Waste Management 34 (2014) 1035–1040
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Waste Management journal homepage: www.elsevier.com/locate/wasman
Ultrasound pretreatment of filamentous algal biomass for enhanced biogas production Kwanyong Lee a, Phrompol Chantrasakdakul a, Daegi Kim a, Mingeun Kong b, Ki Young Park a,⇑ a b
Department of Civil and Environmental System Engineering, Konkuk University, 120 Neungdong-ro, Gwangjin-gu, Seoul 143-701, Republic of Korea EPS Solution Ltd., 126-1 Pyeongchon-dong, Dongan-gu, Anyang, Gyeonggi-do 431-755, Republic of Korea
a r t i c l e
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Article history: Available online 22 November 2013 Keywords: Algae Biogas production Hydrodictyon reticulatum Pretreatment Ultrasound
a b s t r a c t The filamentous alga Hydrodictyon reticulatum harvested from a bench-scale wastewater treatment pond was used to evaluate biogas production after ultrasound pretreatment. The effects of ultrasound pretreatment at a range of 10–5000 J/mL were tested with harvested H. reticulatum. Cell disruption by ultrasound was successful and showed a higher degree of disintegration at a higher applied energy. The range of 10–5000 J/mL ultrasound was able to disintegrated H. reticulatum and the soluble COD was increased from 250 mg/L to 1000 mg/L at 2500 J/mL. The disintegrated algal biomass was digested for biogas production in batch experiments. Both cumulative gas generation and volatile solids reduction data were obtained during the digestion. Cell disintegration due to ultrasound pretreatment increased the specific biogas production and degradation rates. Using the ultrasound approach, the specific methane production at a dose of 40 J/mL increased up to 384 mL/g-VS fed that was 2.3 times higher than the untreated sample. For disintegrated samples, the volatile solids reduction was greater with increased energy input, and the degradation increased slightly to 67% at a dose of 50 J/mL. The results also indicate that disintegration of the algal cells is the essential step for efficient anaerobic digestion of algal biomass. Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction The exhaustion of fossil fuels and the global warming situation are strongly motivating research in alternative energies (Berndes et al., 2003). Many countries are interested in renewable energy sources, especially in sustainable forms of energy, i.e., geothermal power, wind power, small-scale hydropower, solar energy, biomass energy, tidal power, and wave power. Biomass energy is gaining increasing importance because of its environmentally sound and energy-saving production methods (Zheng et al., 2012). Various biomasses derived from the carbonaceous waste of human and natural activities could be utilized as renewable energy resources. Algae have been identified as a promising biomass feedstock because of their high biomass productivity and their non-food-source properties (Mandal and Mallick, 2009). Algae have attracted increasing attention as a sustainable process component for nutrient removal and biofuel production, as well as for mitigation of excessive CO2 production. Biofuel generated from algal treatment of wastewater is a more sustainable fuel while using significantly less energy (Rawat et al., 2011; Satyanarayana et al., 2011; Ehimen et al., 2013). Biogas production from algal biomass by anaerobic digestion is one of the most environmentally beneficial technologies, based on both total produced biomass and ⇑ Corresponding author. Tel.: +82 2 450 3736. E-mail address: [email protected] (K.Y. Park). 0956-053X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.wasman.2013.10.012
the residuals remaining after conversion to biofuel. Anaerobic digestion is a process wherein anaerobic bacteria convert organic matter into biogas. Biogas is a mixture of methane gas (CH4) and carbon dioxide gas (CO2). Natural gas consists of approximately 90–95% methane, but biogas is composed of approximately 50–65% methane, signifying a low-grade natural gas. This biogas can be used as a fuel for heating, in gas engines for electricity and heating, or for upgrading to natural gas quality. Thus, biogas production is an interesting alternative energy because it contributes to not only energy production but also to reducing organic wastes. Methane production from algal biomass has been discussed in the literature, with an emphasis on anaerobic digestion of singlecelled species such as Chlorella spp. (Oswald and Goluke, 1960). However, the practicality of energy generation from algae has been limited because of the economic and energy costs associated with cultivating and harvesting unicellular microalgae species (Sialve et al., 2009). Filamentous algae are easier and less expensive to harvest compared with unicellular algae because of their physical characteristics. Low-cost filtration methods could be used to harvest filamentous algae strains described high rate algae by microstrainer to retain larger cells and washing out smaller non-filamentous algae (Logan and Ronald, 2011). Thus, the use of filamentous algae could potentially improve the economics of energy generation from algae. There are only a few reports on CH4 production using filamentous algae (Samson and LeDuy, 1983). H. reticulatum is a diverse filamentous alga that can be found in
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almost all aquatic environments, and this alga is usually observed as green, thread-like, mat-forming structures floating close to the surface of non-turbulent water bodies (Flory and Hawley, 1994). Owing to its widespread availability, H. reticulatum was used as the biomass in this investigation. In the case of methane fermentation of solid organic materials such as microbial cells, the methane yield is significantly affected by the mass transfer of each biological step as well as by food availability (Izumi et al., 2010). Up to date, however, the literature on anaerobic digestion of microalgae is limited (Passos et al., 2013a). Anaerobic biogas production from algal biomass is impeded by the hard cell wall of microalgae (Chen and Oswald, 1998). Golueke et al. (1957) reported that digested sludge provides a noticeable green color during anaerobic digestion because of the persistence of chlorophyll, which is an intracellular material. This observation suggests that cellular lysis was not completed during digestion, thereby reducing the total biogas production. To improve the overall substrate degradability, pretreatment or disintegration of the algal biomass is required. Various disintegration techniques, including ultrasonography, have been successfully applied as pretreatment methods to enhance anaerobic digestibility (Nickel and Neis, 2007). Biomass disintegration improves the solubility of the sludge particles by disrupting the sludge/flocs in the aqueous phase. The dissolved components can be readily degraded and utilized as substrates in the biological process, thus resulting in increased bioavailability (Park et al., 2004; Lee et al., 2005). Recently, sonication was applied to break down unicellular algal biomass and improve methane production, achieving up to a 60% increase in methane yield (González-Fernández et al., 2012; Alzate et al., 2012). Passos et al., (2013a, 2013b) employed microwave irradiation and thermal treatment to enhance the disintegration and digestibility of microalgae. However, little information is available regarding filamentous algal biomass-disintegrating methods for anaerobic digestion. This technical study aimed at providing preliminary information on methane yields from the anaerobic digestion of the filamentous alga H. reticulatum. An additional aim of this work was to study the effects of different ultrasound doses to algal feed on anaerobic digestion of the filamentous alga H. reticulatum. 2. Materials and methods 2.1. Biomass sources The filamentous alga used in this study, H. reticulatum, was supplied by the Korean Research Institute of Chemical Technology, Daejeon, Korea, and was cultivated in secondary effluent of treated wastewater collected from the municipal wastewater treatment plant in Ansan, Korea (Table 1). H. reticulatum was cultivated in a bench-scale raceway pond with secondary wastewater (Fig. 1). The capacity of the raceway pond was 100 L (width, 0.4 m; length, 1.4 m; depth, 0.5 m) and the pond was constructed and operated under indoor conditions with agitation using a stainless steel paddle wheel. An artificially illuminated raceway reactor was used, and light sources were positioned on both sides of the reactor. Illumination was provided by a light-emitting diode (LED) array. The incident average light intensity was 120 ± 20 lmol m2 s with a 12-h light/dark cycle. The
growth was manually harvested once daily (7.25 g/m2 d) using a sieve. Although the harvested samples contained non-H. reticulatum biomass because of the wastewater cultivation conditions, the H. reticulatum biomass accounted for more than 99% of the total biomass dry weight due to sifting out of small organisms. The harvested samples were initially used in the pretreatment process without cleaning. Prior to their use in the digestion process, the concentration of total solids (TS), total suspended solids (TSS), volatile solids (VS), volatile suspended solids (VSS), soluble COD, and ammonia nitrogen were analyzed (see Fig. 2). 2.2. Pretreatment Blended H. reticulatum samples were further subjected to mechanical disruption using a low frequency ultrasound homogenizer (STH-750S; Sonitopia, Korea). A constant frequency of 20 kHz and an ultrasonic power of 150 W were used. The homogenizer was equipped with a horn (20 123 mm in diameter). The ultrasound dose is related to the amount of energy supplied per unit volume of substrate (expressed in J/mL). However, the dose does not depend on the TS concentration. The ultrasound dose cannot be used to compare substrates with different TS contents. When the TS content remains constant, the ultrasound density is a practical method of expressing the energy input for the disintegration of algal cells on a volume basis. The different ultrasound doses applied to 100 mL of H. reticulatum (10,000 mg/L TS) were 10 J/mL, 20 J/mL, 30 J/mL, 40 J/mL, 50 J/mL, 500 J/mL, 1000 J/mL, 2500 J/mL, and 5000 J/mL. Soluble COD release was used as a direct measurement of H. reticulatum cell disintegration. When the H. reticulatum cells were sonicated, the intracellular contents were released into the aqueous phase. Increased soluble COD after ultrasonic disintegration was an indicator of the cell disintegration efficiency. Samples were filtered through a 0.45 lm membrane and then used for soluble COD measurements. 2.3. Biochemical methane potential (BMP) Batch digestion was performed in a series of BMP assays by incubating algal biomass inoculated with anaerobic bacteria. The active anaerobic inoculum operating at methophilic conditions was obtained from the anaerobic digester at the Ansan municipal wastewater treatment plant in Korea. Feed sludge was also taken to compare digestibility with algal biomass. The inoculum was then filtered by stainless steel filter mesh to prevent inorganic foreign materials, mixed thoroughly, and used for the digestion trials. A nutrient/mineral/buffer (NMB) medium prepared according to Young and Tabak (1993). H. reticulatum samples thawed to room temperature were used in the BMP assays. The BMP assays were performed using sealed 160-mL serum bottles at 35 °C. The produced biogas was measured and was used to represent the BMP. Duplicate units of the digestion setup were used for all pretreatment schemes in this study. Biogas production from the inoculum and medium was recorded and used as the blank. Inoculum and substrate were used at a ratio of 1:1 using the TS mass. Nutrients required for the growth of anaerobic microorganisms were added to each BMP serum bottle (NMB medium) at a volume that was
Table 1 Secondary effluent composition as an algae growth medium.
Concentration (mg/L)
*
Standard deviation.
COD
T-N
Nitrate N
Ammonia N
Nitrite N
T-P
Ortho-phosphate
12.8 (1.9)*
7.7 (0.4)*
3.7 (0.5)*
0.2 (0.1)*
0.04 (0.02)*
1.1 (0.5)*
0.8 (0.2)*
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K. Lee et al. / Waste Management 34 (2014) 1035–1040
3. Results and discussion 3.1. Algal growth Continuous experiments were conducted to study the growth of H. reticulatum in secondary effluent of treated wastewater. The concentrations of nitrogen and phosphorous species over time during the experiments are shown in Fig. 3. Algal treatment offers a cost-effective approach to removing nutrients from tertiary wastewater treatment (Tang et al., 1997). Throughout the experiments, the nitrogen levels of the effluent ranged from 1.5 mg/L to 3.5 mg/L given an initial nitrogen concentration of approximately 7.7 mg/L. Effluent phosphorus showed a value of below 0.4 mg/L, with an initial phosphorus concentration of 1.1 mg/L. The average efficiencies of nitrogen and phosphorus
Concentration (mg/L)
The concentration of TS, TSS, VS, VSS, Nitrate nitrogen, Nitrite nitrogen, Total phosphorus (T-P), ortho-phosphate and COD were analyzed following standard methods (APHA, 1998). Total nitrogen (T-N) was determined in H. reticulatum before ultrasonic disintegration by Persulfate Digestion Method in HACH methods 10072. To study the release of ammonia nitrogen concentration at different ultrasonic dose, the ammonia nitrogen (ammonia N) release after ultrasonic disintegration of H. reticulatum cell was determined by salicylate method. To evaluate the effect of ultrasonication on H. reticulatum cell disintegration, sonicated samples were examined at the cellular level using light microscope. Cell morphology of sonicated samples was compared with that of non-sonicated samples using an optical microscope (DP71-SETModel, Olympus). The composition of the headspace was analyzed toward the end of the experiment via gas chromatography (HP 5890, PA, USA) using a thermal conductivity detector, by injecting gas samples into a packed column (hayesep 3 m 1/8 in. 100/120). The carrier gas was Helium in split less mode (column flow: 19 mL/min). The oven temperature was 35 °C with a retention time of 1.5 min. Injector and detector temperature were 150 °C and 180 °C, respectively. The gas composition was measured toward the end of the experiment.
Harvested algae (g)
2.4. Analytical methods
7 6 5 4 3 2 1 0
(a)
T-N Nitrate N Ammonia N Nitrite N
0.7 0.6 0.5 0.4 0.3 0.2 0.1 0
10000 8000
(b)
T-P
(c)
Harvested algae
Phosphate P
6000 4000 2000 0 0
5
10
15
20
25
Elapsed time (day) Fig. 2. Nitrogen and phosphorus removal and harvested algae during algal treatment.
Cumulative methane production (mL/g-VS)
approximately 3 times the total volume of the added inoculum (Table 1). The serum bottle was then filled to 100 mL with distilled water. Data for both cumulative methane generation and volatile solids reduction (VSR) were obtained during the digestion. The gas production was measured daily by inserting a needle attached to a frictionless syringe through the septum and allowing the headspace to equilibrate to atmospheric pressure. The composition of the headspace was analyzed toward the end of the experiment via gas chromatography.
Concentration (mg/L)
Fig. 1. Image of bench-scale raceway pond. (a) Before operation and (b) during operation.
400 350
Sludge
Algae
300 250 200 150 100 50 0 0
5
10
15
20
25
30
Elapsed time (day) Fig. 3. Methane production from algae and sludge.
removal were 70.5% and 62.9%, respectively. The daily harvested algal biomass was approximately 7.95 g m2 d1. Algal biomass is a potentially important biomass for biofuel production. Algal biomass has widely varying lipid contents, and technologies for lipid
extraction are still under development (Woertz et al., 2009; Fabiana et al., 2013; Alzate et al. 2012). Although relatively few studies have been published on the anaerobic digestion of filamentous microalgae, anaerobic digestion is a promising process that could solve waste issues and provide an economical and energy-balanced technology (Sialve et al., 2009). Fig. 3 shows that batch digestion, using the alga H. reticulatum as feed, was not effective in producing methane, compared with wastewater sludge feed. In this experiment, the methane yield produced by H. reticulatum was 170 mL/g of VS fed, which was lower than that from wastewater sludge. The earliest work on this topic compared the digestion of domestic wastewater sludge and green microalgal biomass, Scenedesmus spp. and Chlorella spp. harvested from wastewater ponds (Golueke et al., 1957), and this early study showed a 32% lower yield from the microalgal biomass compared with the wastewater sludge. 3.2. Effect of ultrasound pretreatment on disintegration of H. reticulatum Ultrasound disintegration technologies focus on H. reticulatum to improve anaerobic digestion in this study. Sonication of H. reticulatum prior to anaerobic digestion significantly enhanced the biodegradability of these cells. Figs. 4 and 5 show the effects of ultrasound pretreatment of filamentous algae: Fig. 4 is a photograph of the microalgae with pretreatment, before (a) and after (b) ultrasound application. A microscopic image of the microalgae shows significant disruption of the H. reticulatum cells. We used 10 different ultrasound doses: 0 J/mL, 10 J/mL, 20 J/mL, 30 J/mL, 40 J/mL, 50 J/mL, 500 J/mL, 1000 J/mL, 2500 J/mL, and 5000 J/mL. The concentration of algae used for the sonication was approximately 10,000 mg/L of TS (8700 mg/L of VS). Both TS and VS of each sample followed a similar trend with different ultrasound doses. The results showed that the concentrations of total suspended solids (TSS) and volatile suspended solids (VSS) decreased by sonication (Fig. 5). When an ultrasound dose of 5000 J/mL was used, the TSS concentration was decreased to 6200 mg/L. These results prove that the insoluble particulate organic matter is transformed into a soluble state by ultrasonic disintegration. The relationship between the ultrasound dose and soluble COD concentration is shown in Fig. 5. The soluble COD concentration increased until an ultrasound dose of 2500 J/mL was used, and the concentration then decreased when the highest dose (5000 J/mL) was applied. This result shows that the concentration of soluble COD increases by sonication and proves that intracellular organic matter is released because of algal cell wall disruption by ultrasound, and then soluble COD destroyed to inorganic at the higher dose of sonication. Ammonia nitrogen released from H. reticulatum was analyzed during sonication. The effects of the ultrasound treatment on the concentration of ammonia N released at different ultrasound doses are shown in Fig. 5. The results show that the ammonia N concentration increases with an increase in the
10000
TSS SCOD
VSS Ammonia N
1500
8000 1000 6000 4000 500 2000 0 0
1000
2000
3000
4000
5000
0 6000
SCOD and Ammonia N (mg/L)
K. Lee et al. / Waste Management 34 (2014) 1035–1040
TSS and VSS (mg/L)
1038
Ultrasound dose (J/mL) Fig. 5. TSS and VSS concentrations using different ultrasound doses.
ultrasound dose. For ultrasound doses of 0 J/mL and 5000 J/mL, the concentrations of ammonia nitrogen released were 80 mg/L and 110 mg/L, respectively. 3.3. Effects of ultrasound pretreatment on the biochemical methane potential The BMP experiment was performed at 35 °C in 160 mL serum bottles. BMP assays were conducted to determine the specific conditions required for optimal methane production of filamentous algae using different ultrasound doses. The 1% TS (8700 mg/L VS) of H. reticulatum was subjected to ultrasound pretreatment before the BMP assay was performed. The inoculum and substrate added to each serum bottle were at a 1:1 ratio based on the TS mass. Fig. 6 shows the methane generation of H. reticulatum. In the first 3 days of the BMP assay, the methane generation rate was low for all tested ultrasound doses because of the lag phase of anaerobic digestion. The rate of biogas generation increased exponentially from the initial day until the generation slowed. At 10 days, the accumulated methane generation was maximal and methane was thereafter produced at a steady generation rate. When the ultrasound dose in range from 10 J/mL to 5000 J/mL, methane gas accumulated up to 313–384 mL/g-VS fed, which is approximately 1.9–2.3 times higher than that obtained with the non-sonicated sample (0 J/mL). Most disintegration technologies focus on sludge pretreatment to increase the overall biodegradability of the substrate. The relative improvement by ultrasound disintegration with respect to gas production from sludge varies from 0% to 60% (Palmowski et al., 2006), and indicates that compared with wastewater sludge, it is easy to decompose H. reticulatum using ultrasound pretreatment. Graph fitting was performed to investigate the BMP and estimated parameters using the modified Gompertz Eq. (1).
Rm e M ¼ P exp exp ðk t Þ þ 1 P
Fig. 4. Image of algae with sonication. (a) Before and (b) after application of an ultrasound dose of 5000 J/mL.
ð1Þ
K. Lee et al. / Waste Management 34 (2014) 1035–1040
Cumulative methane production (mL-CH4/g-VS)
500
results indicate that pretreatment of filamentous algal cells need to optimize anaerobic digestion efficiency of algal biomass.
0 J/mL 10 J/mL 20 J/mL 30 J/mL 40 J/mL 50 J/mL 500 J/mL 1,000 J/mL 2,500 J/mL 5,000 J/mL
400
300
4. Conclusions
200
100
0
0
10
20
30
40
50
Elapsed time (days) Fig. 6. Accumulated biogas production profile with different ultrasound doses.
Table 2 Effect of ultrasound pretreatment on BMP. Ultrasound dose (J/mL)
P Rm k
1039
0
50
500
1000
2500
5000
165.9 8.0 6.2
348.3 21.3 5.1
326.2 20.9 4.9
318.9 18.7 5.5
313.5 16.8 6.1
333.2 20.5 5.2
Methods that use algae have attracted increasing attention as sustainable processes for nutrient removal and biofuel production as well as for mitigation of CO2 generation. Biogas production from algal biomass is one of the most environmentally beneficial technologies, whether using the total produced biomass or the residual fraction remaining after biofuel production. However, biogas production from anaerobic processes is impeded by the rigid algal cell wall. In this study, the alga H. reticulatum was used to treat secondary wastewater and was harvested as a residual. Ultrasonic pretreatment was successfully used for improving the disintegration and anaerobic biodegradability of H. reticulatum. The effect of ultrasonic pretreatment at doses from 10 J/mL to 5000 J/mL applied to H. reticulatum was investigated. The cell disruption efficiency in terms of disintegration degree increased as the applied energy increased. The methane formation efficiency was improved by up to 2.3 times when the ultrasound changed the chemical composition and enhanced the substrate solubility at an energy dose of 40 J/mL. The VS reduction data were also obtained during the batch digestion. The VS reduction observed for the digester using a feed of disintegrated samples was greater than in the control reactor without biomass pretreatment, and the reduction reached up to 67% at an energy dose of 50 J/mL.
⁄
P = methane production potential, Rm = methane production rate, k = lag-phase time.
80 75
400
70 300 65 200 60
Methane Yield VS reduction
100
VS reduction (%)
Methane yield (mL/g-VS)
500
55
0 0
1000
2000
3000
4000
5000
50 6000
Ultrasound dose (J/mL) Fig. 7. Effects of ultrasound pretreatment of VS reduction.
where M = cumulative methane production, k = lag-phase time, P = methane production potential, Rm = methane production rate, and e = exp(1). To compare the progression of digestion of different samples, the determined biogas volume was calculated by non-linear regression analysis using the modified Gompertz equation and SigmaPlot 10.0 (Table 2). The curves showed a good fit between the experimental data and calculated values in all cases (R2 was between 0.984 and 0.997, and p-value was below 0.0001). The cumulative methane production showed that sonication at 40–50 J/mL was adequate for H. reticulatum in terms of maximum methane production potential and methane production rate. These 2 parameters increased by approximately 2-fold while the lag time was shortened. The VS reduction data were also obtained during the digestion (Fig. 7). For the disintegrated samples, VS reduction increased according to the energy input, from 60% to 67%. These
Acknowledgements This study was supported by the Korea Ministry of the Environment (MOE) as an ‘‘Eco-Innovation Project’’ (Project No.: E21240005-0035-0). This work was supported by the Waste to Energy and Recycling Human Resource Development Project (YL-WE-12-001) funded by the Korea MOE. References Alzate, M.E., Munoz, R., Rogalla, F., Fdz-Polanco, F., Pérez-Elvira, S.I., 2012. Biochemical methane potential of microalgae: influence of substrate to inoculum ratio, biomass concentration and pretreatment. Bioresour. Technol. 123, 488–494. APHA, 1998. Standard Methods for Water and Wastewater Examination. American Public Health Association, Washington, DC. Berndes, G., Hoogwijk, M., van den Broek, R., 2003. The contribution of biomass in the future global energy supply: A review of 17 studies. Biomass Bioenerg. 25, 1–28. Chen, P.H., Oswald, W.J., 1998. Thermochemical treatment for algal fermentation. Environ. Int. 24, 889–897. Ehimen, E.A., Holm-Nielsen, J.B., Poulsen, M., Boelsmand, J.E., 2013. Influence of different pre-treatment routs on the anaerobic digestion of a filamentous algae. Renew. Energy 50, 476–480. Fabiana, P., Maria, S., Joan, G., Ivet, F., 2013. Biogas production from microalgae grown in wastewater: effect of microwave pretreatment. Appl. Energy 108, 168–175. Flory, J.E., Hawley, G.R.W., 1994. A Hydrodictyon reticulatum bloom at Loe Pool. Cornwall. Eur. J. Phycol. 29, 17–20. Golueke, C.G., Oswald, W.J., Gottas, H.B., 1957. Anaerobic digestion of algae. Appl. Environ. Microbiol. 5, 47–55. González-Fernández, C., Sialve, B., Bernet, N., Steyer, J.P., 2012. Comparison of ultrasound and thermal pretreatment of Scenedesmus biomass on methane production. Bioresour. Technol. 110, 610–616. Izumi, K., Okishio, Y., Nagao, N., Niwa, C., Yamamoto, S., Toda, T., 2010. Effects of particle size on anaerobic digestion of food waste. Int. Biodeterior. Biodegrad. 64, 601–608. Lee, J.W., Cha, H.Y., Park, K.Y., Song, K.G., Ahn, K.H., 2005. Operational strategies for an activated sludge process in conjunction with ozone oxidation for zero excess sludge production during winter season. Water Res. 39, 1199–1204. Logan, C., Ronald, S., 2011. Production and harvesting of microalgae for wastewater treatment, biofuels, and bioproducts. Biotechnol. Adv. 29, 686–702. Mandal, S., Mallick, N., 2009. Microalga Scenedesmus obliquus as a potential source for biodiesel production. Appl. Microbiol. Biotechnol. 84 (2), 281–291. Nickel, K., Neis, U., 2007. Ultrosonic disintegration of biosolids for improved biodegradation. Ultrasonics sonochemistry 14, 450–455.
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Contents lists available at ScienceDirect
Waste Management journal homepage: www.elsevier.com/locate/wasman
Ultrasound pretreatment of filamentous algal biomass for enhanced biogas production Kwanyong Lee a, Phrompol Chantrasakdakul a, Daegi Kim a, Mingeun Kong b, Ki Young Park a,⇑ a b
Department of Civil and Environmental System Engineering, Konkuk University, 120 Neungdong-ro, Gwangjin-gu, Seoul 143-701, Republic of Korea EPS Solution Ltd., 126-1 Pyeongchon-dong, Dongan-gu, Anyang, Gyeonggi-do 431-755, Republic of Korea
a r t i c l e
i n f o
Article history: Available online 22 November 2013 Keywords: Algae Biogas production Hydrodictyon reticulatum Pretreatment Ultrasound
a b s t r a c t The filamentous alga Hydrodictyon reticulatum harvested from a bench-scale wastewater treatment pond was used to evaluate biogas production after ultrasound pretreatment. The effects of ultrasound pretreatment at a range of 10–5000 J/mL were tested with harvested H. reticulatum. Cell disruption by ultrasound was successful and showed a higher degree of disintegration at a higher applied energy. The range of 10–5000 J/mL ultrasound was able to disintegrated H. reticulatum and the soluble COD was increased from 250 mg/L to 1000 mg/L at 2500 J/mL. The disintegrated algal biomass was digested for biogas production in batch experiments. Both cumulative gas generation and volatile solids reduction data were obtained during the digestion. Cell disintegration due to ultrasound pretreatment increased the specific biogas production and degradation rates. Using the ultrasound approach, the specific methane production at a dose of 40 J/mL increased up to 384 mL/g-VS fed that was 2.3 times higher than the untreated sample. For disintegrated samples, the volatile solids reduction was greater with increased energy input, and the degradation increased slightly to 67% at a dose of 50 J/mL. The results also indicate that disintegration of the algal cells is the essential step for efficient anaerobic digestion of algal biomass. Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction The exhaustion of fossil fuels and the global warming situation are strongly motivating research in alternative energies (Berndes et al., 2003). Many countries are interested in renewable energy sources, especially in sustainable forms of energy, i.e., geothermal power, wind power, small-scale hydropower, solar energy, biomass energy, tidal power, and wave power. Biomass energy is gaining increasing importance because of its environmentally sound and energy-saving production methods (Zheng et al., 2012). Various biomasses derived from the carbonaceous waste of human and natural activities could be utilized as renewable energy resources. Algae have been identified as a promising biomass feedstock because of their high biomass productivity and their non-food-source properties (Mandal and Mallick, 2009). Algae have attracted increasing attention as a sustainable process component for nutrient removal and biofuel production, as well as for mitigation of excessive CO2 production. Biofuel generated from algal treatment of wastewater is a more sustainable fuel while using significantly less energy (Rawat et al., 2011; Satyanarayana et al., 2011; Ehimen et al., 2013). Biogas production from algal biomass by anaerobic digestion is one of the most environmentally beneficial technologies, based on both total produced biomass and ⇑ Corresponding author. Tel.: +82 2 450 3736. E-mail address: [email protected] (K.Y. Park). 0956-053X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.wasman.2013.10.012
the residuals remaining after conversion to biofuel. Anaerobic digestion is a process wherein anaerobic bacteria convert organic matter into biogas. Biogas is a mixture of methane gas (CH4) and carbon dioxide gas (CO2). Natural gas consists of approximately 90–95% methane, but biogas is composed of approximately 50–65% methane, signifying a low-grade natural gas. This biogas can be used as a fuel for heating, in gas engines for electricity and heating, or for upgrading to natural gas quality. Thus, biogas production is an interesting alternative energy because it contributes to not only energy production but also to reducing organic wastes. Methane production from algal biomass has been discussed in the literature, with an emphasis on anaerobic digestion of singlecelled species such as Chlorella spp. (Oswald and Goluke, 1960). However, the practicality of energy generation from algae has been limited because of the economic and energy costs associated with cultivating and harvesting unicellular microalgae species (Sialve et al., 2009). Filamentous algae are easier and less expensive to harvest compared with unicellular algae because of their physical characteristics. Low-cost filtration methods could be used to harvest filamentous algae strains described high rate algae by microstrainer to retain larger cells and washing out smaller non-filamentous algae (Logan and Ronald, 2011). Thus, the use of filamentous algae could potentially improve the economics of energy generation from algae. There are only a few reports on CH4 production using filamentous algae (Samson and LeDuy, 1983). H. reticulatum is a diverse filamentous alga that can be found in
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almost all aquatic environments, and this alga is usually observed as green, thread-like, mat-forming structures floating close to the surface of non-turbulent water bodies (Flory and Hawley, 1994). Owing to its widespread availability, H. reticulatum was used as the biomass in this investigation. In the case of methane fermentation of solid organic materials such as microbial cells, the methane yield is significantly affected by the mass transfer of each biological step as well as by food availability (Izumi et al., 2010). Up to date, however, the literature on anaerobic digestion of microalgae is limited (Passos et al., 2013a). Anaerobic biogas production from algal biomass is impeded by the hard cell wall of microalgae (Chen and Oswald, 1998). Golueke et al. (1957) reported that digested sludge provides a noticeable green color during anaerobic digestion because of the persistence of chlorophyll, which is an intracellular material. This observation suggests that cellular lysis was not completed during digestion, thereby reducing the total biogas production. To improve the overall substrate degradability, pretreatment or disintegration of the algal biomass is required. Various disintegration techniques, including ultrasonography, have been successfully applied as pretreatment methods to enhance anaerobic digestibility (Nickel and Neis, 2007). Biomass disintegration improves the solubility of the sludge particles by disrupting the sludge/flocs in the aqueous phase. The dissolved components can be readily degraded and utilized as substrates in the biological process, thus resulting in increased bioavailability (Park et al., 2004; Lee et al., 2005). Recently, sonication was applied to break down unicellular algal biomass and improve methane production, achieving up to a 60% increase in methane yield (González-Fernández et al., 2012; Alzate et al., 2012). Passos et al., (2013a, 2013b) employed microwave irradiation and thermal treatment to enhance the disintegration and digestibility of microalgae. However, little information is available regarding filamentous algal biomass-disintegrating methods for anaerobic digestion. This technical study aimed at providing preliminary information on methane yields from the anaerobic digestion of the filamentous alga H. reticulatum. An additional aim of this work was to study the effects of different ultrasound doses to algal feed on anaerobic digestion of the filamentous alga H. reticulatum. 2. Materials and methods 2.1. Biomass sources The filamentous alga used in this study, H. reticulatum, was supplied by the Korean Research Institute of Chemical Technology, Daejeon, Korea, and was cultivated in secondary effluent of treated wastewater collected from the municipal wastewater treatment plant in Ansan, Korea (Table 1). H. reticulatum was cultivated in a bench-scale raceway pond with secondary wastewater (Fig. 1). The capacity of the raceway pond was 100 L (width, 0.4 m; length, 1.4 m; depth, 0.5 m) and the pond was constructed and operated under indoor conditions with agitation using a stainless steel paddle wheel. An artificially illuminated raceway reactor was used, and light sources were positioned on both sides of the reactor. Illumination was provided by a light-emitting diode (LED) array. The incident average light intensity was 120 ± 20 lmol m2 s with a 12-h light/dark cycle. The
growth was manually harvested once daily (7.25 g/m2 d) using a sieve. Although the harvested samples contained non-H. reticulatum biomass because of the wastewater cultivation conditions, the H. reticulatum biomass accounted for more than 99% of the total biomass dry weight due to sifting out of small organisms. The harvested samples were initially used in the pretreatment process without cleaning. Prior to their use in the digestion process, the concentration of total solids (TS), total suspended solids (TSS), volatile solids (VS), volatile suspended solids (VSS), soluble COD, and ammonia nitrogen were analyzed (see Fig. 2). 2.2. Pretreatment Blended H. reticulatum samples were further subjected to mechanical disruption using a low frequency ultrasound homogenizer (STH-750S; Sonitopia, Korea). A constant frequency of 20 kHz and an ultrasonic power of 150 W were used. The homogenizer was equipped with a horn (20 123 mm in diameter). The ultrasound dose is related to the amount of energy supplied per unit volume of substrate (expressed in J/mL). However, the dose does not depend on the TS concentration. The ultrasound dose cannot be used to compare substrates with different TS contents. When the TS content remains constant, the ultrasound density is a practical method of expressing the energy input for the disintegration of algal cells on a volume basis. The different ultrasound doses applied to 100 mL of H. reticulatum (10,000 mg/L TS) were 10 J/mL, 20 J/mL, 30 J/mL, 40 J/mL, 50 J/mL, 500 J/mL, 1000 J/mL, 2500 J/mL, and 5000 J/mL. Soluble COD release was used as a direct measurement of H. reticulatum cell disintegration. When the H. reticulatum cells were sonicated, the intracellular contents were released into the aqueous phase. Increased soluble COD after ultrasonic disintegration was an indicator of the cell disintegration efficiency. Samples were filtered through a 0.45 lm membrane and then used for soluble COD measurements. 2.3. Biochemical methane potential (BMP) Batch digestion was performed in a series of BMP assays by incubating algal biomass inoculated with anaerobic bacteria. The active anaerobic inoculum operating at methophilic conditions was obtained from the anaerobic digester at the Ansan municipal wastewater treatment plant in Korea. Feed sludge was also taken to compare digestibility with algal biomass. The inoculum was then filtered by stainless steel filter mesh to prevent inorganic foreign materials, mixed thoroughly, and used for the digestion trials. A nutrient/mineral/buffer (NMB) medium prepared according to Young and Tabak (1993). H. reticulatum samples thawed to room temperature were used in the BMP assays. The BMP assays were performed using sealed 160-mL serum bottles at 35 °C. The produced biogas was measured and was used to represent the BMP. Duplicate units of the digestion setup were used for all pretreatment schemes in this study. Biogas production from the inoculum and medium was recorded and used as the blank. Inoculum and substrate were used at a ratio of 1:1 using the TS mass. Nutrients required for the growth of anaerobic microorganisms were added to each BMP serum bottle (NMB medium) at a volume that was
Table 1 Secondary effluent composition as an algae growth medium.
Concentration (mg/L)
*
Standard deviation.
COD
T-N
Nitrate N
Ammonia N
Nitrite N
T-P
Ortho-phosphate
12.8 (1.9)*
7.7 (0.4)*
3.7 (0.5)*
0.2 (0.1)*
0.04 (0.02)*
1.1 (0.5)*
0.8 (0.2)*
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3. Results and discussion 3.1. Algal growth Continuous experiments were conducted to study the growth of H. reticulatum in secondary effluent of treated wastewater. The concentrations of nitrogen and phosphorous species over time during the experiments are shown in Fig. 3. Algal treatment offers a cost-effective approach to removing nutrients from tertiary wastewater treatment (Tang et al., 1997). Throughout the experiments, the nitrogen levels of the effluent ranged from 1.5 mg/L to 3.5 mg/L given an initial nitrogen concentration of approximately 7.7 mg/L. Effluent phosphorus showed a value of below 0.4 mg/L, with an initial phosphorus concentration of 1.1 mg/L. The average efficiencies of nitrogen and phosphorus
Concentration (mg/L)
The concentration of TS, TSS, VS, VSS, Nitrate nitrogen, Nitrite nitrogen, Total phosphorus (T-P), ortho-phosphate and COD were analyzed following standard methods (APHA, 1998). Total nitrogen (T-N) was determined in H. reticulatum before ultrasonic disintegration by Persulfate Digestion Method in HACH methods 10072. To study the release of ammonia nitrogen concentration at different ultrasonic dose, the ammonia nitrogen (ammonia N) release after ultrasonic disintegration of H. reticulatum cell was determined by salicylate method. To evaluate the effect of ultrasonication on H. reticulatum cell disintegration, sonicated samples were examined at the cellular level using light microscope. Cell morphology of sonicated samples was compared with that of non-sonicated samples using an optical microscope (DP71-SETModel, Olympus). The composition of the headspace was analyzed toward the end of the experiment via gas chromatography (HP 5890, PA, USA) using a thermal conductivity detector, by injecting gas samples into a packed column (hayesep 3 m 1/8 in. 100/120). The carrier gas was Helium in split less mode (column flow: 19 mL/min). The oven temperature was 35 °C with a retention time of 1.5 min. Injector and detector temperature were 150 °C and 180 °C, respectively. The gas composition was measured toward the end of the experiment.
Harvested algae (g)
2.4. Analytical methods
7 6 5 4 3 2 1 0
(a)
T-N Nitrate N Ammonia N Nitrite N
0.7 0.6 0.5 0.4 0.3 0.2 0.1 0
10000 8000
(b)
T-P
(c)
Harvested algae
Phosphate P
6000 4000 2000 0 0
5
10
15
20
25
Elapsed time (day) Fig. 2. Nitrogen and phosphorus removal and harvested algae during algal treatment.
Cumulative methane production (mL/g-VS)
approximately 3 times the total volume of the added inoculum (Table 1). The serum bottle was then filled to 100 mL with distilled water. Data for both cumulative methane generation and volatile solids reduction (VSR) were obtained during the digestion. The gas production was measured daily by inserting a needle attached to a frictionless syringe through the septum and allowing the headspace to equilibrate to atmospheric pressure. The composition of the headspace was analyzed toward the end of the experiment via gas chromatography.
Concentration (mg/L)
Fig. 1. Image of bench-scale raceway pond. (a) Before operation and (b) during operation.
400 350
Sludge
Algae
300 250 200 150 100 50 0 0
5
10
15
20
25
30
Elapsed time (day) Fig. 3. Methane production from algae and sludge.
removal were 70.5% and 62.9%, respectively. The daily harvested algal biomass was approximately 7.95 g m2 d1. Algal biomass is a potentially important biomass for biofuel production. Algal biomass has widely varying lipid contents, and technologies for lipid
extraction are still under development (Woertz et al., 2009; Fabiana et al., 2013; Alzate et al. 2012). Although relatively few studies have been published on the anaerobic digestion of filamentous microalgae, anaerobic digestion is a promising process that could solve waste issues and provide an economical and energy-balanced technology (Sialve et al., 2009). Fig. 3 shows that batch digestion, using the alga H. reticulatum as feed, was not effective in producing methane, compared with wastewater sludge feed. In this experiment, the methane yield produced by H. reticulatum was 170 mL/g of VS fed, which was lower than that from wastewater sludge. The earliest work on this topic compared the digestion of domestic wastewater sludge and green microalgal biomass, Scenedesmus spp. and Chlorella spp. harvested from wastewater ponds (Golueke et al., 1957), and this early study showed a 32% lower yield from the microalgal biomass compared with the wastewater sludge. 3.2. Effect of ultrasound pretreatment on disintegration of H. reticulatum Ultrasound disintegration technologies focus on H. reticulatum to improve anaerobic digestion in this study. Sonication of H. reticulatum prior to anaerobic digestion significantly enhanced the biodegradability of these cells. Figs. 4 and 5 show the effects of ultrasound pretreatment of filamentous algae: Fig. 4 is a photograph of the microalgae with pretreatment, before (a) and after (b) ultrasound application. A microscopic image of the microalgae shows significant disruption of the H. reticulatum cells. We used 10 different ultrasound doses: 0 J/mL, 10 J/mL, 20 J/mL, 30 J/mL, 40 J/mL, 50 J/mL, 500 J/mL, 1000 J/mL, 2500 J/mL, and 5000 J/mL. The concentration of algae used for the sonication was approximately 10,000 mg/L of TS (8700 mg/L of VS). Both TS and VS of each sample followed a similar trend with different ultrasound doses. The results showed that the concentrations of total suspended solids (TSS) and volatile suspended solids (VSS) decreased by sonication (Fig. 5). When an ultrasound dose of 5000 J/mL was used, the TSS concentration was decreased to 6200 mg/L. These results prove that the insoluble particulate organic matter is transformed into a soluble state by ultrasonic disintegration. The relationship between the ultrasound dose and soluble COD concentration is shown in Fig. 5. The soluble COD concentration increased until an ultrasound dose of 2500 J/mL was used, and the concentration then decreased when the highest dose (5000 J/mL) was applied. This result shows that the concentration of soluble COD increases by sonication and proves that intracellular organic matter is released because of algal cell wall disruption by ultrasound, and then soluble COD destroyed to inorganic at the higher dose of sonication. Ammonia nitrogen released from H. reticulatum was analyzed during sonication. The effects of the ultrasound treatment on the concentration of ammonia N released at different ultrasound doses are shown in Fig. 5. The results show that the ammonia N concentration increases with an increase in the
10000
TSS SCOD
VSS Ammonia N
1500
8000 1000 6000 4000 500 2000 0 0
1000
2000
3000
4000
5000
0 6000
SCOD and Ammonia N (mg/L)
K. Lee et al. / Waste Management 34 (2014) 1035–1040
TSS and VSS (mg/L)
1038
Ultrasound dose (J/mL) Fig. 5. TSS and VSS concentrations using different ultrasound doses.
ultrasound dose. For ultrasound doses of 0 J/mL and 5000 J/mL, the concentrations of ammonia nitrogen released were 80 mg/L and 110 mg/L, respectively. 3.3. Effects of ultrasound pretreatment on the biochemical methane potential The BMP experiment was performed at 35 °C in 160 mL serum bottles. BMP assays were conducted to determine the specific conditions required for optimal methane production of filamentous algae using different ultrasound doses. The 1% TS (8700 mg/L VS) of H. reticulatum was subjected to ultrasound pretreatment before the BMP assay was performed. The inoculum and substrate added to each serum bottle were at a 1:1 ratio based on the TS mass. Fig. 6 shows the methane generation of H. reticulatum. In the first 3 days of the BMP assay, the methane generation rate was low for all tested ultrasound doses because of the lag phase of anaerobic digestion. The rate of biogas generation increased exponentially from the initial day until the generation slowed. At 10 days, the accumulated methane generation was maximal and methane was thereafter produced at a steady generation rate. When the ultrasound dose in range from 10 J/mL to 5000 J/mL, methane gas accumulated up to 313–384 mL/g-VS fed, which is approximately 1.9–2.3 times higher than that obtained with the non-sonicated sample (0 J/mL). Most disintegration technologies focus on sludge pretreatment to increase the overall biodegradability of the substrate. The relative improvement by ultrasound disintegration with respect to gas production from sludge varies from 0% to 60% (Palmowski et al., 2006), and indicates that compared with wastewater sludge, it is easy to decompose H. reticulatum using ultrasound pretreatment. Graph fitting was performed to investigate the BMP and estimated parameters using the modified Gompertz Eq. (1).
Rm e M ¼ P exp exp ðk t Þ þ 1 P
Fig. 4. Image of algae with sonication. (a) Before and (b) after application of an ultrasound dose of 5000 J/mL.
ð1Þ
K. Lee et al. / Waste Management 34 (2014) 1035–1040
Cumulative methane production (mL-CH4/g-VS)
500
results indicate that pretreatment of filamentous algal cells need to optimize anaerobic digestion efficiency of algal biomass.
0 J/mL 10 J/mL 20 J/mL 30 J/mL 40 J/mL 50 J/mL 500 J/mL 1,000 J/mL 2,500 J/mL 5,000 J/mL
400
300
4. Conclusions
200
100
0
0
10
20
30
40
50
Elapsed time (days) Fig. 6. Accumulated biogas production profile with different ultrasound doses.
Table 2 Effect of ultrasound pretreatment on BMP. Ultrasound dose (J/mL)
P Rm k
1039
0
50
500
1000
2500
5000
165.9 8.0 6.2
348.3 21.3 5.1
326.2 20.9 4.9
318.9 18.7 5.5
313.5 16.8 6.1
333.2 20.5 5.2
Methods that use algae have attracted increasing attention as sustainable processes for nutrient removal and biofuel production as well as for mitigation of CO2 generation. Biogas production from algal biomass is one of the most environmentally beneficial technologies, whether using the total produced biomass or the residual fraction remaining after biofuel production. However, biogas production from anaerobic processes is impeded by the rigid algal cell wall. In this study, the alga H. reticulatum was used to treat secondary wastewater and was harvested as a residual. Ultrasonic pretreatment was successfully used for improving the disintegration and anaerobic biodegradability of H. reticulatum. The effect of ultrasonic pretreatment at doses from 10 J/mL to 5000 J/mL applied to H. reticulatum was investigated. The cell disruption efficiency in terms of disintegration degree increased as the applied energy increased. The methane formation efficiency was improved by up to 2.3 times when the ultrasound changed the chemical composition and enhanced the substrate solubility at an energy dose of 40 J/mL. The VS reduction data were also obtained during the batch digestion. The VS reduction observed for the digester using a feed of disintegrated samples was greater than in the control reactor without biomass pretreatment, and the reduction reached up to 67% at an energy dose of 50 J/mL.
⁄
P = methane production potential, Rm = methane production rate, k = lag-phase time.
80 75
400
70 300 65 200 60
Methane Yield VS reduction
100
VS reduction (%)
Methane yield (mL/g-VS)
500
55
0 0
1000
2000
3000
4000
5000
50 6000
Ultrasound dose (J/mL) Fig. 7. Effects of ultrasound pretreatment of VS reduction.
where M = cumulative methane production, k = lag-phase time, P = methane production potential, Rm = methane production rate, and e = exp(1). To compare the progression of digestion of different samples, the determined biogas volume was calculated by non-linear regression analysis using the modified Gompertz equation and SigmaPlot 10.0 (Table 2). The curves showed a good fit between the experimental data and calculated values in all cases (R2 was between 0.984 and 0.997, and p-value was below 0.0001). The cumulative methane production showed that sonication at 40–50 J/mL was adequate for H. reticulatum in terms of maximum methane production potential and methane production rate. These 2 parameters increased by approximately 2-fold while the lag time was shortened. The VS reduction data were also obtained during the digestion (Fig. 7). For the disintegrated samples, VS reduction increased according to the energy input, from 60% to 67%. These
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