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Enhanced the energy outcomes from microalgal biomass by the novel biopretreatment
Energy Conversion and Management 135 (2017) 291–296
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Energy Conversion and Management journal homepage: www.elsevier.com/locate/enconman
Enhanced the energy outcomes from microalgal biomass by the novel biopretreatment Shuai He a,b, Xiaolei Fan a, Shengjun Luo a, Naveen Reddy Katukuri a,b, Rongbo Guo a,⇑ a Shandong Industrial Engineering Laboratory of Biogas Production & Utilization, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao, Shandong Province 266101, PR China b University of Chinese Academy of Sciences, Beijing 100049, PR China
a r t i c l e
i n f o
Article history: Received 27 September 2016 Received in revised form 27 November 2016 Accepted 19 December 2016
Keywords: Microalgae Anaerobic digestion Micro-aerobic bio-pretreatment Energy outcomes
a b s t r a c t Microalgae have been considered as one of the most promising biomass for the generation of biofuels. The anaerobic digestion (AD) has been proved to be a promising technique to transfer the microalgal biomass into biofuels. Previous study demonstrated that anaerobic pretreatment of microalgae biomass by Bacillus licheniformis could improve methane production. In this study micro-aerobic bio-pretreatment of microalgal biomass by the facultative anaerobic bacteria Bacillus licheniformis was invested with different loads of oxygen supplied. The bio-hydrogen and biomethane productions were tested to calculate total energy outcomes. The transmission electron microscope (TEM) photographs suggested that the novel micro-aerobic bio-pretreatment (MBP) could effectively damage the firm cell wall of algal cells. The processing time of the novel method (24 h) was less than the previous anaerobic pretreatment (60 h). Results showed that the group with 5 mL oxygen/g VSfed had the highest total energy outcomes, which was 17.6% higher than that of the anaerobic pretreatment. Ó 2016 Published by Elsevier Ltd.
1. Introduction Fossil fuels have been meeting the increasing energy needs of the world for centuries [1], whereas the overuses result in exacerbating climate change, energy crisis and environmental pollution [2]. As biofuels generated from biomass are excellent for the sustainable and renewable energy producing potential, they are considered as a potential alternative of fossil fuels in the long term [2]. Microalgae, capable of fixing carbon dioxide and providing algal biomass concurrently, have aroused considerable interest among researchers [3,4]. Biofuels, such as biodiesel, bioethanol, bio-hydrogen, and biomethane, have been successfully generated from microalgal and macroalgal biomass [5–10]. However, indispensable fertilizers and energy consumptions have impeded the application of the microalgal biofuels [11]. It was demonstrated that AD would be a necessary technique to generate more biofuels from microalgae and realize the recycling nutritive elements [12,13]. Besides, the microalgal cell walls are the main barriers for maximizing energy conversion efficiency [14]. Generally, breakup of ⇑ Corresponding author at: Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, No. 189 Songling Road, Qingdao, Shandong Province 266101, PR China. E-mail address: [email protected] (R. Guo). http://dx.doi.org/10.1016/j.enconman.2016.12.049 0196-8904/Ó 2016 Published by Elsevier Ltd.
the rigid and tough cell walls by different kinds of pretreatment techniques is an effective strategy to improve the bioavailability of organic matters in the algae cells [15]. Currently, the physical, chemical and enzymatic methods are the most widely used solutions [16]. Mendez et al. validated that thermal pretreatments could increase microalgal biodegradability by 50% [17]. Spiden et al. revealed that acidic and thermal treatments damaged microalgal cell walls [18]. Passos et al. proved that mixenzymolysis improved methane production by 15% [19]. But, physicochemical methods are constrained by high cost or high energy consumption, and studies on novel, economical methods are scarce [16]. Some researchers have been making attempts to reduce the microalgal pretreatments cost and energy consumption [20]. Prajapati et al. verified that fungal crude enzymes including cellulase and xylanase were successfully used to elevate the methane production from Chroococcus sp. [21]. However, to produce fungal enzymes, the long-time cultivation of fungi was necessary. Muñoz et al. found that ‘‘whole-cell” cellulolytic pretreatment by some marine bacteria could effectively improve the biogas production from Nannochloropsis gaditana [22]. Ahmed showed that the methane productions of Chlorella vulgaris were 190.6 mL CH4 g CODfed without hydrolytic enzymes pretreatment and 299.6 mL CH4 g CODfed with pretreatment [23]. Notwithstanding these
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attempts, to date, reports about using proteolytic bacteria to enhance methane production from Chlorella sp. are few. In addition, some studies have evidenced that the methane production can be enhanced by adding certain amount of oxygen through a pretreatment process [24–26]. Previous researches demonstrated that micro-aerobic bio-pretreatment (MBP) improved the hydrolytic enzymes activities [25], promoted the growth of facultative bacteria [27] and stimulated the formation of some metabolics benificial for the anaerobes [26]. Our previous work demonstrated that microbial pretreatment with proteolytic Bacillus licheniformis was an eco-friendly method to break the microalgal cell and to enhance corresponding methane yields [28]. But, for this kind of bio-pretreatment under anaerobic condition, the process time was as long as 60 h, which was much longer than the cost by physical, chemical and enzymatic methods, in which, the treatment time was only several h or scores of min [29–31]. Therefore, exploring an efficient solution is crucial for practical application of the novel microbial pretreatment [32]. As the facultative anaerobic bacteria, B. licheniformis grows fast and needs less doubling time in the presence of oxygen than under the anaerobic atmosphere [33]. Amutha verified that B. licheniformis could ferment biomass and produce biohydrogen [34]. In the present study, we investigated the effects of MBP by B. licheniformis on biohydrogen and biomethane production from Chlorella sp. For the first time to our knowledge, little studies on the enhancing effects of MBP by the pure bacteria B. licheniformis on energy outcome from microalgae were conducted [23,35]. 2. Methods 2.1. Substrates, inocula and microorganisms Chlorella sp powder (Fangsheng Co., Shanxi, China) was used as substrates. The inocula were anaerobic digested mesophilic granular sludge taken from a brewery plant (Qingdao, China). The granular sludge was stored at 4 °C refrigerator and was activated under 37 °C before inoculation. The chemical parameters of substrates and inocula were tested through the following methods and the results were shown in Table 1. TS and VS were determined by standard method [36]. Protein content in microalgae was estimated according to the total elemental nitrogen measurement with the conversion factor 6.35 [37]. Carbohydrate and lipid concentration were analyzed through phenol sulfuric acid method [38] and cold extraction using chloroform/methanol [39], respectively. The ratio of inoculum to substrate (VS/VS) was set as 1:1. Bacillus licheniformis 21,886 was kindly provided by Prof. Xiangzhao Mao (Ocean University of China). The B. licheniformis 21,886 was cultivated in the modified nutrient broth medium as described previously [28]. 2.2. MBP of microalgae biomass with B. licheniformis The optimal concentration of 8% (v/v, bacterial culture/working volume) was set as the dosage of bacteria [28]. For pretreatment,
Table 1 Chemical characterization of Chlorella sp. and granular sludge (Average ± standard deviation). Chemical parameters
Chlorella sp. (%)
Granular sludge (%)
TS VS (based on TS) Protein (based on TS) Carbohydrate (based on TS) Lipids (based on TS)
92.9 ± 0.2 83.9 ± 3.4 65.8 ± 0.3 16.0 ± 0.6 17.0 ± 0.3
8.8 ± 0.1 77.5 ± 0.1 – – –
the 8 mL bacteria culture, 1.283 g microalgae powder and 92 mL sterile water were mixed in the 250 mL bottles with 100 mL working volume. The bottles only containing bacteria culture and sterile water were set as the blanks. The pH value was adjusted to 7.1 with 2.0 M HCl and NaOH. In order to quantitatively investigate the oxygen effects on the pretreatment, firstly, all bottles were sealed with butyl rubber stoppers and flushed with pure nitrogen to replace the air in bottles. And then, A syringe (5 mL) with fine needle was used to inject 0, 1, 2, 5, 10 mL of oxygen into each bottle to reach the oxygen loads of 0, 1, 2, 5, 10 mL/g VS substrate (marked as M0, M1, M2, M5, M10), respectively. Batch pretreatments were conducted at 37 °C for 140 rpm. After the total 24 h MBP process, biogas production was measured. To analyze the morphological change of Chlorella sp. cells, 1 mL solution were sampled before and after the pretreatment. Net H2 (mL/g VS added) produced from microalgae was calculated by the following equation:
H2 ðmL=g VSÞ ¼
H2 ðmL; algae and bacteria cultureÞ H2 ðmL;bacteria cultureÞ microalgae ðg VSÞ ð1Þ
2.3. Batch AD experiments After the MBP, 5.87 g granular sludge was added into these bottles. To obtain the biogas yield from the non-treated microalgae (marked as N), another group consisting of crude algae powder and corresponding inoculum blanks were employed. The working volume and initial pH value of all bottles was adjusted to 100 mL and 7.1 with 2.0 M HCl or NaOH. Then, all bottles were sealed and flushed with N2:CO2 (80:20, v/v) to keep anaerobic conditions. All bottles were placed at 37 °C constant temperature incubator without shaking. Biogas production was measured periodically (the 1st, 3rd, 5th, 7th, 10th, 13th, 21th, 25th day). All groups were performed in duplicates. Net CH4 (mL/g VS added) produced from microalgae was calculated by the following equation [40]:
CH4 ðmL=g VSÞ ¼
CH4 ðmL;algae and bacteria cultureÞ CH4 ðmL;bacteria cultureÞ microalgae ðg VSÞ ð2Þ
The VS degradation efficiency of algal biomass was calculated as the formula [24]:
VS degradation efficiencyð%Þ ¼
Initial VSðgÞ Final VSðgÞ Initial VSðgÞ
ð3Þ
2.4. Analytical methods The volume of biogas was measured by water displacement method and its composition was detected by a gas chromatograph (SP 6890, Lunan Inc., China), equipped with Porapak Q stainless steel column (180 cm Ø 3 mm) and a TCD (thermal conductivity detector). The temperatures of injector, detector and oven were 50, 100 and 100 °C, respectively. For the morphological observation, samples were centrifuged at 1000g for 3 min and the precipitate was washed three times with 0.1 M PBS buffer (pH 7.2). Then, the precipitate was immersed into 2.5% glutaraldehyde dissolved in 0.1 M PBS buffer for two h. After washing with PBS buffer for three times, algal cells were further subjected to osmic acid fixation (2%, 1 h). Again algal cells were washed for three times. Then, the gradient 30%, 50%, 70%, 80%, 90%, 95% concentrations of acetone were used to dehydrate algae
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cells for fifteen min each time, followed by the 100% acetone for three times. The algal cells were entrapped in different concentrations of epoxy resin (epoxy resin: acetone = 1:1; epoxy resin: acetone = 4:1) successively. Then the ultra-thin slices (70–80 nm) were examined under H-7650 TEM (Hitachi - Science & Technology, Japan) at 120 kV. After centrifugation at 12000g for 3 min, the supernatant filtered through 0.45 lm micro-filters was used to test the soluble components. TS, VS, pH were analyzed according to the standard method [36]. The volatile fatty acid (VFA) was measured by the methods previously stated [28].
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O2/g VSfed, the biogas outputs improved from 39.5 ± 22.5 to 90.0 ± 1.0 mL/g VSfed with the hydrogen productions from 21.3 ± 12.3 to 48.2 ± 2.7 mL/g VSfed. When the oxygen exceeded 2 mL/g VS, the biogas and bio-hydrogen production showed a modest decrease. This indicated that with the limited oxygen provided, the B. licheniformis utilized more biomass and might destroy the algal cells very well. For hydrogen yields, there were significant differences between the M0 and the M2 groups (p < 0.05), and between the M0 and the M10 groups (p < 0.05). While there was no significant differences between other groups (p > 0.05). 3.2. Morphological observation
2.5. Data analysis The cumulative methane production was fitted using modified Gompertz model to estimate the kinetic parameters: the potential methane production (Pm), the maximum rate of methane production (Rm), and lag-phase time (k). The modified Gompertz equation is expressed as
Rm e ðk tÞ þ 1 P ¼ Pm exp exp Pm
ð4Þ
where P represents cumulative methane production (mL CH4/g VS at time t, and e is the base of the natural logarithms. The origin (8.0) software was used to fit the experimental data in the model (Eq. (3)). And the SPSS 21.0 software was used to do the ANOVA test, along with the post hoc Fisher’s least-significant difference t test (PValue of 0.05, 95% confidence). fed)
3. Results and discussion 3.1. The biogas and hydrogen production during pretreatment The hydrogen was detected after the pretreatment, for the facultative bacteria could produce hydrogen under anaerobic conditions [41]. As shown in Fig. 1, after the 24 h pretreatment, the yields of biogas (consisting of hydrogen, carbon dioxide and nitrogen) in all groups through MBP were higher than that of absolute anaerobic group. Compared with results from Fu et al. [25], the hydrogen production was higher in the absolute anaerobic group than that of the micro-aerobic group. In that experiment, the biogas slurry was used to treat the corn straw [25], while the pure facultative bacteria was used to treat the microalgal biomass in this study. As the oxygen supply increased in the range of 0 to 2 mL
By comparing TEM photographs of the initial and the pretreated cells under the condition of 2 mL oxygen/g VS, it was found that a round of dark, thick and rigid cell wall surrounded the initial cell (Fig. 2a), which played an important role in protecting the intracellular components. However, after the pretreatment, the cell wall was partly dissolved and cell constituents along with cytoplasm were released into the surrounding medium (Fig. 2b). In view of the firm cell wall of Chlorella sp. composed of polysaccharide and glycoprotein matrix, the degradation of cell wall might be resulted from the proteolytic degradation. In addition, the chloroplasts turned into fragments after the pretreatment. Based on observing the morphological observation, it was confirmed that the MBP effectively degraded the tough cell wall. Therefore, the intracellular organic matters without cell wall protection were available for bacteria. And the MBP would be beneficial for the following AD. 3.3. The Properties of microalgal biomass feedstock after the MBP The TCOD, SCOD, TVFAs and VS reduction were tested before and after the MBP. As shown in Table 2, the TCOD decreased from 18,141 mg/L to around 16,000 mg/L in the five groups, which reflected that the after 24 h MBP, organic matters were consumed partly. However, there was no significant difference between the five groups. As for the SCOD, there was decrease in the M0 group, which was in accordance with the anaerobic pretreatment research [28]. Under the anaerobic conditions, bacteria consumed SCOD for growth in earlier stage and couldn’t degrade the algal biomass very fast [28]. Compared with the initial SCOD, the SCOD of M1, M2, M5 and M10 groups increased, but not significantly. This might be resulted from the biogas generation. In the range from 0 to 2 mL oxygen/g VS fed, the VFAs yields increased with the oxygen increasing, while in the range from 2 to 10 mL oxygen/g VSfed, the VFAs productions decreased with the oxygen increasing. This decrease of VFAs might result from the exceeded oxygen, which could consume some organic matters (including the VFAs) [24]. And after pretreatment, the VS degradation efficiency was tested. It showed that during the MBP, the highest VS reduction was obtained in the M2 group. With the presence of limited oxygen, the VS reduction was higher than the M0 group without oxygen. 3.4. The cumulative biomethane production of algal biomass and Gompertz model analysis
Fig. 1. The bio-hydrogen and biogas production during pretreatment.
During the 25 days’ AD, the accumulated methane productions were shown in Fig. 3. The final methane productions of N, M0, M1, M2, M5 and M10 groups were 315.8 ± 0.1, 297.0 ± 0.4, 332.0 ± 1.0, 335.1 ± 3.0, 344.1 ± 1.0 and 336.6 ± 4.0 mL/g VS fed, respectively. And the methane percent of the N group was 62.4%, with the M0 group 64.5%, the M1 group 65.6%, M2 group 66.5%, M5 group 65.6% and M10 group 67.0%. Compared with the N group, the methane production of M0 group decreased by 6.0%, and methane of M1, M2, M5 and M10 increased by 5.1%, 6.1%, 9.0% and 6.6%,
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Fig. 2. The transmission electron microscopy (TEM) photographs of algal cell before and after pretreatment. (a) TEM photos of initial algal cells; (b) TEM photos of algal cells pretreated with the B. licheniformis under the condition of 2 mL oxygen/g VS.
Table 2 The TCOD, SCOD, TVFAs and VS reduction before and after the MBP. Groups
TCOD (mg/L)
SCOD (mg/L)
TVFAs (mg/L)
VS reduction (%)
Initial M0 M1 M2 M5 M10
18,141 ± 676 16,108 ± 156 16,124 ± 225 16,000 ± 113 16,226 ± 113 16,112 ± 113
8788 ± 253 7690 ± 140 9098 ± 84 9408 ± 56 9316 ± 69 9014 ± 112
0 3352 4308 4458 4112 3527
0 6.55 ± 0.25 8.95 ± 0.75 10.8 ± 0.48 8.72 ± 0.33 10.1 ± 0.54
Mahdy et al. [42] demonstrated that through 2% NaOH pretreatment at 50 °C for 48 h, methane productions from C. vulgaris were improved by 17%. Passos et al. [19] showed that after 6 h enzymatic pretreatment with enzyme mix, 15% improvement of methane yield was obtained. Therefore, the MBP method improved methane production 9.0% in 24 h seemed to be efficient. And the differences between the N group and all the pretreated groups were verified (p < 0.05). The accumulated methane productions were fitted with the modified Gompertz model very well (As shown in Table 3). For the biomethane production, there was no lag-phase time during the AD process. The maximum methane producing rates of all the other groups were higher than the N group. With more oxygen supplied, higher Rm was achieved. And the Rm improved by 4.7% to 31.2% compared with the N group. 3.5. Effects of the pretreatment on the VFAs and VS degradation After three days’ AD, the concentrations and variety of VFAs are shown in Fig. 4. As the dominant intermediate metabolites, the contents and kinds of VFAs played significant role in methane production. The M2 group obtained the highest concentration of VFAs, which were 802.9 mg/L and 71.3% higher than that of the N group. For the M0, M1, M5 and M10 groups, VFAs were 62.7%, 57.1%, 55.4%, and 54.7% higher than the N group. The contents of VFAs
Fig. 3. The cumulative biomethane production of treated and non-treated algal biomass.
respectively. The lower methane production of the M0 group might be caused by the decrease of SCOD after 24 h anaerobic pretreatment. As He et al. [28] reported that after 60 h anaerobic pretreatment, the methane yield from Chlorella sp was enhanced by 22.7%.
Table 3 Kinetic parameters of cumulative methane production from Chlorella sp. pretreated with bacterial culture under different oxygen supplying. Groups
Pm (mL CH4/g VS)
Rm (mL CH4/(g VS/d)
k (d)
R2
N M0 M1 M2 M5 M10
318.7 297.7 333.0 337.8 344.7 337.0
23.4 24.5 28.2 28.3 29.5 30.7
0 0 0 0 0 0
0.97 0.99 0.99 0.99 0.98 0.99
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limiting factors for methane producing [43] were 54.4% in the N group, 52.9% for the M0, 50.1% for the M1, 42.4% for the M2, 51.1% for the M5 and 46.7% for the M10. The results indicated that more beneficial VFAs and less limiting VFAs for methane production were produced during AD after the MBP. The VS degradation efficiency was also an important parameter to appraise the effects of pretreatment on the AD process. On one hand, the more VS were degraded, the more organic matters were utilized to generate biogas. On the other hand, the higher VS degradation efficiency benefitted reducing the biogas residues. In the range of 1–5 mL oxygen/g VS fed, with the oxygen loads increasing, the VS degradation efficiency was improved (Fig. 5). However, it decreased when the oxygen reached to 10 mL/g VS fed. The highest VS degradation efficiency of the group of 5 mL/g VS fed was up to 82.1%, and was 11.4% higher than that of the M0 group and 8.5% higher than that of the N group. The VS degradation efficiency was kept consistent in the final biomethane production. Fig. 4. The contents and varieties of VFAs during AD (the third day).
3.6. Energy calculation
Fig. 5. The VS degradation efficiency of algal biomass.
did not increase with the oxygen loads increasing during pretreatment, which agreed with the results reported by Fu et al. [24]. Besides, there was almost no acetate in all groups. The proportions of butyrate and isobutyrate which would be desirable to maximize the methane yields [43] were 44.1%, 46.3%, 49.0%, 56.7%, 48.1% and 52.7% in the N, M0, M1, M2, M5, and M10 respectively. On the contrary, the propionate, valerate and isovalerate which might be
The total energy outcomes (E t) during the whole process including the pretreatment and the subsequent AD were consisted of two different parts. The energy of hydrogen and methane were obtained by multiplying the production of biofuels by the relevant specific heat content (Table 4). The group added with 5 mL oxygen/ g VS fed obtained the highest total E outcome, which was 12,091 MJ/ton VS fed and was 12.3% significant higher than the N group (p < 0.05). These results indicated that the established MBP method could effectively improve the E t with a short duration. In our previous report, after 60 h anaerobic pretreatment, the methane production was increased by 22.7% (in terms of methane). Through the novel 24 h MBP, 12.3% improvement of the total energy outcomes was achieved. The E H2 means the energy outcomes in the form of H2 (specific heat content of H2 = 10 MJ/Nm3 [44]), and the E CH4 means the energy outcomes in the form of CH4 (specific heat content of CH4 = 35 MJ/Nm3 [44]), and the E t represents the total energy outcomes. Table 5 showed a comparative study on improvements of energy outcomes from Chlorella sp. by different pretreatment methods. The 12.3% increase of energy outcomes by the novel pretreatment was a little lower than thermal, thermal-alkali, and carbohydrolase pretreatment reported by Cho et al. [45] and Mahdy et al. [23,42]. Compared with the anaerobic method, pretreatment time of MBP was decreased from 60 h to 24 h. Therefore, the novel MBP would be an exploration for improving the energy outcomes by an environmental friendly method.
Table 4 The energy outcome of the whole process. Groups/MJ/ton VSfed
N
M0
M1
M2
M5
M10
E H2 E CH4 Et
0 10,766 10,766
213 10,071 10,283
359 11,259 11,617
482 11,302 11,784
349 11,742 12,091
423 11,491 11,915
Table 5 Comparative study on improvements of energy outcomes from Chlorella sp. by different pretreatment methods. Microalgae species
Pretreatment method
Operation temperature/time
Energy outcomes increase (%)
Reference
C. C. C. C.
Thermal-alkali Thermal
2% NaOH at 50 °C/48 h 120 °C/30 min 0.585 AU/g at 50 °C/3 h 0.3 mL/g at 50 °C/5 h 0.2 mL/g at 50 °C/5 h Anaerobic 37 °C/60 h Microaerobic 37 °C/24 h
17 20 64 14 51 22.7 12.3
[42] [45] [46] [23]
vulgaris sp. vulgaris vulgaris
C. sp. C. sp.
Carbohydrolase Protease B. licheniformis B. licheniformis
[28] This study
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4. Conclusion The novel micro-aerobic bio-pretreatment, except the M0 group, significantly enhanced both the methane production and the total energy outcomes. The facultative anaerobic bacteria B. licheniformis produced hydrogen during the pretreatment. And the TEM results indicated that the MBP could disrupt the tough cell wall of algal cells. The increased SCOD and TVFAs after pretreatment also demonstrated that the MBP improved the soluble organic matters. In AD process, the cumulative methane production, the concentrations of VFAs and the VS degradation efficiency reached to the highest value at the loads of 5 mL oxygen/g VSfed, while the maximum methane producing rates of the 10 mL oxygen/g VSfed was the highest one and 31.2% higher than that of the N group. This MBP method could improve the energy outcomes from the Chlorella sp. within a short duration. Acknowledgements This work was supported by the National Natural Science Foundation of China (41276143); Key Research & Development Project of Shandong No. 2015GSF115037 and No. 2015GSF117016); Technology development for energy utilization of municipal and rural organic waste, science and technology plan projects for benefiting the people, Ministry of Science and Technology of the People’s Republic of China (2013GS460202); the National Key Technology Support Program of China [2015BAL04B02]. References [1] Georgianna DR, Mayfield SP. Exploiting diversity and synthetic biology for the production of algal biofuels. Nature 2012;488:329–35. [2] Peralta-Yahya PP, Zhang FZ, del Cardayre SB, Keasling JD. Microbial engineering for the production of advanced biofuels. Nature 2012;488:320–8. [3] Zhao Y, Sun S, Hu C, Zhang H, Xu J, Ping L. Performance of three microalgal strains in biogas slurry purification and biogas upgrade in response to various mixed light-emitting diode light wavelengths. Bioresour Technol 2015;187:338–45. [4] Hallenbeck PC, Grogger M, Mraz M, Veverka D. Solar biofuels production with microalgae. Appl Energy 2016;179:136–45. [5] Tan CH, Show PL, Chang JS, Ling TC, Lan JC. Novel approaches of producing bioenergies from microalgae: a recent review. Biotechnol Adv 2015;33:1219–27. [6] Sivagurunathan P, Kumar G, Park JH, Park JH, Park HD, Yoon JJ, et al. Feasibility of enriched mixed cultures obtained by repeated batch transfer in continuous hydrogen fermentation. Int J Hydrogen Energy 2016;41:4393–403. [7] Park JH, Hong JY, Jang HC, Oh SG, Kim SH, Yoon JJ, et al. Use of Gelidium amansii as a promising resource for bioethanol: a practical approach for continuous dilute-acid hydrolysis and fermentation. Bioresource Technol 2012;108:83–8. [8] Park JH, Cheon HC, Yoon JJ, Park HD, Kim SH. Optimization of batch dilute-acid hydrolysis for biohydrogen production from red algal biomass. Int J Hydrogen Energy 2013;38:6130–6. [9] Kumar G, Zhen GY, Kobayashi T, Sivagurunathan P, Kim SH, Xu KQ. Impact of pH control and heat pre-treatment of seed inoculum in dark H2 fermentation: a feasibility report using mixed microalgae biomass as feedstock. Int J Hydrogen Energy 2016;41:4382–92. [10] Kumar G, Sivagurunathan P, Park JH, Park JH, Park HD, Yoon JJ, et al. HRT dependent performance and bacterial community population of granular hydrogen-producing mixed cultures fed with galactose. Bioresour Technol 2016;206:188–94. [11] Hlavova M, Turoczy Z, Bisova K. Improving microalgae for biotechnology from genetics to synthetic biology. Biotechnol Adv 2015;33:1194–203. [12] Bai X, Lant PA, Jensen PD, Astals S, Pratt S. Enhanced methane production from algal digestion using free nitrous acid pre-treatment. Renewable Energy 2016;88:383–90. [13] Sialve B, Bernet N, Bernard O. Anaerobic digestion of microalgae as a necessary step to makemicroalgal biodiesel sustainable. Biotechnol Adv 2009;27:409–16. [14] Domozych DS. Algal cell walls. Chichester: eLS. John Wiley & Sons, Ltd; 2011. [15] Seo JY, Praveenkumar R, Kim B, Seo JC, Park JY, Na JG, et al. Downstream integration of microalgae harvesting and cell disruption by means of cationic surfactant-decorated Fe3O4 nanoparticles. Green Chem 2016;18:3981–9. [16] Passos F, Uggetti E, Carrere H, Ferrer I. Pretreatment of microalgae to improve biogas production: a review. Bioresour Technol 2014;172:403–12. [17] Mendez L, Mahdy A, Ballesteros M, Gonzalez-Fernandez C. Methane production of thermally pretreated Chlorella vulgaris and Scenedesmus sp. biomass at increasing biomass loads. Appl Energy 2014;129:238–42.
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Energy Conversion and Management journal homepage: www.elsevier.com/locate/enconman
Enhanced the energy outcomes from microalgal biomass by the novel biopretreatment Shuai He a,b, Xiaolei Fan a, Shengjun Luo a, Naveen Reddy Katukuri a,b, Rongbo Guo a,⇑ a Shandong Industrial Engineering Laboratory of Biogas Production & Utilization, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao, Shandong Province 266101, PR China b University of Chinese Academy of Sciences, Beijing 100049, PR China
a r t i c l e
i n f o
Article history: Received 27 September 2016 Received in revised form 27 November 2016 Accepted 19 December 2016
Keywords: Microalgae Anaerobic digestion Micro-aerobic bio-pretreatment Energy outcomes
a b s t r a c t Microalgae have been considered as one of the most promising biomass for the generation of biofuels. The anaerobic digestion (AD) has been proved to be a promising technique to transfer the microalgal biomass into biofuels. Previous study demonstrated that anaerobic pretreatment of microalgae biomass by Bacillus licheniformis could improve methane production. In this study micro-aerobic bio-pretreatment of microalgal biomass by the facultative anaerobic bacteria Bacillus licheniformis was invested with different loads of oxygen supplied. The bio-hydrogen and biomethane productions were tested to calculate total energy outcomes. The transmission electron microscope (TEM) photographs suggested that the novel micro-aerobic bio-pretreatment (MBP) could effectively damage the firm cell wall of algal cells. The processing time of the novel method (24 h) was less than the previous anaerobic pretreatment (60 h). Results showed that the group with 5 mL oxygen/g VSfed had the highest total energy outcomes, which was 17.6% higher than that of the anaerobic pretreatment. Ó 2016 Published by Elsevier Ltd.
1. Introduction Fossil fuels have been meeting the increasing energy needs of the world for centuries [1], whereas the overuses result in exacerbating climate change, energy crisis and environmental pollution [2]. As biofuels generated from biomass are excellent for the sustainable and renewable energy producing potential, they are considered as a potential alternative of fossil fuels in the long term [2]. Microalgae, capable of fixing carbon dioxide and providing algal biomass concurrently, have aroused considerable interest among researchers [3,4]. Biofuels, such as biodiesel, bioethanol, bio-hydrogen, and biomethane, have been successfully generated from microalgal and macroalgal biomass [5–10]. However, indispensable fertilizers and energy consumptions have impeded the application of the microalgal biofuels [11]. It was demonstrated that AD would be a necessary technique to generate more biofuels from microalgae and realize the recycling nutritive elements [12,13]. Besides, the microalgal cell walls are the main barriers for maximizing energy conversion efficiency [14]. Generally, breakup of ⇑ Corresponding author at: Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, No. 189 Songling Road, Qingdao, Shandong Province 266101, PR China. E-mail address: [email protected] (R. Guo). http://dx.doi.org/10.1016/j.enconman.2016.12.049 0196-8904/Ó 2016 Published by Elsevier Ltd.
the rigid and tough cell walls by different kinds of pretreatment techniques is an effective strategy to improve the bioavailability of organic matters in the algae cells [15]. Currently, the physical, chemical and enzymatic methods are the most widely used solutions [16]. Mendez et al. validated that thermal pretreatments could increase microalgal biodegradability by 50% [17]. Spiden et al. revealed that acidic and thermal treatments damaged microalgal cell walls [18]. Passos et al. proved that mixenzymolysis improved methane production by 15% [19]. But, physicochemical methods are constrained by high cost or high energy consumption, and studies on novel, economical methods are scarce [16]. Some researchers have been making attempts to reduce the microalgal pretreatments cost and energy consumption [20]. Prajapati et al. verified that fungal crude enzymes including cellulase and xylanase were successfully used to elevate the methane production from Chroococcus sp. [21]. However, to produce fungal enzymes, the long-time cultivation of fungi was necessary. Muñoz et al. found that ‘‘whole-cell” cellulolytic pretreatment by some marine bacteria could effectively improve the biogas production from Nannochloropsis gaditana [22]. Ahmed showed that the methane productions of Chlorella vulgaris were 190.6 mL CH4 g CODfed without hydrolytic enzymes pretreatment and 299.6 mL CH4 g CODfed with pretreatment [23]. Notwithstanding these
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attempts, to date, reports about using proteolytic bacteria to enhance methane production from Chlorella sp. are few. In addition, some studies have evidenced that the methane production can be enhanced by adding certain amount of oxygen through a pretreatment process [24–26]. Previous researches demonstrated that micro-aerobic bio-pretreatment (MBP) improved the hydrolytic enzymes activities [25], promoted the growth of facultative bacteria [27] and stimulated the formation of some metabolics benificial for the anaerobes [26]. Our previous work demonstrated that microbial pretreatment with proteolytic Bacillus licheniformis was an eco-friendly method to break the microalgal cell and to enhance corresponding methane yields [28]. But, for this kind of bio-pretreatment under anaerobic condition, the process time was as long as 60 h, which was much longer than the cost by physical, chemical and enzymatic methods, in which, the treatment time was only several h or scores of min [29–31]. Therefore, exploring an efficient solution is crucial for practical application of the novel microbial pretreatment [32]. As the facultative anaerobic bacteria, B. licheniformis grows fast and needs less doubling time in the presence of oxygen than under the anaerobic atmosphere [33]. Amutha verified that B. licheniformis could ferment biomass and produce biohydrogen [34]. In the present study, we investigated the effects of MBP by B. licheniformis on biohydrogen and biomethane production from Chlorella sp. For the first time to our knowledge, little studies on the enhancing effects of MBP by the pure bacteria B. licheniformis on energy outcome from microalgae were conducted [23,35]. 2. Methods 2.1. Substrates, inocula and microorganisms Chlorella sp powder (Fangsheng Co., Shanxi, China) was used as substrates. The inocula were anaerobic digested mesophilic granular sludge taken from a brewery plant (Qingdao, China). The granular sludge was stored at 4 °C refrigerator and was activated under 37 °C before inoculation. The chemical parameters of substrates and inocula were tested through the following methods and the results were shown in Table 1. TS and VS were determined by standard method [36]. Protein content in microalgae was estimated according to the total elemental nitrogen measurement with the conversion factor 6.35 [37]. Carbohydrate and lipid concentration were analyzed through phenol sulfuric acid method [38] and cold extraction using chloroform/methanol [39], respectively. The ratio of inoculum to substrate (VS/VS) was set as 1:1. Bacillus licheniformis 21,886 was kindly provided by Prof. Xiangzhao Mao (Ocean University of China). The B. licheniformis 21,886 was cultivated in the modified nutrient broth medium as described previously [28]. 2.2. MBP of microalgae biomass with B. licheniformis The optimal concentration of 8% (v/v, bacterial culture/working volume) was set as the dosage of bacteria [28]. For pretreatment,
Table 1 Chemical characterization of Chlorella sp. and granular sludge (Average ± standard deviation). Chemical parameters
Chlorella sp. (%)
Granular sludge (%)
TS VS (based on TS) Protein (based on TS) Carbohydrate (based on TS) Lipids (based on TS)
92.9 ± 0.2 83.9 ± 3.4 65.8 ± 0.3 16.0 ± 0.6 17.0 ± 0.3
8.8 ± 0.1 77.5 ± 0.1 – – –
the 8 mL bacteria culture, 1.283 g microalgae powder and 92 mL sterile water were mixed in the 250 mL bottles with 100 mL working volume. The bottles only containing bacteria culture and sterile water were set as the blanks. The pH value was adjusted to 7.1 with 2.0 M HCl and NaOH. In order to quantitatively investigate the oxygen effects on the pretreatment, firstly, all bottles were sealed with butyl rubber stoppers and flushed with pure nitrogen to replace the air in bottles. And then, A syringe (5 mL) with fine needle was used to inject 0, 1, 2, 5, 10 mL of oxygen into each bottle to reach the oxygen loads of 0, 1, 2, 5, 10 mL/g VS substrate (marked as M0, M1, M2, M5, M10), respectively. Batch pretreatments were conducted at 37 °C for 140 rpm. After the total 24 h MBP process, biogas production was measured. To analyze the morphological change of Chlorella sp. cells, 1 mL solution were sampled before and after the pretreatment. Net H2 (mL/g VS added) produced from microalgae was calculated by the following equation:
H2 ðmL=g VSÞ ¼
H2 ðmL; algae and bacteria cultureÞ H2 ðmL;bacteria cultureÞ microalgae ðg VSÞ ð1Þ
2.3. Batch AD experiments After the MBP, 5.87 g granular sludge was added into these bottles. To obtain the biogas yield from the non-treated microalgae (marked as N), another group consisting of crude algae powder and corresponding inoculum blanks were employed. The working volume and initial pH value of all bottles was adjusted to 100 mL and 7.1 with 2.0 M HCl or NaOH. Then, all bottles were sealed and flushed with N2:CO2 (80:20, v/v) to keep anaerobic conditions. All bottles were placed at 37 °C constant temperature incubator without shaking. Biogas production was measured periodically (the 1st, 3rd, 5th, 7th, 10th, 13th, 21th, 25th day). All groups were performed in duplicates. Net CH4 (mL/g VS added) produced from microalgae was calculated by the following equation [40]:
CH4 ðmL=g VSÞ ¼
CH4 ðmL;algae and bacteria cultureÞ CH4 ðmL;bacteria cultureÞ microalgae ðg VSÞ ð2Þ
The VS degradation efficiency of algal biomass was calculated as the formula [24]:
VS degradation efficiencyð%Þ ¼
Initial VSðgÞ Final VSðgÞ Initial VSðgÞ
ð3Þ
2.4. Analytical methods The volume of biogas was measured by water displacement method and its composition was detected by a gas chromatograph (SP 6890, Lunan Inc., China), equipped with Porapak Q stainless steel column (180 cm Ø 3 mm) and a TCD (thermal conductivity detector). The temperatures of injector, detector and oven were 50, 100 and 100 °C, respectively. For the morphological observation, samples were centrifuged at 1000g for 3 min and the precipitate was washed three times with 0.1 M PBS buffer (pH 7.2). Then, the precipitate was immersed into 2.5% glutaraldehyde dissolved in 0.1 M PBS buffer for two h. After washing with PBS buffer for three times, algal cells were further subjected to osmic acid fixation (2%, 1 h). Again algal cells were washed for three times. Then, the gradient 30%, 50%, 70%, 80%, 90%, 95% concentrations of acetone were used to dehydrate algae
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cells for fifteen min each time, followed by the 100% acetone for three times. The algal cells were entrapped in different concentrations of epoxy resin (epoxy resin: acetone = 1:1; epoxy resin: acetone = 4:1) successively. Then the ultra-thin slices (70–80 nm) were examined under H-7650 TEM (Hitachi - Science & Technology, Japan) at 120 kV. After centrifugation at 12000g for 3 min, the supernatant filtered through 0.45 lm micro-filters was used to test the soluble components. TS, VS, pH were analyzed according to the standard method [36]. The volatile fatty acid (VFA) was measured by the methods previously stated [28].
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O2/g VSfed, the biogas outputs improved from 39.5 ± 22.5 to 90.0 ± 1.0 mL/g VSfed with the hydrogen productions from 21.3 ± 12.3 to 48.2 ± 2.7 mL/g VSfed. When the oxygen exceeded 2 mL/g VS, the biogas and bio-hydrogen production showed a modest decrease. This indicated that with the limited oxygen provided, the B. licheniformis utilized more biomass and might destroy the algal cells very well. For hydrogen yields, there were significant differences between the M0 and the M2 groups (p < 0.05), and between the M0 and the M10 groups (p < 0.05). While there was no significant differences between other groups (p > 0.05). 3.2. Morphological observation
2.5. Data analysis The cumulative methane production was fitted using modified Gompertz model to estimate the kinetic parameters: the potential methane production (Pm), the maximum rate of methane production (Rm), and lag-phase time (k). The modified Gompertz equation is expressed as
Rm e ðk tÞ þ 1 P ¼ Pm exp exp Pm
ð4Þ
where P represents cumulative methane production (mL CH4/g VS at time t, and e is the base of the natural logarithms. The origin (8.0) software was used to fit the experimental data in the model (Eq. (3)). And the SPSS 21.0 software was used to do the ANOVA test, along with the post hoc Fisher’s least-significant difference t test (PValue of 0.05, 95% confidence). fed)
3. Results and discussion 3.1. The biogas and hydrogen production during pretreatment The hydrogen was detected after the pretreatment, for the facultative bacteria could produce hydrogen under anaerobic conditions [41]. As shown in Fig. 1, after the 24 h pretreatment, the yields of biogas (consisting of hydrogen, carbon dioxide and nitrogen) in all groups through MBP were higher than that of absolute anaerobic group. Compared with results from Fu et al. [25], the hydrogen production was higher in the absolute anaerobic group than that of the micro-aerobic group. In that experiment, the biogas slurry was used to treat the corn straw [25], while the pure facultative bacteria was used to treat the microalgal biomass in this study. As the oxygen supply increased in the range of 0 to 2 mL
By comparing TEM photographs of the initial and the pretreated cells under the condition of 2 mL oxygen/g VS, it was found that a round of dark, thick and rigid cell wall surrounded the initial cell (Fig. 2a), which played an important role in protecting the intracellular components. However, after the pretreatment, the cell wall was partly dissolved and cell constituents along with cytoplasm were released into the surrounding medium (Fig. 2b). In view of the firm cell wall of Chlorella sp. composed of polysaccharide and glycoprotein matrix, the degradation of cell wall might be resulted from the proteolytic degradation. In addition, the chloroplasts turned into fragments after the pretreatment. Based on observing the morphological observation, it was confirmed that the MBP effectively degraded the tough cell wall. Therefore, the intracellular organic matters without cell wall protection were available for bacteria. And the MBP would be beneficial for the following AD. 3.3. The Properties of microalgal biomass feedstock after the MBP The TCOD, SCOD, TVFAs and VS reduction were tested before and after the MBP. As shown in Table 2, the TCOD decreased from 18,141 mg/L to around 16,000 mg/L in the five groups, which reflected that the after 24 h MBP, organic matters were consumed partly. However, there was no significant difference between the five groups. As for the SCOD, there was decrease in the M0 group, which was in accordance with the anaerobic pretreatment research [28]. Under the anaerobic conditions, bacteria consumed SCOD for growth in earlier stage and couldn’t degrade the algal biomass very fast [28]. Compared with the initial SCOD, the SCOD of M1, M2, M5 and M10 groups increased, but not significantly. This might be resulted from the biogas generation. In the range from 0 to 2 mL oxygen/g VS fed, the VFAs yields increased with the oxygen increasing, while in the range from 2 to 10 mL oxygen/g VSfed, the VFAs productions decreased with the oxygen increasing. This decrease of VFAs might result from the exceeded oxygen, which could consume some organic matters (including the VFAs) [24]. And after pretreatment, the VS degradation efficiency was tested. It showed that during the MBP, the highest VS reduction was obtained in the M2 group. With the presence of limited oxygen, the VS reduction was higher than the M0 group without oxygen. 3.4. The cumulative biomethane production of algal biomass and Gompertz model analysis
Fig. 1. The bio-hydrogen and biogas production during pretreatment.
During the 25 days’ AD, the accumulated methane productions were shown in Fig. 3. The final methane productions of N, M0, M1, M2, M5 and M10 groups were 315.8 ± 0.1, 297.0 ± 0.4, 332.0 ± 1.0, 335.1 ± 3.0, 344.1 ± 1.0 and 336.6 ± 4.0 mL/g VS fed, respectively. And the methane percent of the N group was 62.4%, with the M0 group 64.5%, the M1 group 65.6%, M2 group 66.5%, M5 group 65.6% and M10 group 67.0%. Compared with the N group, the methane production of M0 group decreased by 6.0%, and methane of M1, M2, M5 and M10 increased by 5.1%, 6.1%, 9.0% and 6.6%,
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Fig. 2. The transmission electron microscopy (TEM) photographs of algal cell before and after pretreatment. (a) TEM photos of initial algal cells; (b) TEM photos of algal cells pretreated with the B. licheniformis under the condition of 2 mL oxygen/g VS.
Table 2 The TCOD, SCOD, TVFAs and VS reduction before and after the MBP. Groups
TCOD (mg/L)
SCOD (mg/L)
TVFAs (mg/L)
VS reduction (%)
Initial M0 M1 M2 M5 M10
18,141 ± 676 16,108 ± 156 16,124 ± 225 16,000 ± 113 16,226 ± 113 16,112 ± 113
8788 ± 253 7690 ± 140 9098 ± 84 9408 ± 56 9316 ± 69 9014 ± 112
0 3352 4308 4458 4112 3527
0 6.55 ± 0.25 8.95 ± 0.75 10.8 ± 0.48 8.72 ± 0.33 10.1 ± 0.54
Mahdy et al. [42] demonstrated that through 2% NaOH pretreatment at 50 °C for 48 h, methane productions from C. vulgaris were improved by 17%. Passos et al. [19] showed that after 6 h enzymatic pretreatment with enzyme mix, 15% improvement of methane yield was obtained. Therefore, the MBP method improved methane production 9.0% in 24 h seemed to be efficient. And the differences between the N group and all the pretreated groups were verified (p < 0.05). The accumulated methane productions were fitted with the modified Gompertz model very well (As shown in Table 3). For the biomethane production, there was no lag-phase time during the AD process. The maximum methane producing rates of all the other groups were higher than the N group. With more oxygen supplied, higher Rm was achieved. And the Rm improved by 4.7% to 31.2% compared with the N group. 3.5. Effects of the pretreatment on the VFAs and VS degradation After three days’ AD, the concentrations and variety of VFAs are shown in Fig. 4. As the dominant intermediate metabolites, the contents and kinds of VFAs played significant role in methane production. The M2 group obtained the highest concentration of VFAs, which were 802.9 mg/L and 71.3% higher than that of the N group. For the M0, M1, M5 and M10 groups, VFAs were 62.7%, 57.1%, 55.4%, and 54.7% higher than the N group. The contents of VFAs
Fig. 3. The cumulative biomethane production of treated and non-treated algal biomass.
respectively. The lower methane production of the M0 group might be caused by the decrease of SCOD after 24 h anaerobic pretreatment. As He et al. [28] reported that after 60 h anaerobic pretreatment, the methane yield from Chlorella sp was enhanced by 22.7%.
Table 3 Kinetic parameters of cumulative methane production from Chlorella sp. pretreated with bacterial culture under different oxygen supplying. Groups
Pm (mL CH4/g VS)
Rm (mL CH4/(g VS/d)
k (d)
R2
N M0 M1 M2 M5 M10
318.7 297.7 333.0 337.8 344.7 337.0
23.4 24.5 28.2 28.3 29.5 30.7
0 0 0 0 0 0
0.97 0.99 0.99 0.99 0.98 0.99
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limiting factors for methane producing [43] were 54.4% in the N group, 52.9% for the M0, 50.1% for the M1, 42.4% for the M2, 51.1% for the M5 and 46.7% for the M10. The results indicated that more beneficial VFAs and less limiting VFAs for methane production were produced during AD after the MBP. The VS degradation efficiency was also an important parameter to appraise the effects of pretreatment on the AD process. On one hand, the more VS were degraded, the more organic matters were utilized to generate biogas. On the other hand, the higher VS degradation efficiency benefitted reducing the biogas residues. In the range of 1–5 mL oxygen/g VS fed, with the oxygen loads increasing, the VS degradation efficiency was improved (Fig. 5). However, it decreased when the oxygen reached to 10 mL/g VS fed. The highest VS degradation efficiency of the group of 5 mL/g VS fed was up to 82.1%, and was 11.4% higher than that of the M0 group and 8.5% higher than that of the N group. The VS degradation efficiency was kept consistent in the final biomethane production. Fig. 4. The contents and varieties of VFAs during AD (the third day).
3.6. Energy calculation
Fig. 5. The VS degradation efficiency of algal biomass.
did not increase with the oxygen loads increasing during pretreatment, which agreed with the results reported by Fu et al. [24]. Besides, there was almost no acetate in all groups. The proportions of butyrate and isobutyrate which would be desirable to maximize the methane yields [43] were 44.1%, 46.3%, 49.0%, 56.7%, 48.1% and 52.7% in the N, M0, M1, M2, M5, and M10 respectively. On the contrary, the propionate, valerate and isovalerate which might be
The total energy outcomes (E t) during the whole process including the pretreatment and the subsequent AD were consisted of two different parts. The energy of hydrogen and methane were obtained by multiplying the production of biofuels by the relevant specific heat content (Table 4). The group added with 5 mL oxygen/ g VS fed obtained the highest total E outcome, which was 12,091 MJ/ton VS fed and was 12.3% significant higher than the N group (p < 0.05). These results indicated that the established MBP method could effectively improve the E t with a short duration. In our previous report, after 60 h anaerobic pretreatment, the methane production was increased by 22.7% (in terms of methane). Through the novel 24 h MBP, 12.3% improvement of the total energy outcomes was achieved. The E H2 means the energy outcomes in the form of H2 (specific heat content of H2 = 10 MJ/Nm3 [44]), and the E CH4 means the energy outcomes in the form of CH4 (specific heat content of CH4 = 35 MJ/Nm3 [44]), and the E t represents the total energy outcomes. Table 5 showed a comparative study on improvements of energy outcomes from Chlorella sp. by different pretreatment methods. The 12.3% increase of energy outcomes by the novel pretreatment was a little lower than thermal, thermal-alkali, and carbohydrolase pretreatment reported by Cho et al. [45] and Mahdy et al. [23,42]. Compared with the anaerobic method, pretreatment time of MBP was decreased from 60 h to 24 h. Therefore, the novel MBP would be an exploration for improving the energy outcomes by an environmental friendly method.
Table 4 The energy outcome of the whole process. Groups/MJ/ton VSfed
N
M0
M1
M2
M5
M10
E H2 E CH4 Et
0 10,766 10,766
213 10,071 10,283
359 11,259 11,617
482 11,302 11,784
349 11,742 12,091
423 11,491 11,915
Table 5 Comparative study on improvements of energy outcomes from Chlorella sp. by different pretreatment methods. Microalgae species
Pretreatment method
Operation temperature/time
Energy outcomes increase (%)
Reference
C. C. C. C.
Thermal-alkali Thermal
2% NaOH at 50 °C/48 h 120 °C/30 min 0.585 AU/g at 50 °C/3 h 0.3 mL/g at 50 °C/5 h 0.2 mL/g at 50 °C/5 h Anaerobic 37 °C/60 h Microaerobic 37 °C/24 h
17 20 64 14 51 22.7 12.3
[42] [45] [46] [23]
vulgaris sp. vulgaris vulgaris
C. sp. C. sp.
Carbohydrolase Protease B. licheniformis B. licheniformis
[28] This study
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4. Conclusion The novel micro-aerobic bio-pretreatment, except the M0 group, significantly enhanced both the methane production and the total energy outcomes. The facultative anaerobic bacteria B. licheniformis produced hydrogen during the pretreatment. And the TEM results indicated that the MBP could disrupt the tough cell wall of algal cells. The increased SCOD and TVFAs after pretreatment also demonstrated that the MBP improved the soluble organic matters. In AD process, the cumulative methane production, the concentrations of VFAs and the VS degradation efficiency reached to the highest value at the loads of 5 mL oxygen/g VSfed, while the maximum methane producing rates of the 10 mL oxygen/g VSfed was the highest one and 31.2% higher than that of the N group. This MBP method could improve the energy outcomes from the Chlorella sp. within a short duration. Acknowledgements This work was supported by the National Natural Science Foundation of China (41276143); Key Research & Development Project of Shandong No. 2015GSF115037 and No. 2015GSF117016); Technology development for energy utilization of municipal and rural organic waste, science and technology plan projects for benefiting the people, Ministry of Science and Technology of the People’s Republic of China (2013GS460202); the National Key Technology Support Program of China [2015BAL04B02]. References [1] Georgianna DR, Mayfield SP. Exploiting diversity and synthetic biology for the production of algal biofuels. Nature 2012;488:329–35. [2] Peralta-Yahya PP, Zhang FZ, del Cardayre SB, Keasling JD. Microbial engineering for the production of advanced biofuels. Nature 2012;488:320–8. [3] Zhao Y, Sun S, Hu C, Zhang H, Xu J, Ping L. Performance of three microalgal strains in biogas slurry purification and biogas upgrade in response to various mixed light-emitting diode light wavelengths. Bioresour Technol 2015;187:338–45. [4] Hallenbeck PC, Grogger M, Mraz M, Veverka D. Solar biofuels production with microalgae. Appl Energy 2016;179:136–45. [5] Tan CH, Show PL, Chang JS, Ling TC, Lan JC. Novel approaches of producing bioenergies from microalgae: a recent review. Biotechnol Adv 2015;33:1219–27. [6] Sivagurunathan P, Kumar G, Park JH, Park JH, Park HD, Yoon JJ, et al. Feasibility of enriched mixed cultures obtained by repeated batch transfer in continuous hydrogen fermentation. Int J Hydrogen Energy 2016;41:4393–403. [7] Park JH, Hong JY, Jang HC, Oh SG, Kim SH, Yoon JJ, et al. Use of Gelidium amansii as a promising resource for bioethanol: a practical approach for continuous dilute-acid hydrolysis and fermentation. Bioresource Technol 2012;108:83–8. [8] Park JH, Cheon HC, Yoon JJ, Park HD, Kim SH. Optimization of batch dilute-acid hydrolysis for biohydrogen production from red algal biomass. Int J Hydrogen Energy 2013;38:6130–6. [9] Kumar G, Zhen GY, Kobayashi T, Sivagurunathan P, Kim SH, Xu KQ. Impact of pH control and heat pre-treatment of seed inoculum in dark H2 fermentation: a feasibility report using mixed microalgae biomass as feedstock. Int J Hydrogen Energy 2016;41:4382–92. [10] Kumar G, Sivagurunathan P, Park JH, Park JH, Park HD, Yoon JJ, et al. HRT dependent performance and bacterial community population of granular hydrogen-producing mixed cultures fed with galactose. Bioresour Technol 2016;206:188–94. [11] Hlavova M, Turoczy Z, Bisova K. Improving microalgae for biotechnology from genetics to synthetic biology. Biotechnol Adv 2015;33:1194–203. [12] Bai X, Lant PA, Jensen PD, Astals S, Pratt S. Enhanced methane production from algal digestion using free nitrous acid pre-treatment. Renewable Energy 2016;88:383–90. [13] Sialve B, Bernet N, Bernard O. Anaerobic digestion of microalgae as a necessary step to makemicroalgal biodiesel sustainable. Biotechnol Adv 2009;27:409–16. [14] Domozych DS. Algal cell walls. Chichester: eLS. John Wiley & Sons, Ltd; 2011. [15] Seo JY, Praveenkumar R, Kim B, Seo JC, Park JY, Na JG, et al. Downstream integration of microalgae harvesting and cell disruption by means of cationic surfactant-decorated Fe3O4 nanoparticles. Green Chem 2016;18:3981–9. [16] Passos F, Uggetti E, Carrere H, Ferrer I. Pretreatment of microalgae to improve biogas production: a review. Bioresour Technol 2014;172:403–12. [17] Mendez L, Mahdy A, Ballesteros M, Gonzalez-Fernandez C. Methane production of thermally pretreated Chlorella vulgaris and Scenedesmus sp. biomass at increasing biomass loads. Appl Energy 2014;129:238–42.
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