge and waste paper to produce methane. Bioresour. Technol. 98, 130–134.