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Effect of thermal–alkaline pretreatment on the anaerobic digestion of streptomycin bacterial residues for methane production
Bioresource Technology 151 (2014) 436–440
Contents lists available at ScienceDirect
Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Short Communication
Effect of thermal–alkaline pretreatment on the anaerobic digestion of streptomycin bacterial residues for methane production Weizhang Zhong a, Zaixing Li a,⇑, Jingliang Yang a, Chun Liu a, Baokuo Tian a, Yongjun Wang b, Ping Chen b a b
School of Environmental Science and Engineering, Hebei University of Science and Technology, Yuhua East Road, Shijiazhuang 050018, China Environmental Protection Institute of NCPC, Shijiazhuang 050015, China
h i g h l i g h t s Anaerobic digestion was a solution with streptomycin bacterial residue treatments. Thermal–alkaline pretreatment can enhanced methane production significantly. Thermal–alkaline pretreatment can enhance reactor OLR by more than double.
a r t i c l e
i n f o
Article history: Received 1 September 2013 Received in revised form 26 October 2013 Accepted 28 October 2013 Available online 6 November 2013 Keywords: Streptomycin bacterial residue Anaerobic digestion Methane Thermal–alkaline pretreatment
a b s t r a c t The anaerobic digestion of streptomycin bacterial residues, solutions with hazardous waste treatments and bioenergy recovery, was tested in laboratory-scale digesters at 35 °C at various organic loading rates (OLRs). The methane production and biomass digestion were efficient at OLRs below 2.33 gVS L1 d1 but were deteriorated as OLR increased because of the increased total ammonia nitrogen (TAN) concentration from cell protein decay. The thermal–alkaline pretreatment with 0.10 NaOH/TS at 70 °C for 2 h significantly improved the digestion performance. With the thermal–alkaline pretreatment, the volumetric reactor productivity and specific methane yield of the pretreated streptomycin bacterial residue increased by 22.08–27.08% compared with those of the unpretreated streptomycin bacterial residue at an OLR of 2.33 gVS L1 d1. The volatile solid removal was 64.09%, with less accumulation of TAN and total volatile fatty acid. Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction The biopharmaceutical industry has important contributions to the national economy of China. In 2009, China produced 14.7 million tons of antibiotics, accounting for >70% of the total global market and ranking first in the world. Bulk (90%) of the antibiotics globally produced by China is streptomycin. A mass of waste bacterial residues is generated during the production of streptomycin. Bacterial residues were previously used as food additives and fertilizers. However, doubts have been raised on the suitability of streptomycin bacterial residues as feedstock and fertilizers because of their small amount of antibiotics and considerable amount of degradation products. Streptomycin bacterial residues are considered hazardous wastes in China. Therefore, bacterial residues should be managed strictly in accordance with the hazardous waste regulations (Guo et al., 2012; Li et al., 2012a,b). Incineration and landfill are currently the main treatment and disposal
⇑ Corresponding author. Tel./fax: +86 0311 88632210. E-mail address: [email protected] (Z. Li). 0960-8524/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biortech.2013.10.100
technologies for bacterial residues. However, these methods have significant disadvantages, such as high cost and serious secondary environmental pollution (Cassone et al., 2012). Therefore, the development of safe and effective treatments for streptomycin bacterial residues is important. Anaerobic digestion, coupled with energy production in the form of methane and waste treatment, has been employed to treat several types of organic wastes. Bacterial residues contain rich high-quality protein, fat, cellulose, enzymes, and other components, which can be used for methane by anaerobic digestion (Guo et al., 2012). The feasibility of the anaerobic digestion of various bacterial residues has been investigated by many researchers (Li et al., 1992; Li and He, 1988). However, microbial cell walls contain glycan strands crosslinked by peptide chains, causing resistance to biodegradation. Thermal–alkaline pretreatment promotes the dissolution and decomposition of microbial cells and organic material to improve anaerobic digestibility (Bougrier et al., 2008; Skiadas et al., 2005). Our previous study (Li et al., 2012a) showed that the optimal conditions of the thermal–alkaline pretreatment of streptomycin bacterial residues are 0.10 NaOH/TS, 70 °C, and 2 h.
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W. Zhong et al. / Bioresource Technology 151 (2014) 436–440
In this study, we investigated the effect of thermal–alkaline pretreatment on the anaerobic digestion of streptomycin bacterial residues at various organic loading rates (OLRs) in laboratory-scale digesters with continuous feed mode. The results demonstrated that thermal–alkaline pretreatment can significantly enhance methane yield. The loading rate limitation of the anaerobic digestion of streptomycin bacterial residues was due to ammonia accumulation. The optimal hydraulic retention time (HRT) and OLR of the anaerobic digestion of streptomycin bacterial residues were identified for further application and reactor configuration modification.
3
1
2
4
5
6 2. Methods 2.1. Feedstock Streptomycin bacterial residues were obtained from the North China Pharmaceutical Company, Hebei, China and were stored in a refrigerator at 4 °C prior to use. According to our previous study (Li et al., 2012a), we performed the thermal–alkaline pretreatment under the optimal conditions of 0.10 NaOH/TS, 70 °C, and 2 h. The characteristics of the streptomycin bacterial residues are listed in Table 1.
7
Fig. 1. Laboratory scale CSTR digesters with 5.0 L working volume. (1. Time controller; 2. Speed governor; 3. Stirrer; 4. Wet gas flow meter; 5. Temperature controller; 6. Outlet).
2.2. Reactor and operational procedures
2.3. Analytical methods
Two identically sized completely stirred tank reactor (CSTR) digesters with a working volume of 5 L (14 cm ID 45 cm height) were fabricated with a double-wall Perspex cylinder (15 mm thickness) (Fig. 1). The temperature was maintained at 35 °C by recirculating the temperature-controlled water through the water jacket of the reactor. The reactors were sealed with a Perspex cover, which had three ports on the top. The first port was for substrate feeding. The second port was connected to a wet gas flow meter (LML-2, Jinzhiye Watch Industry Co., Beijing, China). The gas sample was collected via a silicone tube using a 10 lL pressure-tight gas syringe (104, Techcomp, China). The third port was inserted with a high-power magnetic stirrer (JB-2, Jintan Ronghua Instrument Factory, China) for intermittent mixing at 50 rpm for 24 min every 2 h. The daily biogas production was recorded using the gas flow meter (Fig. 1). The inoculum of the anaerobic reactor was anaerobic granular sludge from the streptomycin wastewater treatment plants of North China Pharmaceutical Company, Hebei, China. The sludge contained 50.76 g L1 total solid (TS) and 35.95 g L1 volatile solid (VS). The experimental results (average values of the two reactors) were collected at the steady state, i.e., a constant CH4 production (<10% of deviation) for 5 d. The samples removed from the reactors were stored at 4 °C prior to analysis. The desired OLRs were 1.11, 2.33, and 3.68 gVS L1 d1, and the related HRTs were 50, 25, and 12.5 d, respectively. Experiments were conducted for 75, 125, and 42 d for each test condition. Table 1 Characteristics of the streptomycin bacterial residue. Constituent
Streptomycin bacterial residue
TS (g/L) VS (%-TS) pH TNK (mg/L) Protein (%-TS) Total chemical oxygen demand (mg/L) Soluble chemical oxygen demand (mg/L) Moisture content (%) TAN (mg/L)
55.62 (2.64) 84.90 (7.34) 2.90 (0.28) 5373 (379) 60.37 (5.76) 53,854 (6204) 5593 (745) 93.87 (1.20) 168 (23)
Biogas volume was monitored daily using the wet gas flow meter. The measured volume was converted to that at standard temperature (0 °C) and pressure (1 atm) conditions. CH4 and CO2 were analyzed using a gas chromatograph (GC7900, Techcomp, China) equipped with a packed column (Shimadzu, 60 1.800 OD, 80/100, Japan) and a thermal conductivity detector. CH4 production was determined by multiplying the CH4 content with the volume of the biogas produced. Gas samples were injected directly using the 10 lL pressure-tight gas syringe after sampling. The pH value was directly measured with a Mettler-Toledo pH/conductivity meter using a combined pH electrode LE409 (FE20, Mettler-Toledo, Switzerland). TS, VS, total nitrogen Kjeldahl (TNK), and total ammonia nitrogen (TAN) were measured following previously described methods (Li et al., 2012a). The total chemical oxygen demand (COD) and soluble COD were measured with COD analysis systems (Lianhua Technology Company, China) composed of a spectrophotometer (5B-3), a COD reactor (5B-1), and a COD digestion reagent following the manufacturer’s instructions. Analysis of the liquid composition of digester content, including total volatile fatty acids (TVFAs) and ammonia, was conducted in triplicate using the supernatant of mixed slurry after centrifugation (SIGMA, 10,000 rpm for 30 min at 2 °C). Volatile fatty acid (VFA) analysis using gas chromatography was performed as previously described (Zhong et al., 2012). The concentration of TVFA was calculated by summarizing all VFAs. TAN was measured in triplicate according to the APHA standard methods (APHA, 1998). Each analytical result was the mean value of at least three measurements. 3. Results and discussion 3.1. Characterization of biomass The characteristics of the streptomycin bacterial residues are summarized in Table 1. The streptomycin bacterial residue biomass generally contains 85% VS in TS and is an attractive substrate for anaerobic digestion. The TNK (5373 mg L1) and protein
W. Zhong et al. / Bioresource Technology 151 (2014) 436–440
Anaerobic digestion with unpretreated and pretreated streptomycin bacterial residues as feedstock was performed in the two reactors at different OLRs of 1.11, 2.33, and 3.68 gVS L1 d1. The loading rates were maintained for 75, 125, and 42 d to collect data under steady state condition. The major operational results are summarized in Fig. 2 and Table 2. Volumetric reactor productivity (VRP), expressed in mL CH4 L1 d1, indicates the productivity of digester in term of the volume of methane per unit reactor working volume per day. As shown in Fig. 2(a), the VRP of the unpretreated and pretreated streptomycin bacterial residues at an OLR of 2.33 gVS L1 d1 were 412 and 496 mL CH4 L1 d1, respectively. These values were respectively 56.65% and 5.64% higher than those of the residues at an OLR of 1.11 gVS L1 d1 and 55.25% and 1.41% higher than those of the residues at an OLR of 3.68 gVS L1 d1. The VRPs of the pretreated streptomycin bacterial residue at each OLR were all larger than those of the unpretreated one. Specific methane yield (SMY), expressed in mL CH4 gVS1, was used to determine the rate of biomass converted to methane. Higher VRP and SMY mean better performance of an anaerobic digester (Park and Li, 2012). As shown in Fig. 2(b), SMY decreased as OLR increased from 1.11 gVS L1 d1 to 3.68 gVS L1 d1 for both unpretreated and pretreated streptomycin bacterial residues. The highest SMYs of the unpretreated and pretreated streptomycin bacterial residues were 237 and 292 mL CH4 gVS1 d1 at an OLR of 1.11 gVS L1 d1, respectively. At an OLR of 2.33 gVS L1 d1, the SMYs of the unpretreated and pretreated streptomycin bacterial residues were 177 and 216 mL CH4 gVS1 d1, respectively. The thermal–alkaline pretreatment increased the VRP and SMY of the pretreated streptomycin bacterial residue by 22.08–27.08% compared with those of the unpretreated streptomycin bacterial residue. The maximum possible methane yield is 350 mL CH4 gCOD1 during the anaerobic treatment of wastes at standard temperature and pressure (Zhang et al., 2010). Methane was generated from the biological conversion of substrate. The amount of organic compounds, which are represented by VS, will be reduced. A close relationship exists between the methane yield and VS reduction. The VS removal efficiency indicates the biodegradation extent of the feedstock added. The % VS reduction is presented in Fig. 2(c). The VS removal efficiency was the highest (80%) at 1.11 gVS L1 d1 but gradually declined as the OLR increased. This result suggests that the hydrolysis-fermentative activities may approach their limitation. The mass ratio of mL methane produced per gCOD removed ranged from 322 mL CH4 gCOD1 to 378 mL CH4 gCOD1. This result supports the initial hypothesis that the thermal–alkaline pretreatment at 0.10 NaOH/TS, 70 °C, and 2 h enhances the solubilization of solids, thereby increasing the digestibility of streptomycin bacterial residues. During the entire experimental period, the pH value was between 7.3 and 7.9 (Table 2), which agreed with the normal growth of anaerobic microorganisms. The high buffering capacity of the system could sustain the volatile acid production and the high and stable pH (7.0–7.4) over a wide range of experimental conditions. The energy contained in biogas was determined by both biogas volume and methane content. The methane contents for all
VRP (mL CH4 L–1 d–1)
3.2. Effects of thermal–alkaline pretreatment on methane production yields in the anaerobic digestion of streptomycin bacterial residues at various OLRs
(a)
600 Unpretreated Pretreated
500 400 300 200 100 0
(b)
Unpretreated Pretreated
300 250
SMY (mL CH4 gVS–1)
contents in the streptomycin bacterial residue biomass were high, in which the latter was estimated to be 60.37% of the TS based on the conversion factor of 6.25 g protein g1 TNK. High protein content likely causes significant ammonia inhibition if the protein is completely degraded.
200 150 100 50
(c)
0 100 Unpretreated Pretreated
80 VS removal (%)
438
60
40
20
0
1.11
2.33
3.68
Organic loading rate (gVS L–1 d–1) Fig. 2. The VRP (a), SMY (b) and VS removal (c) of unpretreated and pretreated streptomycin bacterial residue at different OLR and HRT. VRP = Volumetric reactor methane productivity. SMY = Specific methane yield.
digesters were measured during the entire digestion period. The total methane production for each feedstock was calculated by adding the daily methane production, which was calculated by multiplying the daily biogas production by the corresponding methane content. The methane content in biogas slightly declined from 60% to 55% at the OLR range of 1.11–3.68 gVS L1 d1 (Table 2). The methane contents from the pretreated streptomycin bacterial residue at each OLR were all higher than those from the unpretreated streptomycin bacterial residue. As one of the most important parameters for the accurate control of anaerobic digestion, the concentrations of TVFAs have a direct correlation with the digester performance (Siegert and Banks, 2005). As shown in Table 2, the VFAs increased with increasing OLR. The TVFA concentration increased sharply from
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W. Zhong et al. / Bioresource Technology 151 (2014) 436–440 Table 2 The reactor performance with the streptomycin bacterial residue as feedstock at different organic loading rates (OLRs). OLR (gVS L1 d1) 1.11 2.33 3.68
pH Unpretreated Pretreated Unpretreated Pretreated Unpretreated Pretreated
7.69 7.83 7.34 7.53 7.45 7.55
(0.27) (0.27) (0.15) (0.12) (0.27) (0.21)
approximately 1800–5000 mg L1 as OLR increased from 1.11 gVS L1 d1 to 2.33 gVS L1 d1 and up to approximately 6000 mg L1 at 3.68 gVS L1 d1. The TVFA levels at all OLRs did not change significantly and varied below 5000 mg L1, suggesting no inhibition of their degradation. Further increase in OLR would push the VFA concentration to reach toxicity level. Although the inhibitory concentrations of ammonia differ per experiment, the concentration range of 1.7–5 g TAN L1 shows the highest inhibitory effect on anaerobic digestion (Stams et al., 2003). As shown in Table 2, the concentration of TAN was approximately 1600 mg L1 at an OLR of 1.11 gVS L1 d1, increased to approximately 2200 mg L1 at an OLR of 2.33 gVS L1 d1, and then further increased to 2900 mg L1 at an OLR of 3.68 gVS L1 d1. These values belong to the inhibitory level for methanogenesis at 4.0 g VS L1 d1 (Appels et al., 2008). The anaerobic digestion of organic matter with a high nitrogen content (or low C/N ratio) could be susceptible to ammonia inhibition because of the accumulation of ammonia from protein degradation, causing reaction failure. Depending on pH, TAN in digester is composed of different fractions of free ammonia (NH3) and ammonium ions (NHþ 4 ), of which NH3 is the most toxic.
NH3ðaqÞ þ H2 O $ NHþ4 þ OH ðpK b ¼ 4:733 at 35 CÞ
ð1Þ
TAN inhibits methanogens at the concentration range of 15,000–3000 mg L1 at pH 7.2–7.6. The toxicity level increases with increasing pH because of the shift to a higher fraction of NH3 (Appels et al., 2008). In the present study, the increase in TAN was due to more protein in the streptomycin bacterial residue degraded at higher OLR. Longer HRT might improve the residual TVFA and VS removal efficiency of the reactor; however, it might also elicit negative effects because the level of ammonia accumulated not only depends on OLR and TNK content but also on HRT. At the same OLR, long HRT could promote the accumulation of TAN in a CSTR. To avoid ammonia inhibition, a CSTR digester must receive an OLR with low ammonia-producing potential, such as 2.33 gVS L1 d1 in this study, or short HRT. A large reactor size is needed to operate at low OLR, except for the separation of the solid phase from the liquid phase, such as in high-rate upflow anaerobic sludge blanket. The major investment in anaerobic digestion is the reactor capital cost. Therefore, introducing reasonably short HRT and applying high OLR to minimize the reactor volume are economically sound in the design of digestion process. The results of the present study demonstrated the following results. First, anaerobic digestion of the streptomycin bacterial residue can be performed well at a normal OLR of 2.33 gVS L1 d1 without ammonia inhibition. Second, thermal–alkaline pretreatment of the streptomycin bacterial residue can enhance the reactor OLR by more than double. The pretreated streptomycin bacterial residue had higher VS removal efficiency and lower TVFA than the unpretreated streptomycin bacterial residue. The above findings can be used for the scale-up and process design. However, the low C/N ratio of streptomycin bacterial residues is a serious problem that limits anaerobic digestion. The C/N ratio of streptomycin bacterial residues is significantly low for
TVFAs (mg L1)
TAN (mg L1)
CH4 content (%)
1838 1574 4209 5042 5057 6054
1682 1597 2256 2236 2874 2880
58.27 62.19 56.31 59.84 55.07 55.72
(162) (371) (552) (507) (311) (364)
(22) (99) (313) (315) (252) (253)
(3.73) (6.19) (4.26) (4.87) (3.72) (4.03)
anaerobic digestion (<10, 20/1 to 30/1 is the most acceptable range). Low C/N ratio feedstock could result in the release of TAN and the accumulation of VFAs in the digester. Excessive ammonia accumulation can be avoided by adding high carboncontent materials to adjust low feedstock C/N ratios and thus improve the digestion performance. The codigestion of streptomycin bacterial residue with low C/N ratio and crop residues with high C/N ratio is a good solution to the above problem. Crop residues, such as corn and wheat straws, are produced in large quantities in China and other countries every year. These organic crop residues can be valuable alternative feedstock for methane production (Zhong et al., 2012). According to Mata-Alvarez et al. (2000), the digestion of more than one type of substrate in the same digester could establish positive synergism in the digester, i.e., the added nutrients would support the microbial growth. The benefits of codigestion include the dilution of potentially toxic ammonia, allowance for increased loading rate, improved methane yield, economic advantages derived from the sharing of equipment, easier handling of mixed wastes, and synergistic effects. Therefore, further research is still needed to test the cofeedstocks and scale-up of the reactor. 4. Conclusion The anaerobic digestion of streptomycin bacterial residue is feasible in laboratory-scale anaerobic reactors. Thermal–alkaline pretreatment can significantly enhance methane production. The VRP and SMY of the streptomycin bacterial residue thermal–alkaline pretreated at 0.10 NaOH/TS, 70 °C, and 2 h increased by 22.08– 27.08% compared with those of the unpretreated streptomycin bacterial residue. The reactors performed well at an OLR of 2.33 gVS L1 d1 for methane production, with a VS removal of 51.03–64.09% at an HRT of 25 d without ammonia inhibition. At all OLRs, the levels of pH values and TVFAs did not change significantly, suggesting no inhibition of their degradation. Acknowledgement This research was supported by the Science and Technology Department of Hebei Province (No. 12276703Z). References APHA, 1998. Standard Methods for the Examination of Water and Wastewater. 20th ed. American Public Health Association, Washington, DC. Appels, L., Baeyens, J., Degrève, J., Dewil, R., 2008. Principles and potential of the anaerobic digestion of waste-activated sludge. Prog. Energy Combust. Sci. 34 (6), 755–781. Bougrier, C., Delgenès, J.P., Carrère, H., 2008. Effects of thermal treatments on five different waste activated sludge samples solubilisation, physical properties and anaerobic digestion. Chem. Eng. J. 139 (2), 236–244. Cassone, C.G., Vongphachan, V., Chiu, S., Williams, K.L., Letcher, R.J., Pelletier, E., Crump, D., Kennedy, S.W., 2012. In ovo effects of perfluorohexane sulfonate and perfluorohexanoate on pipping success, development, mrna expression, and thyroid hormone levels in chicken embryos. Toxicol. Sci. 127 (1), 216– 224. Guo, B., Gong, L., Duan, E., Liu, R., Ren, A., Han, J., Zhao, W., 2012. Characteristics of penicillin bacterial residue. J. Air Waste Manage. Assoc. 62 (4), 485–488.
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Li, L.M., Chen, Y., Ling, G.C., Wu, Z.Z., 1992. Studies on anaerobic biological treatment of rifampicin mycelia. Chin. J. Liaoning Univ 19 (3), 70–74. Li, S., He, C., 1988. A study of biogas formation of distillers’grains and residue of kanamycin fermentation. J. Microbiol. 8 (2), 11–15. Li, Z., Zuo, J., Tian, B., Yang, J., Yu, X., Chen, P., Zhao, Y., 2012a. Thermal–alkaline pretreatment on the decomposition of the streptomycin bacterial residue. Biotechnol. Biotechnol. Equip. 26 (3), 2971–2975. Li, Z.X., Tian, B.K., Zuo, J.E., Yu, X., Shen, H.Y., Wang, Y.J., Zhao, X.M., 2012b. Progress in treatment and disposal technology of antibiotic bacterial residues. Environ. Eng. 30 (2), 72–75 (in Chinese). Mata-Alvarez, J., Macé, S., Llabrés, P., 2000. Anaerobic digestion of organic solid wastes. An overview of research achievements and perspectives. Bioresour. Technol. 74 (1), 3–16. Park, S., Li, Y., 2012. Evaluation of methane production and macronutrient degradation in the anaerobic co-digestion of algae biomass residue and lipid waste. Bioresour. Technol. 111, 42–48.
Siegert, I., Banks, C., 2005. The effect of volatile fatty acid additions on the anaerobic digestion of cellulose and glucose in batch reactors. Process Biochem. 40 (11), 3412–3418. Skiadas, I.V., Gavala, H.N., Lu, J., Ahring, B.K., 2005. Thermal pre-treatment of primary and secondary sludge at 70 °C prior to anaerobic digestion. Water Sci. Technol. 52 (1–2), 161–166. Stams, A.J.M., Oude Elferink, S.J.W.H., Westermann, P., 2003. Metabolic interactions between methanogenic consortia and anaerobic respiring bacteria. Advances in Biochemical Engineering/Biotechnology 81, 31–56. Zhang, D., Chen, Y.G., Zhao, Y.X., Zhu, X.Y., 2010. New sludge pretreatment method to improve methane production in waste activated sludge digestion. Environ. Sci. Technol. 44 (12), 4802–4808. Zhong, W., Zhang, Z., Luo, Y., Qiao, W., Xiao, M., Zhang, M., 2012. Biogas productivity by co-digesting Taihu blue algae with corn straw as an external carbon source. Bioresour. Technol. 114, 281–286.
Contents lists available at ScienceDirect
Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Short Communication
Effect of thermal–alkaline pretreatment on the anaerobic digestion of streptomycin bacterial residues for methane production Weizhang Zhong a, Zaixing Li a,⇑, Jingliang Yang a, Chun Liu a, Baokuo Tian a, Yongjun Wang b, Ping Chen b a b
School of Environmental Science and Engineering, Hebei University of Science and Technology, Yuhua East Road, Shijiazhuang 050018, China Environmental Protection Institute of NCPC, Shijiazhuang 050015, China
h i g h l i g h t s Anaerobic digestion was a solution with streptomycin bacterial residue treatments. Thermal–alkaline pretreatment can enhanced methane production significantly. Thermal–alkaline pretreatment can enhance reactor OLR by more than double.
a r t i c l e
i n f o
Article history: Received 1 September 2013 Received in revised form 26 October 2013 Accepted 28 October 2013 Available online 6 November 2013 Keywords: Streptomycin bacterial residue Anaerobic digestion Methane Thermal–alkaline pretreatment
a b s t r a c t The anaerobic digestion of streptomycin bacterial residues, solutions with hazardous waste treatments and bioenergy recovery, was tested in laboratory-scale digesters at 35 °C at various organic loading rates (OLRs). The methane production and biomass digestion were efficient at OLRs below 2.33 gVS L1 d1 but were deteriorated as OLR increased because of the increased total ammonia nitrogen (TAN) concentration from cell protein decay. The thermal–alkaline pretreatment with 0.10 NaOH/TS at 70 °C for 2 h significantly improved the digestion performance. With the thermal–alkaline pretreatment, the volumetric reactor productivity and specific methane yield of the pretreated streptomycin bacterial residue increased by 22.08–27.08% compared with those of the unpretreated streptomycin bacterial residue at an OLR of 2.33 gVS L1 d1. The volatile solid removal was 64.09%, with less accumulation of TAN and total volatile fatty acid. Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction The biopharmaceutical industry has important contributions to the national economy of China. In 2009, China produced 14.7 million tons of antibiotics, accounting for >70% of the total global market and ranking first in the world. Bulk (90%) of the antibiotics globally produced by China is streptomycin. A mass of waste bacterial residues is generated during the production of streptomycin. Bacterial residues were previously used as food additives and fertilizers. However, doubts have been raised on the suitability of streptomycin bacterial residues as feedstock and fertilizers because of their small amount of antibiotics and considerable amount of degradation products. Streptomycin bacterial residues are considered hazardous wastes in China. Therefore, bacterial residues should be managed strictly in accordance with the hazardous waste regulations (Guo et al., 2012; Li et al., 2012a,b). Incineration and landfill are currently the main treatment and disposal
⇑ Corresponding author. Tel./fax: +86 0311 88632210. E-mail address: [email protected] (Z. Li). 0960-8524/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biortech.2013.10.100
technologies for bacterial residues. However, these methods have significant disadvantages, such as high cost and serious secondary environmental pollution (Cassone et al., 2012). Therefore, the development of safe and effective treatments for streptomycin bacterial residues is important. Anaerobic digestion, coupled with energy production in the form of methane and waste treatment, has been employed to treat several types of organic wastes. Bacterial residues contain rich high-quality protein, fat, cellulose, enzymes, and other components, which can be used for methane by anaerobic digestion (Guo et al., 2012). The feasibility of the anaerobic digestion of various bacterial residues has been investigated by many researchers (Li et al., 1992; Li and He, 1988). However, microbial cell walls contain glycan strands crosslinked by peptide chains, causing resistance to biodegradation. Thermal–alkaline pretreatment promotes the dissolution and decomposition of microbial cells and organic material to improve anaerobic digestibility (Bougrier et al., 2008; Skiadas et al., 2005). Our previous study (Li et al., 2012a) showed that the optimal conditions of the thermal–alkaline pretreatment of streptomycin bacterial residues are 0.10 NaOH/TS, 70 °C, and 2 h.
437
W. Zhong et al. / Bioresource Technology 151 (2014) 436–440
In this study, we investigated the effect of thermal–alkaline pretreatment on the anaerobic digestion of streptomycin bacterial residues at various organic loading rates (OLRs) in laboratory-scale digesters with continuous feed mode. The results demonstrated that thermal–alkaline pretreatment can significantly enhance methane yield. The loading rate limitation of the anaerobic digestion of streptomycin bacterial residues was due to ammonia accumulation. The optimal hydraulic retention time (HRT) and OLR of the anaerobic digestion of streptomycin bacterial residues were identified for further application and reactor configuration modification.
3
1
2
4
5
6 2. Methods 2.1. Feedstock Streptomycin bacterial residues were obtained from the North China Pharmaceutical Company, Hebei, China and were stored in a refrigerator at 4 °C prior to use. According to our previous study (Li et al., 2012a), we performed the thermal–alkaline pretreatment under the optimal conditions of 0.10 NaOH/TS, 70 °C, and 2 h. The characteristics of the streptomycin bacterial residues are listed in Table 1.
7
Fig. 1. Laboratory scale CSTR digesters with 5.0 L working volume. (1. Time controller; 2. Speed governor; 3. Stirrer; 4. Wet gas flow meter; 5. Temperature controller; 6. Outlet).
2.2. Reactor and operational procedures
2.3. Analytical methods
Two identically sized completely stirred tank reactor (CSTR) digesters with a working volume of 5 L (14 cm ID 45 cm height) were fabricated with a double-wall Perspex cylinder (15 mm thickness) (Fig. 1). The temperature was maintained at 35 °C by recirculating the temperature-controlled water through the water jacket of the reactor. The reactors were sealed with a Perspex cover, which had three ports on the top. The first port was for substrate feeding. The second port was connected to a wet gas flow meter (LML-2, Jinzhiye Watch Industry Co., Beijing, China). The gas sample was collected via a silicone tube using a 10 lL pressure-tight gas syringe (104, Techcomp, China). The third port was inserted with a high-power magnetic stirrer (JB-2, Jintan Ronghua Instrument Factory, China) for intermittent mixing at 50 rpm for 24 min every 2 h. The daily biogas production was recorded using the gas flow meter (Fig. 1). The inoculum of the anaerobic reactor was anaerobic granular sludge from the streptomycin wastewater treatment plants of North China Pharmaceutical Company, Hebei, China. The sludge contained 50.76 g L1 total solid (TS) and 35.95 g L1 volatile solid (VS). The experimental results (average values of the two reactors) were collected at the steady state, i.e., a constant CH4 production (<10% of deviation) for 5 d. The samples removed from the reactors were stored at 4 °C prior to analysis. The desired OLRs were 1.11, 2.33, and 3.68 gVS L1 d1, and the related HRTs were 50, 25, and 12.5 d, respectively. Experiments were conducted for 75, 125, and 42 d for each test condition. Table 1 Characteristics of the streptomycin bacterial residue. Constituent
Streptomycin bacterial residue
TS (g/L) VS (%-TS) pH TNK (mg/L) Protein (%-TS) Total chemical oxygen demand (mg/L) Soluble chemical oxygen demand (mg/L) Moisture content (%) TAN (mg/L)
55.62 (2.64) 84.90 (7.34) 2.90 (0.28) 5373 (379) 60.37 (5.76) 53,854 (6204) 5593 (745) 93.87 (1.20) 168 (23)
Biogas volume was monitored daily using the wet gas flow meter. The measured volume was converted to that at standard temperature (0 °C) and pressure (1 atm) conditions. CH4 and CO2 were analyzed using a gas chromatograph (GC7900, Techcomp, China) equipped with a packed column (Shimadzu, 60 1.800 OD, 80/100, Japan) and a thermal conductivity detector. CH4 production was determined by multiplying the CH4 content with the volume of the biogas produced. Gas samples were injected directly using the 10 lL pressure-tight gas syringe after sampling. The pH value was directly measured with a Mettler-Toledo pH/conductivity meter using a combined pH electrode LE409 (FE20, Mettler-Toledo, Switzerland). TS, VS, total nitrogen Kjeldahl (TNK), and total ammonia nitrogen (TAN) were measured following previously described methods (Li et al., 2012a). The total chemical oxygen demand (COD) and soluble COD were measured with COD analysis systems (Lianhua Technology Company, China) composed of a spectrophotometer (5B-3), a COD reactor (5B-1), and a COD digestion reagent following the manufacturer’s instructions. Analysis of the liquid composition of digester content, including total volatile fatty acids (TVFAs) and ammonia, was conducted in triplicate using the supernatant of mixed slurry after centrifugation (SIGMA, 10,000 rpm for 30 min at 2 °C). Volatile fatty acid (VFA) analysis using gas chromatography was performed as previously described (Zhong et al., 2012). The concentration of TVFA was calculated by summarizing all VFAs. TAN was measured in triplicate according to the APHA standard methods (APHA, 1998). Each analytical result was the mean value of at least three measurements. 3. Results and discussion 3.1. Characterization of biomass The characteristics of the streptomycin bacterial residues are summarized in Table 1. The streptomycin bacterial residue biomass generally contains 85% VS in TS and is an attractive substrate for anaerobic digestion. The TNK (5373 mg L1) and protein
W. Zhong et al. / Bioresource Technology 151 (2014) 436–440
Anaerobic digestion with unpretreated and pretreated streptomycin bacterial residues as feedstock was performed in the two reactors at different OLRs of 1.11, 2.33, and 3.68 gVS L1 d1. The loading rates were maintained for 75, 125, and 42 d to collect data under steady state condition. The major operational results are summarized in Fig. 2 and Table 2. Volumetric reactor productivity (VRP), expressed in mL CH4 L1 d1, indicates the productivity of digester in term of the volume of methane per unit reactor working volume per day. As shown in Fig. 2(a), the VRP of the unpretreated and pretreated streptomycin bacterial residues at an OLR of 2.33 gVS L1 d1 were 412 and 496 mL CH4 L1 d1, respectively. These values were respectively 56.65% and 5.64% higher than those of the residues at an OLR of 1.11 gVS L1 d1 and 55.25% and 1.41% higher than those of the residues at an OLR of 3.68 gVS L1 d1. The VRPs of the pretreated streptomycin bacterial residue at each OLR were all larger than those of the unpretreated one. Specific methane yield (SMY), expressed in mL CH4 gVS1, was used to determine the rate of biomass converted to methane. Higher VRP and SMY mean better performance of an anaerobic digester (Park and Li, 2012). As shown in Fig. 2(b), SMY decreased as OLR increased from 1.11 gVS L1 d1 to 3.68 gVS L1 d1 for both unpretreated and pretreated streptomycin bacterial residues. The highest SMYs of the unpretreated and pretreated streptomycin bacterial residues were 237 and 292 mL CH4 gVS1 d1 at an OLR of 1.11 gVS L1 d1, respectively. At an OLR of 2.33 gVS L1 d1, the SMYs of the unpretreated and pretreated streptomycin bacterial residues were 177 and 216 mL CH4 gVS1 d1, respectively. The thermal–alkaline pretreatment increased the VRP and SMY of the pretreated streptomycin bacterial residue by 22.08–27.08% compared with those of the unpretreated streptomycin bacterial residue. The maximum possible methane yield is 350 mL CH4 gCOD1 during the anaerobic treatment of wastes at standard temperature and pressure (Zhang et al., 2010). Methane was generated from the biological conversion of substrate. The amount of organic compounds, which are represented by VS, will be reduced. A close relationship exists between the methane yield and VS reduction. The VS removal efficiency indicates the biodegradation extent of the feedstock added. The % VS reduction is presented in Fig. 2(c). The VS removal efficiency was the highest (80%) at 1.11 gVS L1 d1 but gradually declined as the OLR increased. This result suggests that the hydrolysis-fermentative activities may approach their limitation. The mass ratio of mL methane produced per gCOD removed ranged from 322 mL CH4 gCOD1 to 378 mL CH4 gCOD1. This result supports the initial hypothesis that the thermal–alkaline pretreatment at 0.10 NaOH/TS, 70 °C, and 2 h enhances the solubilization of solids, thereby increasing the digestibility of streptomycin bacterial residues. During the entire experimental period, the pH value was between 7.3 and 7.9 (Table 2), which agreed with the normal growth of anaerobic microorganisms. The high buffering capacity of the system could sustain the volatile acid production and the high and stable pH (7.0–7.4) over a wide range of experimental conditions. The energy contained in biogas was determined by both biogas volume and methane content. The methane contents for all
VRP (mL CH4 L–1 d–1)
3.2. Effects of thermal–alkaline pretreatment on methane production yields in the anaerobic digestion of streptomycin bacterial residues at various OLRs
(a)
600 Unpretreated Pretreated
500 400 300 200 100 0
(b)
Unpretreated Pretreated
300 250
SMY (mL CH4 gVS–1)
contents in the streptomycin bacterial residue biomass were high, in which the latter was estimated to be 60.37% of the TS based on the conversion factor of 6.25 g protein g1 TNK. High protein content likely causes significant ammonia inhibition if the protein is completely degraded.
200 150 100 50
(c)
0 100 Unpretreated Pretreated
80 VS removal (%)
438
60
40
20
0
1.11
2.33
3.68
Organic loading rate (gVS L–1 d–1) Fig. 2. The VRP (a), SMY (b) and VS removal (c) of unpretreated and pretreated streptomycin bacterial residue at different OLR and HRT. VRP = Volumetric reactor methane productivity. SMY = Specific methane yield.
digesters were measured during the entire digestion period. The total methane production for each feedstock was calculated by adding the daily methane production, which was calculated by multiplying the daily biogas production by the corresponding methane content. The methane content in biogas slightly declined from 60% to 55% at the OLR range of 1.11–3.68 gVS L1 d1 (Table 2). The methane contents from the pretreated streptomycin bacterial residue at each OLR were all higher than those from the unpretreated streptomycin bacterial residue. As one of the most important parameters for the accurate control of anaerobic digestion, the concentrations of TVFAs have a direct correlation with the digester performance (Siegert and Banks, 2005). As shown in Table 2, the VFAs increased with increasing OLR. The TVFA concentration increased sharply from
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W. Zhong et al. / Bioresource Technology 151 (2014) 436–440 Table 2 The reactor performance with the streptomycin bacterial residue as feedstock at different organic loading rates (OLRs). OLR (gVS L1 d1) 1.11 2.33 3.68
pH Unpretreated Pretreated Unpretreated Pretreated Unpretreated Pretreated
7.69 7.83 7.34 7.53 7.45 7.55
(0.27) (0.27) (0.15) (0.12) (0.27) (0.21)
approximately 1800–5000 mg L1 as OLR increased from 1.11 gVS L1 d1 to 2.33 gVS L1 d1 and up to approximately 6000 mg L1 at 3.68 gVS L1 d1. The TVFA levels at all OLRs did not change significantly and varied below 5000 mg L1, suggesting no inhibition of their degradation. Further increase in OLR would push the VFA concentration to reach toxicity level. Although the inhibitory concentrations of ammonia differ per experiment, the concentration range of 1.7–5 g TAN L1 shows the highest inhibitory effect on anaerobic digestion (Stams et al., 2003). As shown in Table 2, the concentration of TAN was approximately 1600 mg L1 at an OLR of 1.11 gVS L1 d1, increased to approximately 2200 mg L1 at an OLR of 2.33 gVS L1 d1, and then further increased to 2900 mg L1 at an OLR of 3.68 gVS L1 d1. These values belong to the inhibitory level for methanogenesis at 4.0 g VS L1 d1 (Appels et al., 2008). The anaerobic digestion of organic matter with a high nitrogen content (or low C/N ratio) could be susceptible to ammonia inhibition because of the accumulation of ammonia from protein degradation, causing reaction failure. Depending on pH, TAN in digester is composed of different fractions of free ammonia (NH3) and ammonium ions (NHþ 4 ), of which NH3 is the most toxic.
NH3ðaqÞ þ H2 O $ NHþ4 þ OH ðpK b ¼ 4:733 at 35 CÞ
ð1Þ
TAN inhibits methanogens at the concentration range of 15,000–3000 mg L1 at pH 7.2–7.6. The toxicity level increases with increasing pH because of the shift to a higher fraction of NH3 (Appels et al., 2008). In the present study, the increase in TAN was due to more protein in the streptomycin bacterial residue degraded at higher OLR. Longer HRT might improve the residual TVFA and VS removal efficiency of the reactor; however, it might also elicit negative effects because the level of ammonia accumulated not only depends on OLR and TNK content but also on HRT. At the same OLR, long HRT could promote the accumulation of TAN in a CSTR. To avoid ammonia inhibition, a CSTR digester must receive an OLR with low ammonia-producing potential, such as 2.33 gVS L1 d1 in this study, or short HRT. A large reactor size is needed to operate at low OLR, except for the separation of the solid phase from the liquid phase, such as in high-rate upflow anaerobic sludge blanket. The major investment in anaerobic digestion is the reactor capital cost. Therefore, introducing reasonably short HRT and applying high OLR to minimize the reactor volume are economically sound in the design of digestion process. The results of the present study demonstrated the following results. First, anaerobic digestion of the streptomycin bacterial residue can be performed well at a normal OLR of 2.33 gVS L1 d1 without ammonia inhibition. Second, thermal–alkaline pretreatment of the streptomycin bacterial residue can enhance the reactor OLR by more than double. The pretreated streptomycin bacterial residue had higher VS removal efficiency and lower TVFA than the unpretreated streptomycin bacterial residue. The above findings can be used for the scale-up and process design. However, the low C/N ratio of streptomycin bacterial residues is a serious problem that limits anaerobic digestion. The C/N ratio of streptomycin bacterial residues is significantly low for
TVFAs (mg L1)
TAN (mg L1)
CH4 content (%)
1838 1574 4209 5042 5057 6054
1682 1597 2256 2236 2874 2880
58.27 62.19 56.31 59.84 55.07 55.72
(162) (371) (552) (507) (311) (364)
(22) (99) (313) (315) (252) (253)
(3.73) (6.19) (4.26) (4.87) (3.72) (4.03)
anaerobic digestion (<10, 20/1 to 30/1 is the most acceptable range). Low C/N ratio feedstock could result in the release of TAN and the accumulation of VFAs in the digester. Excessive ammonia accumulation can be avoided by adding high carboncontent materials to adjust low feedstock C/N ratios and thus improve the digestion performance. The codigestion of streptomycin bacterial residue with low C/N ratio and crop residues with high C/N ratio is a good solution to the above problem. Crop residues, such as corn and wheat straws, are produced in large quantities in China and other countries every year. These organic crop residues can be valuable alternative feedstock for methane production (Zhong et al., 2012). According to Mata-Alvarez et al. (2000), the digestion of more than one type of substrate in the same digester could establish positive synergism in the digester, i.e., the added nutrients would support the microbial growth. The benefits of codigestion include the dilution of potentially toxic ammonia, allowance for increased loading rate, improved methane yield, economic advantages derived from the sharing of equipment, easier handling of mixed wastes, and synergistic effects. Therefore, further research is still needed to test the cofeedstocks and scale-up of the reactor. 4. Conclusion The anaerobic digestion of streptomycin bacterial residue is feasible in laboratory-scale anaerobic reactors. Thermal–alkaline pretreatment can significantly enhance methane production. The VRP and SMY of the streptomycin bacterial residue thermal–alkaline pretreated at 0.10 NaOH/TS, 70 °C, and 2 h increased by 22.08– 27.08% compared with those of the unpretreated streptomycin bacterial residue. The reactors performed well at an OLR of 2.33 gVS L1 d1 for methane production, with a VS removal of 51.03–64.09% at an HRT of 25 d without ammonia inhibition. At all OLRs, the levels of pH values and TVFAs did not change significantly, suggesting no inhibition of their degradation. Acknowledgement This research was supported by the Science and Technology Department of Hebei Province (No. 12276703Z). References APHA, 1998. Standard Methods for the Examination of Water and Wastewater. 20th ed. American Public Health Association, Washington, DC. Appels, L., Baeyens, J., Degrève, J., Dewil, R., 2008. Principles and potential of the anaerobic digestion of waste-activated sludge. Prog. Energy Combust. Sci. 34 (6), 755–781. Bougrier, C., Delgenès, J.P., Carrère, H., 2008. Effects of thermal treatments on five different waste activated sludge samples solubilisation, physical properties and anaerobic digestion. Chem. Eng. J. 139 (2), 236–244. Cassone, C.G., Vongphachan, V., Chiu, S., Williams, K.L., Letcher, R.J., Pelletier, E., Crump, D., Kennedy, S.W., 2012. In ovo effects of perfluorohexane sulfonate and perfluorohexanoate on pipping success, development, mrna expression, and thyroid hormone levels in chicken embryos. Toxicol. Sci. 127 (1), 216– 224. Guo, B., Gong, L., Duan, E., Liu, R., Ren, A., Han, J., Zhao, W., 2012. Characteristics of penicillin bacterial residue. J. Air Waste Manage. Assoc. 62 (4), 485–488.
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