Bioresource Technology 192 (2015) 807–811
Contents lists available at ScienceDirect
Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Short Communication
Effect of NaCl induced floc disruption on biological disintegration of sludge for enhanced biogas production S. Kavitha a, S. Kaliappan b, S. Adish Kumar a, Ick Tae Yeom c, J. Rajesh Banu a,⇑ a
Department of Civil Engineering, Regional Centre of Anna University, Tirunelveli, India Department of Civil Engineering, Thiagarajar College of Engineering, Thiruparankundram, Madurai, India c Department of Civil and Environmental Engineering, Sungkyunkwan University, Seoul, South Korea b
h i g h l i g h t s NaCl induced biological disintegration is a beneficial profitable process. The disintegration was effectual with solubilization of about 23%. The hydrolysis rate of floc disrupted sludge was found to be 0.005 h
1
.
Biodegradability (0.23 gCOD/gCOD) was comparatively greater than other samples. Floc disrupted sludge shows high net profit of about 2.5 USD/per ton SS.
a r t i c l e
i n f o
Article history: Received 25 March 2015 Received in revised form 20 May 2015 Accepted 21 May 2015 Available online 2 June 2015 Keywords: Waste activated sludge Floc disruption Disintegration Extracellular polymeric substance Biochemical methane potential
a b s t r a c t In the present study, the influence of NaCl mediated bacterial disintegration of waste activated sludge (WAS) was evaluated in terms of disintegration and biodegradability of WAS. Floc disruption was efficient at 0.03 g/g SS of NaCl, promoting the shifts of extracellular proteins and carbohydrates from inner layers to extractable – soluble layers (90 mg/L), respectively. Outcomes of sludge disintegration reveal that the maximum solubilization achieved was found to be 23%, respectively. The model elucidating the parameter evaluation, explicates that floc disrupted – bacterially disintegrated sludge (S3) showed superior biodegradability of about 0.23 (gCOD/gCOD) than the bacterially disintegrated (S2) and control (S3) sludges of about 0.13 (gCOD/gCOD) and 0.05 (gCOD/gCOD), respectively. Cost evaluation of the present study affords net profits of approximately 2.5 USD and 21.5 USD in S3 and S2 sludge. Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction A generation of enormous amounts of waste activated sludge (WAS) is an unavoidable shortcoming of activated sludge processes, as sludge management and clearance already accounts for up to 50% of the total treatment costs of wastewater purification (Appels et al., 2010). Anaerobic digestion (AD) is one of the most potential treatments for municipal sludge from the perspective of energy upturn. The efficiency of AD can be amended by a pretreatment process that will hasten the biochemical hydrolysis reactions, prior to anaerobic digestion (Appels et al., 2013). Among the various pretreatments, biological pretreatment provides unique
⇑ Corresponding author at: Department of Civil Engineering, Regional centre of Anna University, Tirunelveli 627007, India. Tel.: +91 9444215544. E-mail address: [email protected] (J. Rajesh Banu). http://dx.doi.org/10.1016/j.biortech.2015.05.071 0960-8524/Ó 2015 Elsevier Ltd. All rights reserved.
advantages, compared to chemical or physical processes, as it is environmentally friendly and causes neither pollution nor needs extraordinary equipment. However, the high-price of industrial enzymes makes the pretreatment economically unviable. Therefore, there is concern in trying to determine an appropriate way in which inoculation of extracellular enzyme secreting strains could be produced (Yu et al., 2013). In addition, the extracellular polymeric substances (EPS), which are the active secretion of biomass of sludge matrix, play a crucial role in flocculation (Kavitha et al., 2013). Therefore, removal of EPS enhances the subsequent pretreatment process. The main aim of this study is: (1) to disrupt flocs and remove EPS with a suitable chemical, NaCl (2) to disintegrate floc disrupted sludge with bacterial consortium (3) to assess the kinetic analysis of bacterial disintegration of WAS (4) to evaluate the anaerobic biodegradability of WAS (5) to investigate the economic feasibility of implementing the process.
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S. Kavitha et al. / Bioresource Technology 192 (2015) 807–811
2. Methods
2.7. Statistical analysis
2.1. Collection of sample and characterization
All the assays were done in triplicate and the outcomes were expressed as an average of three values. Pearson correlation and One way ANOVA analysis, followed by the Student–Newman–Ke uls Method, was employed to test the significance of results where p < 0.05 was measured to be statistically significant.
The sludge used for this work was taken from a municipal waste water treatment plant in karakonam, Kerala. The characteristics of the sample was summarized as: pH = 6.6, total COD = 10100 mg/L, SCOD = 100 mg/L, suspended solids = 7000 mg/L, total solids = 12500 mg/L.
3. Results and discussions
2.2. Bacterial consortium
3.1. Floc disruption of WAS by NaCl
The bacterial consortium employed in the present study contains two bacterial strains (Bacillus jerish 03 Accession number KC597266 and Bacillus jerish 04 Accession number KC597267). These strains were isolated and recognized in the previous work (Lakshmi et al., 2014). The optimal temperature, pH, and time for the growth of these strains were 40 °C, 6.5, and 42 h, respectively.
The fractionation of extracellular polymeric substance (EPS) after floc disruption can offer a thorough understanding of the impact of NaCl on subsequent pretreatment studies. In the present study, DNA was quantified to conclude whether the sludge biomass was lysed by NaCl. Monovalent cations, especially sodium (Na+ ions), transpose the divalent ions within cation bridged floc structure by ion exchange and weaken the floc strength causing disruption of the flocs (Higgins and Novak, 1997). Thus, NaCl was used for EPS removal and floc disruption. The effect of NaCl on floc disruption and fractionation of EPS is depicted in Fig. 1. In raw sludge (without NaCl treatment), the quantity of the soluble LB-EPS and TB-EPS were found to be 10 mg/L, 60 mg/L and 40 mg/L, respectively. The treatment of NaCl shifted the extracellular polymers present in both the layers of TB-EPS and LB-EPS to soluble phase (the amount of EPS got reduced in TB-EPS and LB-EPS and increased in soluble phase). The quantities of extractable exopolymers were estimated to be 100 mg/L by summing up TB and LB EPS. As displayed in Fig. 1, an abrupt decrement in the quantity of both LB (60–23.4 mg/L) and TB EPS (40–10 mg/L) up to 0.03 g/g SS of NaCl dosage was noted. Simultaneously, an increment was noted in the case of soluble EPS, with a quantity of about 84 mg/L, representing an effectual extraction of exocellular extractable EPS. On the other hand, a further increment in NaCl dosage beyond 0.03 g/g SS, and a sheer rise in soluble EPS was noted, which surpassed the maximum extractable exocellular polymers. This construes the prevalence of biomass disintegration with the discharge of intracellular polymers into the supernatant phase. From the above noted facts, it can be concluded that the maximum extraction of EPS, with minimum cell lysis, was achievable only up to 0.03 g/g SS of NaCl. In addition, the quantity of soluble EPS (Slime) was directly proportional to the NaCl dosage. A stepwise increment in soluble EPS and DNA was noted up to 0.03 g/g SS of NaCl. At this NaCl dosage, their measures were found to be 84 mg/L and 22 mg/L, respectively. A further increment in NaCl concentration to 0.04 g/g SS, and the amount of released soluble EPS and DNA were found to be 200 mg/L and 140 mg/L signifying that most of the cells were lysed under these conditions. The outcome of the current work confirmed that 0.03 g/g SS of NaCl was observed to be optimal for floc disruption with negligible biomass disintegration. A significant correlation between the dosage of NaCl and EPS protein, carbohydrate, DNA, and Soluble EPS was observed statistically through Pearson correlation. The P-value for various responses (protein, carbohydrate, DNA, Soluble EPS) was found to be 9 106, 6 106, 2 106, and 1.3 105, respectively. The correlation coefficient for all these responses was found to be 0.993, 0.971, 0.962, and 0.989, respectively. This augurs a significant correlation between the dosage of NaCl and all the other responses.
2.3. Floc disruption experiment The experiment was carried out in eleven identical conical flasks, each having a 100 mL of WAS. The ratio of the NaCl dosage to the suspended solids varied from 0.003 to 0.08 g/g SS. The flasks were kept in an orbital shaker for 2 h at 100 rpm, for complete mixing, at a temperature of 30 °C. The samples were then spin at 10,000g for 15 min. The aqueous portion was filtered to obtain the EPS. 2.4. Disintegration of WAS with bacterial strains In the disintegration experiment, 100 mL of floc disrupted (EPS removed) WAS (S3 sludge) was added with 2.0 g/L of bacteria in a conical flask. The contents were mixed with a slow speed stirrer at 100 rpm for 42 h. Additionally, 100 mL of control (S1) and bacterially pretreated alone (S2) samples were taken in two conical flasks to evaluate the proficiency of floc disruption. 2.5. Biochemical methane potential assay Anaerobic biodegradability assay was done as per the procedure described by Uma et al., 2012a. The inoculum (Bovine rumen fluid) and substrates (S1, S2 and S3 sludges) were taken in a ratio of 3:1. The following first order kinetic model was employed to study the methane production:
YðtÞ ¼ Yðf d Þ ð1 expkhyd t Þ
ð1Þ
where Y(t) is the cumulative methane yielded at digestion time t days (g COD/g COD added), Y(fd) is methane potential of the substrate (fraction of the degradable substrate that can be converted to methane) (g COD/g COD added), khyd is the first order disintegration rate constant (day1), and t is the time (days). The model was executed in a Mat lab 2012a Version. The parameter estimation, and uncertainty with 95% confidence limit, was calculated based on the work of (Batstone et al., 2009). 2.6. Analytical methods
3.2. NaCl mediated biological disintegration Suspended solids, total chemical oxygen demand, and soluble chemical oxygen demand were measured according to Standard methods (APHA, 2005). Biopolymers and DNA were measured by the procedure described by Kavitha et al. (2014).
3.2.1. Solids reduction and SCOD release The effect of biological disintegration on solid reduction for the control (S1) bacterially disintegrated (S2) and floc disrupted
S. Kavitha et al. / Bioresource Technology 192 (2015) 807–811
809
Fig. 1. Optimization of NaCl dosages for floc disruption and fractionation of sludge EPS. The error bars represent the standard errors.
bacterially disintegrated sludges (S3) is depicted in Fig. 2a. From the figure, it is noted that the SS reduction increases with increments in treatment time up to 42 h. At 42 h, the SS reduction was found to be 8%, 14.28% and 26% respectively for S1, S2 and S3 sludges, respectively. The rapid SS reduction up to 42 h could be due to the combinatorial activity of enzymes entrapped in the sludge matrix and also the amount of enzymes (protease and amylase) secreted by the inoculated strain. After 42 h, the increment becomes flat signifying stable SS reduction due to the saturated activity of inoculated bacteria. The solubilization of particulate organics was expressed in terms of SCOD release and solubilization percentages (Fig. 2b). On the basis of bacterial pretreatment time, the release of SCOD can be divided into two phases. A rapid disintegration or release phase from 0 h to 42 h, and a slow decline phase from 56 h to 72 h. This could be attributable to the release of particulate organics into the liquid portion (Uma et al., 2012a,b; Kavitha et al., 2015) by enzyme mediated disintegration of sludge cells. On the other hand, higher liquefaction was noted in S3 sludge than the S2 and S1 sludge. At 42 h, the COD solubilization was found to be 6%, 11% and 23% respectively for S1, S2 and S3 sludges. The disruption floc matrices certainly pave the way for EPS removal and sludge solubilization enhancement due to a greater availability of substrates for the inoculated bacteria and simultaneous release of entrapped organic matter from the sludge (Kavitha et al., 2014).
Fig. 2b. Effect of NaCl mediated biological pretreatment on SCOD release. The error bars represent the standard errors.
The decline phase occurs plausibly after 42 h, which could be due to the consumption of released biopolymers by the inoculated strain. Based on the One way Anova analysis (Students Newman– Keuls Method), the p-value for SS reduction and SCOD release was 0.035 and 0.008, respectively. This indicates a significant difference between S2 and S3 sludges for SS reduction and SCOD release. 3.2.2. Kinetic investigation of disintegration process The effect of bacterial strains on sludge disintegration (SS and Particulate COD degradation) was explored, and the outcomes were analyzed mathematically through first order kinetics. Decrement in SS (Fig. 2a) and PCOD (data not shown) in the S1, S2, and S3 sludges increased up to 42 h. The k value of the S1, S2, and S3 samples were 0.001 h1, 0.003 h1, 0.005 h1, and 0.001 h1, 0.002 h1, 0.005 h1 for SS and PCOD reductions, respectively. The k value of the S3 sample was observed to be higher than the other samples. The correlation coefficient of all three sludges was between 0.92 and 0.99, indicating a better fit between the model and experiment. 3.3. Biochemical methane potential assay
Fig. 2a. Effect of NaCl mediated biological pretreatment on SS reduction. The error bars represent the standard errors.
The outcomes of the BMP tests for all three samples (S1, S2 & S3) with model simulations are given in Fig. 3a. The S3 sample
S. Kavitha et al. / Bioresource Technology 192 (2015) 807–811
showed better biodegradability when compared to others. The disintegrated sludge samples hold larger amounts of soluble organic matters when compared to the raw WAS, which were biodegraded more hastily. Around day 15, the methane yield of the pretreated sludge becomes stabilized due to substrate exhaustion. Methane generation rate was improved initially, whilst the production results decreased progressively over time. Obviously, after 25 days of operation, the control had produced lower methane. For the validation study, the determination coefficient (R2 lies within the ranges 0.95–0.99) revealed that the model exhibited a good fit with the experimental values. Fig. 3b depicts the plot of evaluated parameter confidence region spaces of fd (fraction of degradable substrates- biodegradability) along x-axis and khyd (apparent hydrolysis rate) along y axis. As exposed in Fig. 3b, the S3 sample showed an enhanced degradability when compared to the other samples. The S3 sample was highly reliable with good confidence. On the contrary, S1 sample exposed poorer identifiable parameter and wider confidence region. Biodegradability ranges for S2 and S3 samples were found to be 0.13 and 0.23 (gCOD/gCOD) with narrow confidence regions which indicate higher precision (Batstone et al., 2009). The biodegradability range of S1 was found to be 0.05 (gCOD/gCOD) with wider confidence interval which indicates lower precision. The khyd values of all the three samples – S1, S2 and S3 sludges (0.089 day1, 0.146 day1 and 0.193 day1) varied substantially. The lower value of fd and khyd in S1, designated that the substrates were metabolized more slowly with a lesser degree. The overall findings from the present study supports the general applicability of the proposed method as well as its predictive capability and accuracy.
0.22
0.16
Floc disrupted
Control
0.13
0.1
0.07
0.04 0.03
0.07
0.11
0.15
0.19
0.23
Substra te biodegra da bility (f d) Fig. 3b. Comparison of confidence regions for biodegradability (fd) and hydrolysis disintegration constant (khyd).
Table 1 Evaluation of energy balance and cost assessment. S. No. 1 2 3 4
5 6 7
3.4. Energy balance and cost evaluation To compute the proficiency of S2 and S3 sludges in terms of net energy production, an energy balance investigation was performed and the outcomes are tabulated in Table 1. Table 1 elucidates the energy equilibrium and cost assessment of the S3 and S2 samples based on energy content of the biogas produced from both the samples. To arrive the energy applied, (Table 1, S. No. 6) energy spent for mechanical stirring (EPS removal, bacterial inoculum, pretreatment and anaerobic digestion) and pumping of the sludge was taken into account. The total input energy was calculated to be 422.1 and 419.2 for S3 and S2 sludges, respectively. Concurrently, the energy content of the biogas for S3 and S2 samples were calculated to be 166 kWh and 57.95 kWh, respectively. Besides, in order to evaluate the economic viability of the disintegration process, estimation of operational cost (including consumable chemicals)
Bacterially pretreated
0.19
Hydrolysis rate control (k)
810
8 9 10 11 12
Energy balance (per ton of sludge) Average increase in biogas production Energy content of biogas Energy applied (for EPS removal) Energy applied (for stirring – bacterial growth, pretreatment & anaerobic digestion)a Energy applied (for pumping)a Total energy applied (S. No. 3 + 4 + 5) Net energy production (S. No. 2–6) Cost calculation (per ton of sludge) Energy cost (S. No. 7a0.23 USD/kWh) Decrease in SS to be disposed Reduced cost for sludge disposal (S. No. 9 a 0.28 USD/kg TS) Chemical cost for floc disruption & bacterial growth Net profit
S3 sludge
S2 sludge
Unit
27192 166 2.9 417.6
9500 57.95 – 417.6
L kWh kWh kWh
1.6 422.1 256.1
1.6 419.2 361.3
kWh kWh kWh
58.9 405 113.4
83.1 220 61.6
USD kg USD
52
–
USD
2.5
21.5
USD
a
Calculated based on Metcalf & Eddy (2003), S3 – floc disrupted and bacterially disintegrated, S2 – bacterially disintegrated alone.
and the decreased amount of solids to be disposed was considered in the present study. From Table 1, it was found that higher net profit was achieved for (S3) sludge compared to (S2) sludge. The net profit of S3 and S2 sludges was calculated to be 2.5 USD and 21.5 USD, respectively. Based on the above details, it can be concluded that the phase separated bacterial pretreatment was observed to be cost-effectively viable approach.
4. Conclusion The effects of NaCl mediated bacterial phase separation on biogas production from WAS were investigated and compared to sole bacterial cell disintegration. It was found that floc disruption with 0.03 g/g SS of NaCl and biomass disintegration with bacteria was a very favorable method and the increase of solubilization (23%) was beneficial to thr biodegradability process. The phase separated bacterial disintegration on biochemical methane potential benefited the production of methane efficiently. Acknowledgements
Fig. 3a. Plot showing disintegration.
methane
production
of
NaCl
mediated
biological
Authors are grateful to DST, India for offering economic assistance for this work (SR/FTP/ETA-0021/2010) on behalf of Young Scientist scheme.
S. Kavitha et al. / Bioresource Technology 192 (2015) 807–811
References APHA, 2005. Standard Methods for the Examination of Water and Wastewater, 21st ed. American Public Health Association, Washington, D.C. Appels, L., Degrève, J., Van der Bruggen, B., Van Impe, J., Dewil, R., 2010. Influence of low temperature thermal pre-treatment on sludge solubilization, heavy metal release and anaerobic digestion. Bioresour. Technol. 101, 5743–5748. Appels, L., Houtmeyers, S., Degrève, J., Impe, J.V., Dewil, R., 2013. Influence of microwave pre-treatment on sludge solubilization and pilot scale semicontinuous anaerobic digestion. Bioresour. Technol. 128, 598–603. Batstone, D.J., Tait, S., Starrenburg, D., 2009. Estimation of hydrolysis parameters in full-scale anaerobic digesters. Biotechnol. Bioeng. 102, 1513–1520. Higgins, M.J., Novak, J.T., 1997. The effect of cations on the settling and dewatering of activated sludges. Water Environ. Res. 69, 215–224. Kavitha, S., Adish Kumar, S., Yoga Lakshmi, K.N., Kaliappan, S., Rajesh Banu, J., 2013. Effect of enzyme secreting bacterial pretreatment on enhancement of aerobic digestion potential of waste activated sludge interceded through EDTA. Bioresour. Technol. 150, 210–219. Kavitha, S., Jayashree, C., Adish Kumar, S., Yeom, I.T., Rajesh Banu, J., 2014. Improving the amenability of municipal waste activated sludge for biological
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pretreatment by phase-separated sludge disintegration. Bioresour. Technol. 169, 700–706. Kavitha, S., Saranya, T., Kaliappan, S., Adish Kumar, S., Yeom, I.T., Rajesh, Banu J., 2015. Accelerating the sludge disintegration potential of a novel bacterial strain Planococcus jake 01 by CaCl2 induced deflocculation. Bioresour. Technol. 175, 396–405. Lakshmi, M.V., Merrylin, J., Kavitha, S., Adish, K.S., Yeom, I.T., Rajesh, B.J., 2014. Solubilization of municipal sewage waste activated sludge by novel lytic bacterial strains. Environ. Sci. Pollut. Res. 21, 2733–2743. Metcalf, Eddy, 2003. Waste water Engineering Treatment and Reuse,, McGraw Hill publication, New York, USA. Uma, R.R., Adish, K.S., Kaliappan, S., Yeom, I.T., Rajesh, B.J., 2012a. Low temperature thermo-chemical pretreatment of dairy waste activated sludge for anaerobic digestion process. Bioresour. Technol. 103, 415–424. Uma, R.R., Kaliappan, S., Adish Kumar, S., Rajesh Banu, J., 2012b. Combined treatment of alkaline and disperser for improving solubilization and anaerobic biodegradability of dairy waste activated sludge. Bioresour. Technol. 126, 107–116. Yu, S., Zhang, G., Li, J., Zhao, Z., Kang, X., 2013. Effect of endogenous hydrolytic enzymes pretreatment on the anaerobic digestion of sludge. Bioresour. Technol. 146, 758–761.
Contents lists available at ScienceDirect
Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Short Communication
Effect of NaCl induced floc disruption on biological disintegration of sludge for enhanced biogas production S. Kavitha a, S. Kaliappan b, S. Adish Kumar a, Ick Tae Yeom c, J. Rajesh Banu a,⇑ a
Department of Civil Engineering, Regional Centre of Anna University, Tirunelveli, India Department of Civil Engineering, Thiagarajar College of Engineering, Thiruparankundram, Madurai, India c Department of Civil and Environmental Engineering, Sungkyunkwan University, Seoul, South Korea b
h i g h l i g h t s NaCl induced biological disintegration is a beneficial profitable process. The disintegration was effectual with solubilization of about 23%. The hydrolysis rate of floc disrupted sludge was found to be 0.005 h
1
.
Biodegradability (0.23 gCOD/gCOD) was comparatively greater than other samples. Floc disrupted sludge shows high net profit of about 2.5 USD/per ton SS.
a r t i c l e
i n f o
Article history: Received 25 March 2015 Received in revised form 20 May 2015 Accepted 21 May 2015 Available online 2 June 2015 Keywords: Waste activated sludge Floc disruption Disintegration Extracellular polymeric substance Biochemical methane potential
a b s t r a c t In the present study, the influence of NaCl mediated bacterial disintegration of waste activated sludge (WAS) was evaluated in terms of disintegration and biodegradability of WAS. Floc disruption was efficient at 0.03 g/g SS of NaCl, promoting the shifts of extracellular proteins and carbohydrates from inner layers to extractable – soluble layers (90 mg/L), respectively. Outcomes of sludge disintegration reveal that the maximum solubilization achieved was found to be 23%, respectively. The model elucidating the parameter evaluation, explicates that floc disrupted – bacterially disintegrated sludge (S3) showed superior biodegradability of about 0.23 (gCOD/gCOD) than the bacterially disintegrated (S2) and control (S3) sludges of about 0.13 (gCOD/gCOD) and 0.05 (gCOD/gCOD), respectively. Cost evaluation of the present study affords net profits of approximately 2.5 USD and 21.5 USD in S3 and S2 sludge. Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction A generation of enormous amounts of waste activated sludge (WAS) is an unavoidable shortcoming of activated sludge processes, as sludge management and clearance already accounts for up to 50% of the total treatment costs of wastewater purification (Appels et al., 2010). Anaerobic digestion (AD) is one of the most potential treatments for municipal sludge from the perspective of energy upturn. The efficiency of AD can be amended by a pretreatment process that will hasten the biochemical hydrolysis reactions, prior to anaerobic digestion (Appels et al., 2013). Among the various pretreatments, biological pretreatment provides unique
⇑ Corresponding author at: Department of Civil Engineering, Regional centre of Anna University, Tirunelveli 627007, India. Tel.: +91 9444215544. E-mail address: [email protected] (J. Rajesh Banu). http://dx.doi.org/10.1016/j.biortech.2015.05.071 0960-8524/Ó 2015 Elsevier Ltd. All rights reserved.
advantages, compared to chemical or physical processes, as it is environmentally friendly and causes neither pollution nor needs extraordinary equipment. However, the high-price of industrial enzymes makes the pretreatment economically unviable. Therefore, there is concern in trying to determine an appropriate way in which inoculation of extracellular enzyme secreting strains could be produced (Yu et al., 2013). In addition, the extracellular polymeric substances (EPS), which are the active secretion of biomass of sludge matrix, play a crucial role in flocculation (Kavitha et al., 2013). Therefore, removal of EPS enhances the subsequent pretreatment process. The main aim of this study is: (1) to disrupt flocs and remove EPS with a suitable chemical, NaCl (2) to disintegrate floc disrupted sludge with bacterial consortium (3) to assess the kinetic analysis of bacterial disintegration of WAS (4) to evaluate the anaerobic biodegradability of WAS (5) to investigate the economic feasibility of implementing the process.
808
S. Kavitha et al. / Bioresource Technology 192 (2015) 807–811
2. Methods
2.7. Statistical analysis
2.1. Collection of sample and characterization
All the assays were done in triplicate and the outcomes were expressed as an average of three values. Pearson correlation and One way ANOVA analysis, followed by the Student–Newman–Ke uls Method, was employed to test the significance of results where p < 0.05 was measured to be statistically significant.
The sludge used for this work was taken from a municipal waste water treatment plant in karakonam, Kerala. The characteristics of the sample was summarized as: pH = 6.6, total COD = 10100 mg/L, SCOD = 100 mg/L, suspended solids = 7000 mg/L, total solids = 12500 mg/L.
3. Results and discussions
2.2. Bacterial consortium
3.1. Floc disruption of WAS by NaCl
The bacterial consortium employed in the present study contains two bacterial strains (Bacillus jerish 03 Accession number KC597266 and Bacillus jerish 04 Accession number KC597267). These strains were isolated and recognized in the previous work (Lakshmi et al., 2014). The optimal temperature, pH, and time for the growth of these strains were 40 °C, 6.5, and 42 h, respectively.
The fractionation of extracellular polymeric substance (EPS) after floc disruption can offer a thorough understanding of the impact of NaCl on subsequent pretreatment studies. In the present study, DNA was quantified to conclude whether the sludge biomass was lysed by NaCl. Monovalent cations, especially sodium (Na+ ions), transpose the divalent ions within cation bridged floc structure by ion exchange and weaken the floc strength causing disruption of the flocs (Higgins and Novak, 1997). Thus, NaCl was used for EPS removal and floc disruption. The effect of NaCl on floc disruption and fractionation of EPS is depicted in Fig. 1. In raw sludge (without NaCl treatment), the quantity of the soluble LB-EPS and TB-EPS were found to be 10 mg/L, 60 mg/L and 40 mg/L, respectively. The treatment of NaCl shifted the extracellular polymers present in both the layers of TB-EPS and LB-EPS to soluble phase (the amount of EPS got reduced in TB-EPS and LB-EPS and increased in soluble phase). The quantities of extractable exopolymers were estimated to be 100 mg/L by summing up TB and LB EPS. As displayed in Fig. 1, an abrupt decrement in the quantity of both LB (60–23.4 mg/L) and TB EPS (40–10 mg/L) up to 0.03 g/g SS of NaCl dosage was noted. Simultaneously, an increment was noted in the case of soluble EPS, with a quantity of about 84 mg/L, representing an effectual extraction of exocellular extractable EPS. On the other hand, a further increment in NaCl dosage beyond 0.03 g/g SS, and a sheer rise in soluble EPS was noted, which surpassed the maximum extractable exocellular polymers. This construes the prevalence of biomass disintegration with the discharge of intracellular polymers into the supernatant phase. From the above noted facts, it can be concluded that the maximum extraction of EPS, with minimum cell lysis, was achievable only up to 0.03 g/g SS of NaCl. In addition, the quantity of soluble EPS (Slime) was directly proportional to the NaCl dosage. A stepwise increment in soluble EPS and DNA was noted up to 0.03 g/g SS of NaCl. At this NaCl dosage, their measures were found to be 84 mg/L and 22 mg/L, respectively. A further increment in NaCl concentration to 0.04 g/g SS, and the amount of released soluble EPS and DNA were found to be 200 mg/L and 140 mg/L signifying that most of the cells were lysed under these conditions. The outcome of the current work confirmed that 0.03 g/g SS of NaCl was observed to be optimal for floc disruption with negligible biomass disintegration. A significant correlation between the dosage of NaCl and EPS protein, carbohydrate, DNA, and Soluble EPS was observed statistically through Pearson correlation. The P-value for various responses (protein, carbohydrate, DNA, Soluble EPS) was found to be 9 106, 6 106, 2 106, and 1.3 105, respectively. The correlation coefficient for all these responses was found to be 0.993, 0.971, 0.962, and 0.989, respectively. This augurs a significant correlation between the dosage of NaCl and all the other responses.
2.3. Floc disruption experiment The experiment was carried out in eleven identical conical flasks, each having a 100 mL of WAS. The ratio of the NaCl dosage to the suspended solids varied from 0.003 to 0.08 g/g SS. The flasks were kept in an orbital shaker for 2 h at 100 rpm, for complete mixing, at a temperature of 30 °C. The samples were then spin at 10,000g for 15 min. The aqueous portion was filtered to obtain the EPS. 2.4. Disintegration of WAS with bacterial strains In the disintegration experiment, 100 mL of floc disrupted (EPS removed) WAS (S3 sludge) was added with 2.0 g/L of bacteria in a conical flask. The contents were mixed with a slow speed stirrer at 100 rpm for 42 h. Additionally, 100 mL of control (S1) and bacterially pretreated alone (S2) samples were taken in two conical flasks to evaluate the proficiency of floc disruption. 2.5. Biochemical methane potential assay Anaerobic biodegradability assay was done as per the procedure described by Uma et al., 2012a. The inoculum (Bovine rumen fluid) and substrates (S1, S2 and S3 sludges) were taken in a ratio of 3:1. The following first order kinetic model was employed to study the methane production:
YðtÞ ¼ Yðf d Þ ð1 expkhyd t Þ
ð1Þ
where Y(t) is the cumulative methane yielded at digestion time t days (g COD/g COD added), Y(fd) is methane potential of the substrate (fraction of the degradable substrate that can be converted to methane) (g COD/g COD added), khyd is the first order disintegration rate constant (day1), and t is the time (days). The model was executed in a Mat lab 2012a Version. The parameter estimation, and uncertainty with 95% confidence limit, was calculated based on the work of (Batstone et al., 2009). 2.6. Analytical methods
3.2. NaCl mediated biological disintegration Suspended solids, total chemical oxygen demand, and soluble chemical oxygen demand were measured according to Standard methods (APHA, 2005). Biopolymers and DNA were measured by the procedure described by Kavitha et al. (2014).
3.2.1. Solids reduction and SCOD release The effect of biological disintegration on solid reduction for the control (S1) bacterially disintegrated (S2) and floc disrupted
S. Kavitha et al. / Bioresource Technology 192 (2015) 807–811
809
Fig. 1. Optimization of NaCl dosages for floc disruption and fractionation of sludge EPS. The error bars represent the standard errors.
bacterially disintegrated sludges (S3) is depicted in Fig. 2a. From the figure, it is noted that the SS reduction increases with increments in treatment time up to 42 h. At 42 h, the SS reduction was found to be 8%, 14.28% and 26% respectively for S1, S2 and S3 sludges, respectively. The rapid SS reduction up to 42 h could be due to the combinatorial activity of enzymes entrapped in the sludge matrix and also the amount of enzymes (protease and amylase) secreted by the inoculated strain. After 42 h, the increment becomes flat signifying stable SS reduction due to the saturated activity of inoculated bacteria. The solubilization of particulate organics was expressed in terms of SCOD release and solubilization percentages (Fig. 2b). On the basis of bacterial pretreatment time, the release of SCOD can be divided into two phases. A rapid disintegration or release phase from 0 h to 42 h, and a slow decline phase from 56 h to 72 h. This could be attributable to the release of particulate organics into the liquid portion (Uma et al., 2012a,b; Kavitha et al., 2015) by enzyme mediated disintegration of sludge cells. On the other hand, higher liquefaction was noted in S3 sludge than the S2 and S1 sludge. At 42 h, the COD solubilization was found to be 6%, 11% and 23% respectively for S1, S2 and S3 sludges. The disruption floc matrices certainly pave the way for EPS removal and sludge solubilization enhancement due to a greater availability of substrates for the inoculated bacteria and simultaneous release of entrapped organic matter from the sludge (Kavitha et al., 2014).
Fig. 2b. Effect of NaCl mediated biological pretreatment on SCOD release. The error bars represent the standard errors.
The decline phase occurs plausibly after 42 h, which could be due to the consumption of released biopolymers by the inoculated strain. Based on the One way Anova analysis (Students Newman– Keuls Method), the p-value for SS reduction and SCOD release was 0.035 and 0.008, respectively. This indicates a significant difference between S2 and S3 sludges for SS reduction and SCOD release. 3.2.2. Kinetic investigation of disintegration process The effect of bacterial strains on sludge disintegration (SS and Particulate COD degradation) was explored, and the outcomes were analyzed mathematically through first order kinetics. Decrement in SS (Fig. 2a) and PCOD (data not shown) in the S1, S2, and S3 sludges increased up to 42 h. The k value of the S1, S2, and S3 samples were 0.001 h1, 0.003 h1, 0.005 h1, and 0.001 h1, 0.002 h1, 0.005 h1 for SS and PCOD reductions, respectively. The k value of the S3 sample was observed to be higher than the other samples. The correlation coefficient of all three sludges was between 0.92 and 0.99, indicating a better fit between the model and experiment. 3.3. Biochemical methane potential assay
Fig. 2a. Effect of NaCl mediated biological pretreatment on SS reduction. The error bars represent the standard errors.
The outcomes of the BMP tests for all three samples (S1, S2 & S3) with model simulations are given in Fig. 3a. The S3 sample
S. Kavitha et al. / Bioresource Technology 192 (2015) 807–811
showed better biodegradability when compared to others. The disintegrated sludge samples hold larger amounts of soluble organic matters when compared to the raw WAS, which were biodegraded more hastily. Around day 15, the methane yield of the pretreated sludge becomes stabilized due to substrate exhaustion. Methane generation rate was improved initially, whilst the production results decreased progressively over time. Obviously, after 25 days of operation, the control had produced lower methane. For the validation study, the determination coefficient (R2 lies within the ranges 0.95–0.99) revealed that the model exhibited a good fit with the experimental values. Fig. 3b depicts the plot of evaluated parameter confidence region spaces of fd (fraction of degradable substrates- biodegradability) along x-axis and khyd (apparent hydrolysis rate) along y axis. As exposed in Fig. 3b, the S3 sample showed an enhanced degradability when compared to the other samples. The S3 sample was highly reliable with good confidence. On the contrary, S1 sample exposed poorer identifiable parameter and wider confidence region. Biodegradability ranges for S2 and S3 samples were found to be 0.13 and 0.23 (gCOD/gCOD) with narrow confidence regions which indicate higher precision (Batstone et al., 2009). The biodegradability range of S1 was found to be 0.05 (gCOD/gCOD) with wider confidence interval which indicates lower precision. The khyd values of all the three samples – S1, S2 and S3 sludges (0.089 day1, 0.146 day1 and 0.193 day1) varied substantially. The lower value of fd and khyd in S1, designated that the substrates were metabolized more slowly with a lesser degree. The overall findings from the present study supports the general applicability of the proposed method as well as its predictive capability and accuracy.
0.22
0.16
Floc disrupted
Control
0.13
0.1
0.07
0.04 0.03
0.07
0.11
0.15
0.19
0.23
Substra te biodegra da bility (f d) Fig. 3b. Comparison of confidence regions for biodegradability (fd) and hydrolysis disintegration constant (khyd).
Table 1 Evaluation of energy balance and cost assessment. S. No. 1 2 3 4
5 6 7
3.4. Energy balance and cost evaluation To compute the proficiency of S2 and S3 sludges in terms of net energy production, an energy balance investigation was performed and the outcomes are tabulated in Table 1. Table 1 elucidates the energy equilibrium and cost assessment of the S3 and S2 samples based on energy content of the biogas produced from both the samples. To arrive the energy applied, (Table 1, S. No. 6) energy spent for mechanical stirring (EPS removal, bacterial inoculum, pretreatment and anaerobic digestion) and pumping of the sludge was taken into account. The total input energy was calculated to be 422.1 and 419.2 for S3 and S2 sludges, respectively. Concurrently, the energy content of the biogas for S3 and S2 samples were calculated to be 166 kWh and 57.95 kWh, respectively. Besides, in order to evaluate the economic viability of the disintegration process, estimation of operational cost (including consumable chemicals)
Bacterially pretreated
0.19
Hydrolysis rate control (k)
810
8 9 10 11 12
Energy balance (per ton of sludge) Average increase in biogas production Energy content of biogas Energy applied (for EPS removal) Energy applied (for stirring – bacterial growth, pretreatment & anaerobic digestion)a Energy applied (for pumping)a Total energy applied (S. No. 3 + 4 + 5) Net energy production (S. No. 2–6) Cost calculation (per ton of sludge) Energy cost (S. No. 7a0.23 USD/kWh) Decrease in SS to be disposed Reduced cost for sludge disposal (S. No. 9 a 0.28 USD/kg TS) Chemical cost for floc disruption & bacterial growth Net profit
S3 sludge
S2 sludge
Unit
27192 166 2.9 417.6
9500 57.95 – 417.6
L kWh kWh kWh
1.6 422.1 256.1
1.6 419.2 361.3
kWh kWh kWh
58.9 405 113.4
83.1 220 61.6
USD kg USD
52
–
USD
2.5
21.5
USD
a
Calculated based on Metcalf & Eddy (2003), S3 – floc disrupted and bacterially disintegrated, S2 – bacterially disintegrated alone.
and the decreased amount of solids to be disposed was considered in the present study. From Table 1, it was found that higher net profit was achieved for (S3) sludge compared to (S2) sludge. The net profit of S3 and S2 sludges was calculated to be 2.5 USD and 21.5 USD, respectively. Based on the above details, it can be concluded that the phase separated bacterial pretreatment was observed to be cost-effectively viable approach.
4. Conclusion The effects of NaCl mediated bacterial phase separation on biogas production from WAS were investigated and compared to sole bacterial cell disintegration. It was found that floc disruption with 0.03 g/g SS of NaCl and biomass disintegration with bacteria was a very favorable method and the increase of solubilization (23%) was beneficial to thr biodegradability process. The phase separated bacterial disintegration on biochemical methane potential benefited the production of methane efficiently. Acknowledgements
Fig. 3a. Plot showing disintegration.
methane
production
of
NaCl
mediated
biological
Authors are grateful to DST, India for offering economic assistance for this work (SR/FTP/ETA-0021/2010) on behalf of Young Scientist scheme.
S. Kavitha et al. / Bioresource Technology 192 (2015) 807–811
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