To demonstrate the feasibility of bioaugmentation in enhancement of the biodegradation of pulping effluent, aerobic sludge was intensified with superior mixed flora. The differences between intensified aerobic activated sludge and original sludge were compared. The results showed that the chemical oxygen demand (COD) of pulping effluent treated with the intensified sludge dropped to a much lower level compared with the original sludge, which indicated that the biodegradability of sludge was enhanced by bioaugmentation. The growth kinetics of the sludges were established. The growth rate Vmax of the intensified sludge was elevated from 7.8×10-3 to 7.1×10-3, while the saturation constant Ks decreased from 0.33 to 0.21 after bioaugmentation. In addition, the degradation kinetics showed that the equation coefficient of sludge increased from 4.6×10-3 to 6.4×10-3, confirming the intensification of biodegradation as a result of bioaugmentation.
Enhanced Biodegradability of Aerobic Sludge by Bioaugmentation for Pulping Effluent Treatment
Honglei Chen,* Guihua Yang, Jiachuan Chen, and Yu Liu
To demonstrate the feasibility of bioaugmentation in enhancement of the biodegradation of pulping effluent, aerobic sludge was intensified with superior mixed flora. The differences between intensified aerobic activated sludge and original sludge were compared. The results showed that the chemical oxygen demand (COD) of pulping effluent treated with the intensified sludge dropped to a much lower level compared with the original sludge, which indicated that the biodegradability of sludge was enhanced by bioaugmentation. The growth kinetics of the sludges were established. The growth rate Vmax of the intensified sludge was elevated from 7.8×10-3 to 7.1×10-3, while the saturation constant Ksdecreased from 0.33 to 0.21 after bioaugmentation. In addition, the degradation kinetics showed that the equation coefficient of sludge increased from 4.6×10-3 to 6.4×10-3, confirming the intensification of biodegradation as a result of bioaugmentation.
Keywords: Pulping effluent; Aerobic activated sludge; Kinetics; Bioaugmentation
Contact information: State Key Lab of Pulp and Paper Science and Technology of the Ministry of Education, Qilu University of Technology, Jinan, 250353, Shandong, China;
* Corresponding author: firstname.lastname@example.org
The activated sludge process was developed in the early 1900s for the treatment of domestic wastewater, and it has since been adapted for removing biodegradable organic matter from industrial wastewater (Dufresne et al. 1998; Sarlin et al. 1999; Frolund et al. 1995; Chen and Lo 2006). Currently, it is widely employed in the treatment of both municipal and industrial wastewaters. Activated sludge uses a combination of microorganisms to aerobically treat wastewater. Organic contaminants in the wastewater provide the carbon and energy required to encourage microbial growth and reproduction. In the activated sludge process, wastewater is thoroughly mixed with air or oxygen to develop a biological floc that can reduce the organic matter in the wastewater and convert it into microbial cell tissue and carbon dioxide. At present, the activated sludge process has been given more attention by researchers studying the control of effluents from pulp and paper mills as a popular biodegradation method (Demirbas et al. 1999; Junna and Ruonala 1991; Chandra 2001). Activated sludge has good settling properties, high volume loading, a rich biological phase, and high biological activity. In recent years, activated sludge has been successfully used in the process of treating pulping effluent, such as pulp washing water and bleach effluents.
The bioaugmentation technique can be used to enhance the activity of activated sludge by introduction of a group of natural or genetically engineered microorganisms (Bathe et al. 2009; Wang et al. 2006). Compared with the common biotreatment process, bioaugmentation of specialized-microorganisms can enhance the removal efficiency of target pollutants significantly. This is achieved by strengthening or complementing the metabolic capabilities of the indigenous microbial community (van Limbergen et al. 1998). A superior mixed flora was formed using statistical experimental design in our previous study (Chen et al. 2010). The aerobic granular sludge was intensified with the bacteria flora in this study to improve the biodegrading capability of the sludge. Pulping effluent was treated with both the original and intensified sludges, and advantages of bioaugmentation were demonstrated by comparison of the degradation effects.
Microorganisms and activated sludge
Four strains of bacteria, i.e., Agrobacterium sp., Bacillus sp., Gordonia sp., and P. putid, were used to prepare the inoculum for degrading contaminants in pulping effluents. Anaerobic granular sludge was obtained from Guangxi Guitang Group Co., China. Aerobic granular sludge was taken from Rizhao Jinhe Group Co., China.
The pulping effluent used in this study was a mixture of three-stage bleaching effluents (chlorine dioxide stage 60%, chelate stage 20%, hydrogen peroxide stage 20%) from a bamboo kraft mill, with COD of 1325 mg/L and chroma of 862 C.U. The pH value of the pulping effluent was adjusted to 7.0, and then 200 mL of effluent was placed in a 500 mL conical flask, which was capped and sterilized at 121 °C for 15 min.
Strain culture and bacterial collection
To recover the activity of the stock culture, one loop from each of the four bacteria from the culture-contained agar was separately transferred to the appropriate medium (Agrobacterium sp. and Bacillussp. inoculated Nutrient Broth medium, Gordonia sp. inoculated Yeast Extract Glucose medium, and Pseudomonas putida inoculated bacterium medium) in a glass flask. Each bacterium was activated at 30 oC for 36 h. These activated cells were harvested after centrifugation (5000 rpm for 5 min), resuspended in PBS (NaCl 8 g/L, KCl 0.2 g/L, KH2PO4 0.2 g/L, and K2HPO4 1.15 g/L), and then centrifuged again for cleaning.
Intensification of the aerobic granular sludge
The collected four kinds of bacteria were transferred to separate 300-mL conical flasks. Each flask contained 100 mL of fresh LB medium (tryptone, 10.0 g/L; yeast powder, 5 g/L; NaCl, 10 g/L) and 0.5 g (dry weight) aerobic granular sludge. Microorganisms and sludge were co-cultured in flasks at 30 °C for 24 h to ensure that the newly added bacteria were fixed on the granular sludge. The intensified sludge was collected by centrifugation at a speed of 5000 rpm and stored after washing with PBS buffer.
Anaerobic biological treatment of pulping effluent
First, 200 mL of pulping effluent was placed in a 500-mL conical flask that contained 0.6 g (dry weight) anaerobic granular sludge. The glass flask was subsequently flushed with N2 for 5 min to create anaerobic conditions and was then sealed and incubated at the optimal mesophilic temperature range (30±1 oC) with a stirring speed of 150 rpm. The experiments were performed in triplicate.
Aerobic biological treatment of pulping effluent
Pulping effluent treated by anaerobic sludge was divided into two parts, each 100 mL, which were poured separately into 300 mL conical flasks. The intensified aerobic sludge and the original aerobic sludge were added separately into two glass flasks for the further degradation of pulping effluent. The concentration of sludge was 3 g/L (dry weight), and it was cultured with shaking rate of 150 rpm at 30 °C. Ten milliliters of pulping effluent was taken from each conical flask for the analysis of COD and chroma every 8 h. The experiments were performed in triplicate.
COD and chroma determination
The COD determination was conducted according to the method of the Association of Official Analytical Chemists (1990) in this study (AOAC 1990). The chroma determination was performed according to the CPPA method (CPPA 1974).
RESULTS AND DISCUSSION
Anaerobic Biological Treatment
To degrade the organic contaminants and remove chroma in pulping effluent efficiently, pulping effluent was treated with anaerobic sludge prior to the activated sludge process. Figure 1 shows the UV-vis spectra of the original effluent and the effluent treated with anaerobic sludge.
Fig. 1. UV-visible spectroscopy of original effluent and effluent treated by anaerobic sludge
The spectral signal of the treated pulping effluent is significantly weaker than that of the original effluent, especially in the range of 230 to 450 nm, which means that the organic contaminants in the pulping effluent were degraded greatly in the process of anaerobic treatment. As for the spectral absorption above 550 nm, the treated pulping effluent is slightly stronger than the original pulping effluent, which could be caused by the suspended solids falling off from sludge, because the fine suspended particles in liquid usually have an absorption of around 600 nm (Aoyagi and Yabusaki 2010). The COD and chroma analysis results showed that the COD of the pulping effluent was reduced from 1325 mg/L to 629 mg/L after the anaerobic sludge treatment, with the removal rate of 52.53% (Table 1). Meanwhile, the chroma of the effluent was decreased from 862 C.U. to 118 C.U., with the removal rate of up to 86.31%. Obviously, anaerobic biological treatment can be used as an effective method to decrease the chroma and COD of pulping effluent.
Fig. 2. UV-vis spectroscopy of the pulping effluent treated with different kinds of sludge
Aerobic Biological Treatment
The UV-vis spectra of pulping effluent treated by the original aerobic sludge and the intensified aerobic sludge are shown in Fig. 2. The spectral signal of pulping effluent treated with the intensified sludge is significantly lower than that of pulping effluent treated by original sludge, particularly in the range of 220 nm to 500 nm, which means that the biodegradability of aerobic sludge was improved greatly after bioaugmentation. Table 2 shows the results with respect to the COD and chroma of pulping effluent after treatment by the original sludge and the intensified sludge. For the original sludge, COD decreased from 629 mg/L to 203 mg/L and chroma decreased from 118 C.U. to 91 C.U. In contrast, COD of pulping effluent treated with the intensified sludge was reduced to 146 mg/L, and chroma dropped to 72 C.U. The analysis results suggested the promotion of decontamination capability for the intensified sludge
The appearance of different pulping effluents is shown in Fig. 3. The wastewater discharged from pulp and paper mills usually contains lignin and its derivatives, so raw pulping effluent has a dark color (Fig. 3 (a)). However, the color of pulping effluent was greatly removed after anaerobic and aerobic treatment (Figs. 3 (b) and (c)). A comparison of both the aerobic sludges, it is obvious that the treatment effect was stronger when intensified sludge was used during the aerobic sludge process (Fig. 3 (c)).
Fig. 3. The appearance of raw pulping effluent and treated pulping effluents: (a) raw pulping effluent; (b) pulping effluent treated by anaerobic sludge and original aerobic sludge; (c) pulping effluent treated by anaerobic sludge and intensified aerobic sludge
Reaction Kinetics of the Aerobic Microbial Process
The relationship between the substrate concentration and microbial proliferation rate in the sludge can be described by the Michaelis-Menten equation (Voet and Voet 2001; Nelson and Cox 2000; Hommes 1960):
The rate equation of the bacterial cells’ growth in the activated sludge can be expressed as:
The growth rate of a certain moment is represented by Eq. 3.
The integral of the equation is is (3)
A linear equation was obtained by taking the reciprocal on both sides of Eq. 1:
In the above equations, ν is the microbial growth rate in the sludge; Vmax is the microorganisms’ maximum specific growth rate in sludge; Ks is the saturation constant; Cs is the mass concentration of the substrate, which is the COD of pulping effluent in this study; Cx is the mass concentration of the culture medium in the sludge; and Cx0 is the initial concentration of the culture medium in the sludge.
Pulping effluent was treated by original sludge and intensified sludge respectively, and the analysis results of the kinetic parameters are shown in Table 3.
Figure 4 shows the regression curves of 1/CS vs 1/ν for two kinds of sludge. Based on the corresponding regression curves, some microbial kinetic parameters of the sludge could be calculated. For the original sludge, 1/Vmax1 = 142.05, KS1/Vmax1 = 46.87, ν1 = 7.1×10-3, KS1 = 0.33, and the microbial growth kinetic equation of sludge is
The growth rate equation for the cells is
For reinforced sludge, 1/Vmax2 = 128.84, KS2/Vmax2 = 27.20, ν2 = 7.8×10-3, KS2 = 0.21, and the microbial growth kinetic equation of sludge is
The growth rate equation for the cells is
As can be seen from Eq. (2), Vmax and Ks can indicate the growth characteristics of the activated sludge. A greater Vmax and a smaller KS will result in a larger growth rate of sludge. Equations 5 and 7 show that, after bioaugmentation, Vmax increased from 7.1×10-3 to 7.8×10-3 and Ks decreased from 0.33 to 0.21, which suggested that the growth rate of the intensified sludge was higher than that of the original sludge.
Fig. 4. Regression of 1/CS and1/ν
Degradation Kinetics of Pulping Effluent in the Aerobic Sludge Process
In the substrate consumption kinetic model including endogenous metabolism, the substrate consuming rate (Rs) can be described by Eq. 9 (Voet and Voet 2001; Nelson and Cox 2000).
In the degradation process of pulping effluent, the yield of new cells (YX/S) in activated sludge was calculated according to Eq. 10, and the substrate consuming rate (COD degrading rate) can be associated with the growth rate of microorganisms by the new cell yield coefficient, expressed as Eq. 11.
The specific consuming rate of pulping effluent (Vs) can be defined as:
A new formula can be obtained when Eq. (11) is substituted into Eq. 12,
where Rs is the substrate consuming rate; Vs is the specific consuming rate of pulping effluent; Y2X/S is the maximum cell yield, which means the ratio between the mass of new cells and the mass of the substrate consumed in the cell growth process; YX/S is the new cell yield corresponding to the total substrate consumption; and k is the maintenance coefficient of cell growth.
The data analysis results with respect to the COD consuming kinetics of pulping effluent are shown in Table 4. In the activated sludge, the bacteria maintained their cell growth and metabolism by using the organic contaminants in pulping effluent as the sole carbon source. Therefore, the metabolic process of the organic matter in pulping effluent can be described by the substrate consumption kinetic model including endogenous metabolism. The regression curves of 1/ν and 1/YX/S for the two kinds of sludge are shown in Fig. 5.
Fig. 5. Regression of 1/ν and 1/YX/S
From the curve equations, several kinetic parameters for the two kinds of sludge for pulping effluent degradation were obtained. For the original sludge, k = 0.126, 1/ Y2X/S = 0.652, and the relationship between the COD removal rate and the yield of new cells in activated sludge can be expressed as:
The kinetics equation for the original sludge can be calculated by combining Eq. 6 with Eq. 14:
Similarly, for the intensified sludge, k = 0.083, 1/Y2X/S = 0.815, and the relationship between the COD removal rate and new cell yield can be expressed by Eq. 16:
The degradation kinetics equation for the intensified sludge can be obtained by combining Eq. 8 with Eq. 16:
It can be seen from the degradation kinetics equation that the equation coefficient and KS are two important parameters. A greater equation coefficient and smaller KS are beneficial to the substrate consuming rate (RS). A change in Ks has been described in the growth kinetics equation for sludge, which decreased from 0.33 to 0.21. The equation coefficients of the two kinds sludge are shown in Eqs. (15) and (17): the coefficient of the intensified sludge is 6.4×10-3, which is higher than that of the original sludge (4.6×10-3). Therefore, the COD degrading rate of activated sludge was improved after bioaugmentation with superior mixed flora.
Feasibility Analysis of Bioangmentation
Bioaugmentation, being an economical and eco-friendly approach, has emerged as the most advantageous wastewater clean-up technique for contaminated sites containing organic pollutants and/or heavy metals. Addition of specialized-microorganisms is able to enhance the degradation efficiency of unwanted compounds, shorten the processing time, and reduce the cost of subsequent processing. Therefore, bioangmentation has high economic feasibility for the treatment of wastewater. However, many factors such as strain selection, microbial ecology, type of pollutant, as well as procedures of culture introduction, may affect the result of bioaugmentation or even lead to failure. Several successful studies on bioaugmentation were carried out in laboratorial-scale (Head and Oleszkiewicz 2004; Wang et al. 2002), however the full-scale wastewater treatment process has rarely been tried due to the risk of irrecoverable process failure by unexpected operating problems. For our research, the way in which the full-scale bioaugmentation will be applied in pulping effluents degradation process will be a focus of our future studies.
- By comparing the differences between the intensified sludge and the original sludge in the process of pulping effluent degradation, it was found that bioaugmentation with superior mixed flora not only could enhance COD removal efficiency, but could also improve the decolorization capability.
- The reaction kinetics of the aerobic microbial process and the degradation kinetics of the pulping effluent were established in this study. The kinetics parameter Vmax of the intensified sludge was 7.8×10-3, which was higher than that of the original sludge, and the Ks of sludge changed greatly, decreasing from 0.33 to 0.21 after bioaugmentation. The intensified sludge had many advantages over the original sludge, such as a higher sludge growth rate and higher COD degrading rate for the pulping effluent. In addition, the feasibility of bioangmentation was also analyzed economically and technically.
The authors are grateful for the financial support from the National Science Foundation of China (Grant Nos. 31070525, 31270627, 31270626, and 31170547), the Prior Special Study of 973 Program (Grant No. 2011CB211705), the Project of Shandong Province Higher Education Science and Technology Program (No. 05032501), and the Natural Science Foundation of Shandong Province (Grant Nos. ZR2010CM065, ZR2011CM011, and ZR2010CQ004).
Aoyagi, H., and Yabusaki, K. (2010). “Turbidimetric method for evaluation of photocatalytic activities of suspended fine particles,” Nanotechnology Sci. Appl. 3(1), 85-90.
Association of Official Analytical Chemists (AOAC). (1990). Official Methods of Analysis (15th Ed.), Kenneth Helrick, Arlington.
Bathe, S., Schwarzenbeck, N., and Hausner, M. (2009). “Bioaugmentation of activated sludge towards 3-chloroaniline removal with a mixed bacterial population carrying a degradative plasmid,” Bioresour Technol. 100(12), 2902-2909.
Chandra, R. (2001). “Microbial decolourisation of pulp and paper mill effluent in presence of nitrogen and phosphorus by activated sludge process,” J. Environ. Biol. 22(1), 23-27.
Chen, C. K., and Lo, S. L. (2006). “Treating restaurant wastewater using a combined activated sludge-contact aeration system,” J. Environ. Biol. 27(2), 167-183.
Chen, H. L., Chen, Y. C., Zhan, H. Y., and Fu, S. Y. (2010). “Enhanced biodegradation of pulping effluents by a statistical experimental design using microbial consortia,” BioResources 5(3), 1581-1594.
CPPA. (1974). Technical section standard method H5P: “Color of pulp mill effluents,” Montreal, Canada: Canadian Pulp and Paper Association.
Demirbas, G., Gokcay, C. F., and Dilek, F. B. (1999). “Treatment of organic chlorine in pulping effluents by activated sludge,” Water Sci. Technol. 40(1), 275-279.
Dufresne, R., Lavallee, H. C., Lebrun, R. E., and Lo, S. N. (1998). “Comparison of performance between membrane bioreactor and activated sludge system for the treatment of pulping process wastewaters,” TAPPI J. 81(4), 131-135.
Frolund, B., Griebe, T., and Nielsen, P. H. (1995). “Enzymatic activity in the activated sludge floc matrix,” Appl. Microbiol. Biotechnol. 43(4), 755-761.
Head, M. A., and Oleszkiewicz, J. A. (2004). “Bioaugmentation for nitrification at cold temperatures,” Water Res. 38(3), 523-530.
Hommes, F. A. (1960). A Novel Aspect of the Integrated Michaelis-Menten Equation: A Contribution to the Chemistry of Human Haptoglobins, University of California, USA.
Junna, J., and Ruonala, S. (1991). “Trends and guidelines in water pollution control in the Finnish pulp and paper industry,” TAPPI J. 74(7), 105-111.
Nelson, D. L., and Cox, M. M. (2000). Lehninger Principles of Biochemistry, 3rd Ed., Worth Publishers, New York.
Sarlin, T., Halttunen, S., Vuoriranta, P., and Puhakka, J. (1999). “Effects of chemical spills on activated sludge treatment performance in pulp and paper mills,” Water Sci. Tech. 40(11-12), 319-325.
Van Limbergen, H., Top, E. M., and Verstraete, W. (1998). “Bioaugmentation in activated sludge: current features and future perspectives,” Appl Microb Biotechnol. 50(1), 16-23.
Voet, D., and Voet, J. G. (2001). Biochemistry, 3rd Ed., John Wiley and Sons, New York.
Wang, H., Liu, G., Li, P., and Pan, F. (2006). “The effect of bioaugmentation on the performance of sequencing batch reactor and sludge characteristics in the treatment process of papermaking wastewater,” Bioprocess Biosyst. Eng. 29(5-6), 283-289.
Wang, J. L., Quan X. C., Wu, L. B., Qian, Y., and Hegemann, W. (2002). “Bioaugmentation as a tool to enhance the removal of refractory compound in coke plant wastewater,” Process Biochem. 38(5), 777-781.
Article submitted: November 6, 2013; Peer review completed: December 26, 2013; Revised version received and accepted: February 20, 2014; Published: February 27, 2014.