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Effects of increasing organic loading rate on performance and microbial community shift of an up-flow anaerobic sludge blanket reactor treating diluted pharmaceutical wastewater
Journal of Bioscience and Bioengineering VOL. xx No. xx, 1e5, 2014 www.elsevier.com/locate/jbiosc
Effects of increasing organic loading rate on performance and microbial community shift of an up-flow anaerobic sludge blanket reactor treating diluted pharmaceutical wastewater Zhu Chen,1 Yuguang Wang,1 Kai Li,1 and Hongbo Zhou1, 2, * School of Minerals Processing and Bioengineering, Central South University, Changsha 410083, China1 and Key Laboratory of Biometallurgy of Ministry of Education, Central South University, Changsha 410083, China2 Received 8 October 2013; accepted 27 February 2014 Available online xxx
The performance of an up-flow anaerobic sludge blanket (UASB) reactor was investigated in the treatment of diluted pharmaceutical fermentation wastewater for a continuous operation of 140 days. The dynamics and compositions of the microbial community were monitored using polymerase chain reaction (PCR)-restriction fragment length polymorphism (PCR-RFLP) analysis. Increase of the organic loading rate (OLR) from 2.7 kg COD/m3 d to 7.2 COD/m3 d led to an increase in the COD removal efficiency from 83% to 91%. The dominant bacteria shifted from Proteobacteria (23.8%), Chloroflexi (14.5%) and Firmicutes (4.0%) to Firmicutes (48.4%), Bacteroidetes (9.5%) and Proteobacteria (5.4%). For archeaon, the dominant groups changed from Thermoplasmata (24.4%), Thermoprotei (18.0%) and Methanobacteria (30.8%) to Thermoplasmata (70.4%) and Methanomicrobia (16.8%). Firmicutes, Bacteroidetes, Thermoplasmata and Methanobacteria could outcompete other species and dominated in the reactor under higher OLR. The results indicated that, to some extent, microbial community shift could reflect the performance of the reactor and a significant community shift corresponded to a considerable process event. Ó 2014, The Society for Biotechnology, Japan. All rights reserved. [Key words: Pharmaceutical fermentation wastewater; Anaerobic digestion; Up-flow anaerobic sludge blanket; Polymerase chain reaction-restriction fragment length polymorphism; Microbial community]
Anaerobic digestion is an effective technology to treat different high-strength industrial wastewater. Compared with aerobic digestion, the anaerobic digestion would need less energy and space, produce less sludge, generate biogas and reduce pathogen level (1e5). Various high rate anaerobic bioreactors have been developed, among which the up-flow anaerobic sludge blanket (UASB) is the one that has been most widely used due to its high biomass concentration, microbial diversity, low cost, flexibility and ability to withstand fluctuation of pH and temperature (6e8). The fermentation process in the pharmaceutical industry generates a large volume of wastewater containing different kinds of organic compounds, which would be suitable for anaerobic digestion. However, this kind of wastewater is often subjected to fluctuation in quality and quantity, which results in variation in the organic loading rate (OLR). Increase of the OLR is a main factor that triggers instability of the anaerobic digestion since anaerobic microorganisms are sensitive to organic overloads (9). Better understanding of the structure and dynamic of the microbial community can help us gain deeper insights into the anaerobic digestion process. Previous studies have postulated that monitoring the characteristics of the microbial community could lead to an early
* Corresponding author at: School of Minerals Processing and Bioengineering, Central South University, Changsha 410083, China. Tel.: þ86 73188877216; fax: þ86 7318887216. E-mail address: [email protected] (H. Zhou).
detection of operational problems, making preventive action possible (10,11). Some studies indicated that microbial community change was related to better performance of reactors experiencing increase of the OLR and reduction of the hydraulic residence time (HRT) (12,13), while others demonstrated that no significant change of microbial community structure was observed with deterioration of performance under OLR shocking (14). These conflicting results indicate that under different OLR, microbial community shift and reactor performance, as well as their relationship, may vary according to the types of reactors and characteristics of wastewater treated. Many studies have been conducted to investigate the effects of various OLR on the microbial community shift and performance of the UASB reactor treating nonpharmaceutical wastewater (15,16), or other types of reactors treating pharmaceutical wastewater from chemical synthesis process (14). However, the impacts of increasing OLR on the microbial community change and performance of the UASB reactor treating pharmaceutical wastewater from fermentation process have rarely been reported. Up to now, many culture-free molecular techniques, such as denaturant gradient gel electrophoresis (DGGE) (17), real-time Polymerase chain reaction (RT-PCR) (18) and PCR-restriction fragment length polymorphism (PCR-RFLP) (19) have been used to investigate the microbial community in the anaerobic digesters treating wastewater. These analyses can provide us with comprehensive pictures of microbial compositions and dynamics in anaerobic digestion.
1389-1723/$ e see front matter Ó 2014, The Society for Biotechnology, Japan. All rights reserved. http://dx.doi.org/10.1016/j.jbiosc.2014.02.027
Please cite this article in press as: Chen, Z., et al., Effects of increasing organic loading rate on performance and microbial community shift of an up-flow anaerobic sludge blanket reactor treating diluted..., J. Biosci. Bioeng., (2014), http://dx.doi.org/10.1016/j.jbiosc.2014.02.027
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In this study, we investigated the performance of a UASB reactor treating diluted pharmaceutical fermentation wastewater under different OLR. Meanwhile, the microbial community compositions and dynamics were monitored using the PCR-RFLP analysis.
MATERIALS AND METHODS Seed sludge, source and characteristics of wastewater The sludge inoculated to the UASB reactor was obtained from a plant treating citric acid wastewater in Anhui province, China. The wastewater treated in this study was provided by the Hunan Flag Bio-tech Co. Ltd. Before entering the reactor, the wastewater was diluted to keep the concentration of chemical oxide demand (COD) at about 4000 mg/L. The characteristics of the wastewater are presented in Table 1. The operation of the UASB A laboratory scale UASB reactor with a working volume of 10 L was inoculated with 4 L granular sludge and used to treat the diluted pharmaceutical wastewater. The influent was conducted to the bottom of the reactor by a peristaltic pump. The reactor was incubated in a water jacket to maintain the temperature at 35 C. On the top of the reactor, there is a gas-liquid-solid separator. The volume of the biogas produced was measured by a wet gas flow meter. COD, ammonia nitrogen (NH3eN) and pH were measured every two days. The experiment was divided into five periods (IeVI) based on the reduction of HRT. When a steady state was reached, the HRT was adjusted, in accordance with the desired OLR. Extraction of total genomic DNA and amplification of 16S rDNA The sludge samples were taken from the center of the reactor on Day 0, 58 and 140 when steady state reached. Then the total genomic DNA was extracted from the sludge using the Soil DNA kit (Promega, Madison, WI, USA) following the manufacturer’s instructions. The bacterial and archeal 16S rDNA were amplified with the universal primer sets 1492R (50 -CGGCTACCTTGTTACGACTT-30 ) and 27F (50 -AGAGTTTG ATCCTGGCTCA G-30 ) (20), and 21F (50 -CCGGTTGATCCY GCCGGA-30 ) and 958R (50 YCCGGCGTTGAMTCCAATT-30 ) (21), respectively. The protocol for the amplification was as follows: 94 C for 5 min, 32 cycles of 94 C for 45 s, 55 C for 45 s, 72 C for 1 min, and a final elongation cycle at 72 C for 10 min. PCR products were separated by gel electrophoresis on a 1% agar gel in Tris/acetate buffer and analyzed by staining with ethidium bromide under UV light. The expected band was excised and purified with a commercial kit (Promega). Construction of clone libraries and RFLP analysis The purified 16S rDNA was ligated to the pGEM-T vector (Tiangen, China) and transformed into the Escherichia coli TOP10 (Tiangen, China) in according to the manufacturer’s guidelines for white-blue screen. 160 and 120 positive clones for bacteria and archaeon were randomly selected from each library. The inserted fragments were amplified using the vector specific premiers T7 (50 -TAATACGACTCACTATAGGG-30 ) and SP6 (50 ATTTAGGTGACACTATAG-30 ). Subsequently, the amplicons were digested overnight by the restriction enzyme MspI (Fermentas) and Hin6I (Fermentas) at 37 C. The restriction fragments were separated by electrophoresis on 3% agar gel in Tris/acetate buffer. After being stained by ethidium bromide, the RFLP patterns were visually detected and then used to screen and classify clones into different groups, denominated as operational taxonomic units (OTUs) .The RFLP patterns occurred only once or twice were exempted from sequencing. Then representative from each OTU was chosen for commercial DNA sequencing (GenScript, China). 16S rRNA gene sequences obtained were checked for vector sequence contamination by VecScreen program (National Center for Biotechnology Information, NCBI) and for chimeric characteristics using Bellerophon program (22). Subsequently, the valid sequences were assigned to taxonomical groups using the Ribosomal Database Project (23). Each of the valid sequences was compared to publicly available databases using the basic local alignment search tool (BLAST) to determine approximate phylogenetic affiliations. The obtained sequences were aligned by Clustal X software (24) and the phylogenetic trees were constructed using the Bootstrap Neighbor-Joining algorithm in MEGA 5.1 (25).
FIG. 1. COD removal efficiency under different OLR. Symbols: triangles, COD removal efficiency; squares, OLR.
RESULTS The performance of the UASB During the start-up, the HRT was 38.5 h and the OLR was about 2.8 kg/m3 d (Fig. 1). The COD removal efficiency and biogas yield were relatively low at first, but both increased gradually. At the end of this stage, the removal efficiency maintained at 83% (Fig. 1) with slight fluctuations, and the biogas yield reached about 0.43 L/g CODremoved (Fig. 2). The HRT was changed to 32 h from the 33rd day. Noticeably, a drastic decline of COD removal efficiency was observed from the 44th to 48th day. Since wastewater diluted by a part of the effluent was used as influent in this period, the main factor causing the decline may be the accumulation of ammonia nitrogen and other recalcitrant substance (27,28). As shown in Fig. 3, the concentration of the ammonia nitrogen in the effluent reached up to about 800 mg/L, which might cause the change of the intracellular pH, as well as the inhibition of specific enzyme activity (28). The increase of ammonia nitrogen could partly be attributed to the biological degradation of the nitrogenous matter, mostly in the form of proteins (28). Our result was also in accordance with other studies (29,30). Along with the increase of the ammonia nitrogen, the pH (Fig. 4) increased as well. The optimal pH for the growth of the methanogenic bacteria in the anaerobic digestion is between 6.5
Analytical methods COD, total Kjeldahl nitrogen (TKN), total phosphorus (TP), NH3eN and pH were determined according to the standard method (26). Nucleotide sequence accession numbers The nucleotide sequences of cloned 16S rRNA genes have been deposited in the GenBank database under accession numbers KF564554 to KF564607.
TABLE 1. Characteristics of the pharmaceutical wastewater. Parameter COD/mg L1 TKN/g L1 TP/g L1 NH3eN/mg L1 pH
Average value
Standard deviation
20.140 2.57 0.42 988.6 6.42
8489.58 0.22 0.48 724.99 1.08
FIG. 2. Evolution of COD concentration in the influent and effluent, and biogas yield. Symbols: closed triangles, COD concentration of effluent; closed squares, COD concentration of influent; open squares, biogas yield.
Please cite this article in press as: Chen, Z., et al., Effects of increasing organic loading rate on performance and microbial community shift of an up-flow anaerobic sludge blanket reactor treating diluted..., J. Biosci. Bioeng., (2014), http://dx.doi.org/10.1016/j.jbiosc.2014.02.027
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FIG. 3. Concentration of ammonia nitrogen shift in the influent and effluent. Symbols: open circles, concentration of ammonia nitrogen in effluent; closed squares, concentration of ammonia nitrogen in influent.
and 7.2 (31). The increase of pH would cause the ammonium to transform to the free ammonia, which is more toxic in that the free ammonia can pass through the cell membrane (28,31). In order to mitigate the adverse effect of the ammonia nitrogen, wastewater directly diluted by the tap water was fed in the reactor. As the inhibiting factors were removed, the COD removal efficiency gradually recovered and reached 83% at the end of period II. The HRT was changed to 27 h and the reactor began to experience the third stage on the 59th day. During period III, though the COD removal efficiency and biogas yield declined a little bit in the middle of this stage, the performance of the reactor soon recovered. The reactor entered period IV on the 87th day. A significant deterioration of the bioreactor performance was observed from the 92nd day due to the change in characteristics of original wastewater, which had much higher pH (about 8.0) and concentration of ammonia nitrogen (up to 1200 mg/L). On the 100th day, the COD removal efficiency had dramatically dropped to 50%. Later on, the performance of the bioreactor recovered as the characteristics of the influent was changed back. Eventually, the COD removal efficiency reached about 90%. Hence, the HRT was changed to 13.5 h on the 121st day. In period V, even if the reactor performance experienced a decline at first, it soon recovered. When the reactor performance maintained stable eventually, the COD removal efficiency could reach nearly 91% and the COD concentration of the effluent was about 400 mg/L.
FIG. 4. pH variation in the influent and effluent. Symbols: inverted triangles, pH in effluent; squares, pH in influent.
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The compositions and dynamics of the bacterial and archeal community The bacterial community shift is shown in Fig. 5 (left) and the phylogenetic tree of the bacterial community is presented by Fig. S1A. Before being inoculated to the UASB reactor, the dominated bacteria in the sludge belonged to three phyla: Proteobacteria (23.8%), Chloroflexi (14.5%) and Firmicutes (4.0%). When the reactor had been operated for 58 days and the OLR was about 3 kg/m3 d, a significant change of the compositions of the bacterial community could be observed. The bacterial community of the second sample mainly consisted of three groups, belonging to the phylum Firmicutes (20%), Bacteroidetes (17.1%), and Proteobacteria (5.1%). Firmicutes, replacing Proteobacteria, became dominant in the reactor. Bacteroidetes, which could not be detected at the beginning, also became a dominant group. When the reactor performance remained stable under the OLR of 7.2 kg/m3 d, the structure of the bacterial community drastically changed. The Firmicutes continued to be the most dominant group and its relative abundance increased to 48.4%. Although the Bacteroidetes was still relatively abundant in the reactor, its relative abundance dropped to 9.5%. But, the relative abundance of the Proteobacteria remained almost the same (5.4%). The archeal community shift is shown in Fig. 5 (right) and the phylogenetic tree of the archeal community is presented by Fig. S1B. For archaeon, the identified species through RFLP analysis before the start-up could be assigned to three main classes: Thermoplasmata (24.4%), Thermoprotei(18.0%) and Methanobacteria (30.8%), which are common archaeon found in anaerobic digesters (32,33). At the end of periodⅡ, the Methanomicrobia (35.8%), instead of the Methanobacteria, became the most dominant archaeon in the bioreactor. Meanwhile, the Thermoprotei could not be detected any more. But, the relative abundance of Thermoplasmata in the bioreactor increased to 25.6%. When the OLR was about 7.2 COD/ m3 d, the Thermoplasmata (70.4%), replacing the ever most predominant Methanomicrobia (16.8%), became the most dominant archeal group in the bioreactor.
DISCUSSIONS The results indicated that before the start-up, the predominant bacterial population in the sludge belonged to the phylum Proteobacteria, which could be further divided into Betaproteobacteria (10.3%), Gammaproteobacteria (4.3%) and Deltaproteobacteria (9.2%). For the clones assigned to the phylum Proteobacteria, most of them were closely related to some denitrifying bacteria detected in the anaerobic digesters. For instance, the clone BP0-6 (KF564555) was similar to Acidovorax sp. R-25074 (AM084034), a denitrifying bacteria isolated from sludge (34). The clone BP0-80 (KF564564) assigned to it was very similar to the Thermomonas fusca strain LMG 21739 (AJ519988), a mesophilic species isolated from a denitrification reactor (35). Other clones belonging to the phylum Chloroflexi, such as BP0-1(KF564554) and BP0-92 (KF564558), were significantly related to the Bacterium JN18_A7_F* (DQ168648) which was detected in the anaerobic reactor treating chlorinated polychlorinated biphenyls (36). However, when the OLR increased to 3.0 kg/m3 d, Firmicutes, rather than the Proteobacteria, became the most predominant group in the sludge. The predominance of the Firmicutes was understandable for the reason that Firmicutes are well-known fermenters and syntrophic bacteria to degrade volatile fatty acids (37), which could increase with the concomitant increase of OLR (38). Another dominant phylum Bacteroidetes are proteolytic bacteria (39), which may be involved in the degradation of the proteins. The increased concentration of ammonia nitrogen in the effluent, which derived from the degradation of proteins, was a good indicator of the active role of the Bacteroidetes in the reactor.
Please cite this article in press as: Chen, Z., et al., Effects of increasing organic loading rate on performance and microbial community shift of an up-flow anaerobic sludge blanket reactor treating diluted..., J. Biosci. Bioeng., (2014), http://dx.doi.org/10.1016/j.jbiosc.2014.02.027
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FIG. 5. Bacterial community shift (left) and archeal community shift (right) in different periods.
With the enrichment of Firmicutes and Bacteroidetes, the performance of the bioreactor at the end of the second stage was much better than that at the beginning of the periodⅠ. When the OLR increased to 7.2 kg/m3 d, the relative abundance of the Firmicutes increased to 48.4% and the Bacteroidetes declined to 9.5%. Firmicutes and Bacteroidetes are known for being extremely resistant and versatile microorganisms (40). Previous studies have found that these microorganisms were able to degrade complex organic compounds like vegetable and fruit residues, and abundant in reactors treating such organic compounds (37,40). Hence, Firmicutes and Bacteroidetes can better adapt to the higher OLR and dominated in the reactor. All this together could partly explain the abundance of Firmicutes and Bacteroidetes, and the good COD removal efficiency (nearly 91%, Fig. 1) under higher OLR. For archaeon, the main groups in the original sludge were Thermoplasmata, Thermoprotei and Methanobacteria. When the OLR was approximately 3.0 kg/m3 d, Methanomicrobia was the most abundant group. All the clones belonging to Methanomicrobia were affiliated with the genus Methanosaeta, which is an important acetoclastic methanogen in many high-strength organic wastewater treatment bioreactors (41). The genus Methanosaeta also plays an important role in the onset of granulation and maintenance of stable granules during system perturbations (42). The increased populations of genus Methanosaeta partly account for the good performance and increased biogas yield in the second period. However, at the end of the operation, Thermoplasmata, substituting the Methanomicrobia, became the most dominant archaeon. A similar shift had also been observed in the expanded granular sludge bed (EGSB) reactor treating wastewater from used industrial oils (43). The Thermoplasmata was also found in the anaerobic treatment of complex organic waste and wastewater, such as
polycyclic aromatic hydrocarbons (44) and food waste, wastepaper (40). The clone AP5-44 (KF564607) belonging to the Thermoplasmata was closely related to the uncultured archaeon (AF050616) found in methanogenic core (45). Due to the intolerance to high ammonia for members of the family Methanosaetaceae (41), which was produced in the degradation of protein through anaerobic digestion (46), the Methanomicrobia may gradually diminish in the reactor. Previous work also found out that fewer methanogens dominated in the population when the anaerobic reactors were used to treat protein-rich wastewater (47). Thermoplasmata is grouped into the sulfur reducers and a facultative anaerobe, which respires using sulfur plus organic carbon (48). Early study has found that the Thermoplasmata may be involved in the reduction of sulfate (49), which could be produced in the degradation of protein. It usually appears in extremophilic environments (43) and may outcompete the methanogenic archaeon in the anaerobic reactor. In conclusion, the study indicated that the UASB bioreactor could efficiently treat diluted pharmaceutical fermentation wastewater under the OLR ranging from 2.7 kg COD/m3 d to 7.2 COD/ m3 d. Firmicutes, Bacteroidetes, Thermoplasmata and Methanobacteria could better adapt to higher OLR and gradually played a leading role in the improvement of the reactor performance. Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.jbiosc.2014.02.027.
ACKNOWLEDGMENTS The authors are very grateful to the support provided by Hunan Flag Bio-tech Co., Ltd.
Please cite this article in press as: Chen, Z., et al., Effects of increasing organic loading rate on performance and microbial community shift of an up-flow anaerobic sludge blanket reactor treating diluted..., J. Biosci. Bioeng., (2014), http://dx.doi.org/10.1016/j.jbiosc.2014.02.027
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Collins, G., Woods, A., McHugh, S., Carton, M. W., and O’Flaherty, V.: Microbial community structure and methanogenic activity during start-up of psychrophilic anaerobic digesters treating synthetic industrial wastewaters, FEMS Microbiol. Ecol., 46, 159e170 (2003). Garcia-Mancha, N., Puyol, D., Monsalvo, V. M., Rajhi, H., Mohedano, A. F., and Rodriguez, J. J.: Anaerobic treatment of wastewater from used industrial oil recovery, J. Chem. Technol. Biotechnol., 87, 1320e1328 (2012). Zhang, S. Y., Wang, Q. F., and Xie, S. G.: Molecular characterization of phenanthrene-degrading methanogenic communities in leachate-contaminated aquifer sediment, Int. J. Environ. Sci. Technol., 9, 705e712 (2012). Dojka, M. A., Hugenholtz, P., Haack, S. K., and Pace, N. R.: Microbial diversity in a hydrocarbon- and chlorinated-solvent-contaminated aquifer undergoing intrinsic bioremediation, Appl. Environ. Microbiol., 64, 3869e3877 (1998). Palatsi, J., Vinas, M., Guivernau, M., Fernandez, B., and Flotats, X.: Anaerobic digestion of slaughterhouse waste: main process limitations and microbial community interactions, Bioresour. Technol., 102, 2219e2227 (2011). Acs, N., Kovacs, E., Wirth, R., Bagi, Z., Strang, O., Herbel, Z., Rakhely, G., and Kovacs, KL: Changes in the archaea microbial community when the biogas fermenters are fed with protein-rich substrates, Bioresour. Technol., 131, 121e127 (2013). Chaganti, S. R., Lalman, J. A., and Heath, D. D.: 16S rRNA gene based analysis of the microbial diversity and hydrogen production in three mixed anaerobic cultures, Int. J. Hydrog. Energy, 37, 9002e9017 (2012). Gough, H. L. and Stahl, D. A.: Microbial community structures in anoxic freshwater lake sediment along a metal contamination gradient, ISME J., 5, 543e558 (2011).
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Effects of increasing organic loading rate on performance and microbial community shift of an up-flow anaerobic sludge blanket reactor treating diluted pharmaceutical wastewater Zhu Chen,1 Yuguang Wang,1 Kai Li,1 and Hongbo Zhou1, 2, * School of Minerals Processing and Bioengineering, Central South University, Changsha 410083, China1 and Key Laboratory of Biometallurgy of Ministry of Education, Central South University, Changsha 410083, China2 Received 8 October 2013; accepted 27 February 2014 Available online xxx
The performance of an up-flow anaerobic sludge blanket (UASB) reactor was investigated in the treatment of diluted pharmaceutical fermentation wastewater for a continuous operation of 140 days. The dynamics and compositions of the microbial community were monitored using polymerase chain reaction (PCR)-restriction fragment length polymorphism (PCR-RFLP) analysis. Increase of the organic loading rate (OLR) from 2.7 kg COD/m3 d to 7.2 COD/m3 d led to an increase in the COD removal efficiency from 83% to 91%. The dominant bacteria shifted from Proteobacteria (23.8%), Chloroflexi (14.5%) and Firmicutes (4.0%) to Firmicutes (48.4%), Bacteroidetes (9.5%) and Proteobacteria (5.4%). For archeaon, the dominant groups changed from Thermoplasmata (24.4%), Thermoprotei (18.0%) and Methanobacteria (30.8%) to Thermoplasmata (70.4%) and Methanomicrobia (16.8%). Firmicutes, Bacteroidetes, Thermoplasmata and Methanobacteria could outcompete other species and dominated in the reactor under higher OLR. The results indicated that, to some extent, microbial community shift could reflect the performance of the reactor and a significant community shift corresponded to a considerable process event. Ó 2014, The Society for Biotechnology, Japan. All rights reserved. [Key words: Pharmaceutical fermentation wastewater; Anaerobic digestion; Up-flow anaerobic sludge blanket; Polymerase chain reaction-restriction fragment length polymorphism; Microbial community]
Anaerobic digestion is an effective technology to treat different high-strength industrial wastewater. Compared with aerobic digestion, the anaerobic digestion would need less energy and space, produce less sludge, generate biogas and reduce pathogen level (1e5). Various high rate anaerobic bioreactors have been developed, among which the up-flow anaerobic sludge blanket (UASB) is the one that has been most widely used due to its high biomass concentration, microbial diversity, low cost, flexibility and ability to withstand fluctuation of pH and temperature (6e8). The fermentation process in the pharmaceutical industry generates a large volume of wastewater containing different kinds of organic compounds, which would be suitable for anaerobic digestion. However, this kind of wastewater is often subjected to fluctuation in quality and quantity, which results in variation in the organic loading rate (OLR). Increase of the OLR is a main factor that triggers instability of the anaerobic digestion since anaerobic microorganisms are sensitive to organic overloads (9). Better understanding of the structure and dynamic of the microbial community can help us gain deeper insights into the anaerobic digestion process. Previous studies have postulated that monitoring the characteristics of the microbial community could lead to an early
* Corresponding author at: School of Minerals Processing and Bioengineering, Central South University, Changsha 410083, China. Tel.: þ86 73188877216; fax: þ86 7318887216. E-mail address: [email protected] (H. Zhou).
detection of operational problems, making preventive action possible (10,11). Some studies indicated that microbial community change was related to better performance of reactors experiencing increase of the OLR and reduction of the hydraulic residence time (HRT) (12,13), while others demonstrated that no significant change of microbial community structure was observed with deterioration of performance under OLR shocking (14). These conflicting results indicate that under different OLR, microbial community shift and reactor performance, as well as their relationship, may vary according to the types of reactors and characteristics of wastewater treated. Many studies have been conducted to investigate the effects of various OLR on the microbial community shift and performance of the UASB reactor treating nonpharmaceutical wastewater (15,16), or other types of reactors treating pharmaceutical wastewater from chemical synthesis process (14). However, the impacts of increasing OLR on the microbial community change and performance of the UASB reactor treating pharmaceutical wastewater from fermentation process have rarely been reported. Up to now, many culture-free molecular techniques, such as denaturant gradient gel electrophoresis (DGGE) (17), real-time Polymerase chain reaction (RT-PCR) (18) and PCR-restriction fragment length polymorphism (PCR-RFLP) (19) have been used to investigate the microbial community in the anaerobic digesters treating wastewater. These analyses can provide us with comprehensive pictures of microbial compositions and dynamics in anaerobic digestion.
1389-1723/$ e see front matter Ó 2014, The Society for Biotechnology, Japan. All rights reserved. http://dx.doi.org/10.1016/j.jbiosc.2014.02.027
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In this study, we investigated the performance of a UASB reactor treating diluted pharmaceutical fermentation wastewater under different OLR. Meanwhile, the microbial community compositions and dynamics were monitored using the PCR-RFLP analysis.
MATERIALS AND METHODS Seed sludge, source and characteristics of wastewater The sludge inoculated to the UASB reactor was obtained from a plant treating citric acid wastewater in Anhui province, China. The wastewater treated in this study was provided by the Hunan Flag Bio-tech Co. Ltd. Before entering the reactor, the wastewater was diluted to keep the concentration of chemical oxide demand (COD) at about 4000 mg/L. The characteristics of the wastewater are presented in Table 1. The operation of the UASB A laboratory scale UASB reactor with a working volume of 10 L was inoculated with 4 L granular sludge and used to treat the diluted pharmaceutical wastewater. The influent was conducted to the bottom of the reactor by a peristaltic pump. The reactor was incubated in a water jacket to maintain the temperature at 35 C. On the top of the reactor, there is a gas-liquid-solid separator. The volume of the biogas produced was measured by a wet gas flow meter. COD, ammonia nitrogen (NH3eN) and pH were measured every two days. The experiment was divided into five periods (IeVI) based on the reduction of HRT. When a steady state was reached, the HRT was adjusted, in accordance with the desired OLR. Extraction of total genomic DNA and amplification of 16S rDNA The sludge samples were taken from the center of the reactor on Day 0, 58 and 140 when steady state reached. Then the total genomic DNA was extracted from the sludge using the Soil DNA kit (Promega, Madison, WI, USA) following the manufacturer’s instructions. The bacterial and archeal 16S rDNA were amplified with the universal primer sets 1492R (50 -CGGCTACCTTGTTACGACTT-30 ) and 27F (50 -AGAGTTTG ATCCTGGCTCA G-30 ) (20), and 21F (50 -CCGGTTGATCCY GCCGGA-30 ) and 958R (50 YCCGGCGTTGAMTCCAATT-30 ) (21), respectively. The protocol for the amplification was as follows: 94 C for 5 min, 32 cycles of 94 C for 45 s, 55 C for 45 s, 72 C for 1 min, and a final elongation cycle at 72 C for 10 min. PCR products were separated by gel electrophoresis on a 1% agar gel in Tris/acetate buffer and analyzed by staining with ethidium bromide under UV light. The expected band was excised and purified with a commercial kit (Promega). Construction of clone libraries and RFLP analysis The purified 16S rDNA was ligated to the pGEM-T vector (Tiangen, China) and transformed into the Escherichia coli TOP10 (Tiangen, China) in according to the manufacturer’s guidelines for white-blue screen. 160 and 120 positive clones for bacteria and archaeon were randomly selected from each library. The inserted fragments were amplified using the vector specific premiers T7 (50 -TAATACGACTCACTATAGGG-30 ) and SP6 (50 ATTTAGGTGACACTATAG-30 ). Subsequently, the amplicons were digested overnight by the restriction enzyme MspI (Fermentas) and Hin6I (Fermentas) at 37 C. The restriction fragments were separated by electrophoresis on 3% agar gel in Tris/acetate buffer. After being stained by ethidium bromide, the RFLP patterns were visually detected and then used to screen and classify clones into different groups, denominated as operational taxonomic units (OTUs) .The RFLP patterns occurred only once or twice were exempted from sequencing. Then representative from each OTU was chosen for commercial DNA sequencing (GenScript, China). 16S rRNA gene sequences obtained were checked for vector sequence contamination by VecScreen program (National Center for Biotechnology Information, NCBI) and for chimeric characteristics using Bellerophon program (22). Subsequently, the valid sequences were assigned to taxonomical groups using the Ribosomal Database Project (23). Each of the valid sequences was compared to publicly available databases using the basic local alignment search tool (BLAST) to determine approximate phylogenetic affiliations. The obtained sequences were aligned by Clustal X software (24) and the phylogenetic trees were constructed using the Bootstrap Neighbor-Joining algorithm in MEGA 5.1 (25).
FIG. 1. COD removal efficiency under different OLR. Symbols: triangles, COD removal efficiency; squares, OLR.
RESULTS The performance of the UASB During the start-up, the HRT was 38.5 h and the OLR was about 2.8 kg/m3 d (Fig. 1). The COD removal efficiency and biogas yield were relatively low at first, but both increased gradually. At the end of this stage, the removal efficiency maintained at 83% (Fig. 1) with slight fluctuations, and the biogas yield reached about 0.43 L/g CODremoved (Fig. 2). The HRT was changed to 32 h from the 33rd day. Noticeably, a drastic decline of COD removal efficiency was observed from the 44th to 48th day. Since wastewater diluted by a part of the effluent was used as influent in this period, the main factor causing the decline may be the accumulation of ammonia nitrogen and other recalcitrant substance (27,28). As shown in Fig. 3, the concentration of the ammonia nitrogen in the effluent reached up to about 800 mg/L, which might cause the change of the intracellular pH, as well as the inhibition of specific enzyme activity (28). The increase of ammonia nitrogen could partly be attributed to the biological degradation of the nitrogenous matter, mostly in the form of proteins (28). Our result was also in accordance with other studies (29,30). Along with the increase of the ammonia nitrogen, the pH (Fig. 4) increased as well. The optimal pH for the growth of the methanogenic bacteria in the anaerobic digestion is between 6.5
Analytical methods COD, total Kjeldahl nitrogen (TKN), total phosphorus (TP), NH3eN and pH were determined according to the standard method (26). Nucleotide sequence accession numbers The nucleotide sequences of cloned 16S rRNA genes have been deposited in the GenBank database under accession numbers KF564554 to KF564607.
TABLE 1. Characteristics of the pharmaceutical wastewater. Parameter COD/mg L1 TKN/g L1 TP/g L1 NH3eN/mg L1 pH
Average value
Standard deviation
20.140 2.57 0.42 988.6 6.42
8489.58 0.22 0.48 724.99 1.08
FIG. 2. Evolution of COD concentration in the influent and effluent, and biogas yield. Symbols: closed triangles, COD concentration of effluent; closed squares, COD concentration of influent; open squares, biogas yield.
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FIG. 3. Concentration of ammonia nitrogen shift in the influent and effluent. Symbols: open circles, concentration of ammonia nitrogen in effluent; closed squares, concentration of ammonia nitrogen in influent.
and 7.2 (31). The increase of pH would cause the ammonium to transform to the free ammonia, which is more toxic in that the free ammonia can pass through the cell membrane (28,31). In order to mitigate the adverse effect of the ammonia nitrogen, wastewater directly diluted by the tap water was fed in the reactor. As the inhibiting factors were removed, the COD removal efficiency gradually recovered and reached 83% at the end of period II. The HRT was changed to 27 h and the reactor began to experience the third stage on the 59th day. During period III, though the COD removal efficiency and biogas yield declined a little bit in the middle of this stage, the performance of the reactor soon recovered. The reactor entered period IV on the 87th day. A significant deterioration of the bioreactor performance was observed from the 92nd day due to the change in characteristics of original wastewater, which had much higher pH (about 8.0) and concentration of ammonia nitrogen (up to 1200 mg/L). On the 100th day, the COD removal efficiency had dramatically dropped to 50%. Later on, the performance of the bioreactor recovered as the characteristics of the influent was changed back. Eventually, the COD removal efficiency reached about 90%. Hence, the HRT was changed to 13.5 h on the 121st day. In period V, even if the reactor performance experienced a decline at first, it soon recovered. When the reactor performance maintained stable eventually, the COD removal efficiency could reach nearly 91% and the COD concentration of the effluent was about 400 mg/L.
FIG. 4. pH variation in the influent and effluent. Symbols: inverted triangles, pH in effluent; squares, pH in influent.
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The compositions and dynamics of the bacterial and archeal community The bacterial community shift is shown in Fig. 5 (left) and the phylogenetic tree of the bacterial community is presented by Fig. S1A. Before being inoculated to the UASB reactor, the dominated bacteria in the sludge belonged to three phyla: Proteobacteria (23.8%), Chloroflexi (14.5%) and Firmicutes (4.0%). When the reactor had been operated for 58 days and the OLR was about 3 kg/m3 d, a significant change of the compositions of the bacterial community could be observed. The bacterial community of the second sample mainly consisted of three groups, belonging to the phylum Firmicutes (20%), Bacteroidetes (17.1%), and Proteobacteria (5.1%). Firmicutes, replacing Proteobacteria, became dominant in the reactor. Bacteroidetes, which could not be detected at the beginning, also became a dominant group. When the reactor performance remained stable under the OLR of 7.2 kg/m3 d, the structure of the bacterial community drastically changed. The Firmicutes continued to be the most dominant group and its relative abundance increased to 48.4%. Although the Bacteroidetes was still relatively abundant in the reactor, its relative abundance dropped to 9.5%. But, the relative abundance of the Proteobacteria remained almost the same (5.4%). The archeal community shift is shown in Fig. 5 (right) and the phylogenetic tree of the archeal community is presented by Fig. S1B. For archaeon, the identified species through RFLP analysis before the start-up could be assigned to three main classes: Thermoplasmata (24.4%), Thermoprotei(18.0%) and Methanobacteria (30.8%), which are common archaeon found in anaerobic digesters (32,33). At the end of periodⅡ, the Methanomicrobia (35.8%), instead of the Methanobacteria, became the most dominant archaeon in the bioreactor. Meanwhile, the Thermoprotei could not be detected any more. But, the relative abundance of Thermoplasmata in the bioreactor increased to 25.6%. When the OLR was about 7.2 COD/ m3 d, the Thermoplasmata (70.4%), replacing the ever most predominant Methanomicrobia (16.8%), became the most dominant archeal group in the bioreactor.
DISCUSSIONS The results indicated that before the start-up, the predominant bacterial population in the sludge belonged to the phylum Proteobacteria, which could be further divided into Betaproteobacteria (10.3%), Gammaproteobacteria (4.3%) and Deltaproteobacteria (9.2%). For the clones assigned to the phylum Proteobacteria, most of them were closely related to some denitrifying bacteria detected in the anaerobic digesters. For instance, the clone BP0-6 (KF564555) was similar to Acidovorax sp. R-25074 (AM084034), a denitrifying bacteria isolated from sludge (34). The clone BP0-80 (KF564564) assigned to it was very similar to the Thermomonas fusca strain LMG 21739 (AJ519988), a mesophilic species isolated from a denitrification reactor (35). Other clones belonging to the phylum Chloroflexi, such as BP0-1(KF564554) and BP0-92 (KF564558), were significantly related to the Bacterium JN18_A7_F* (DQ168648) which was detected in the anaerobic reactor treating chlorinated polychlorinated biphenyls (36). However, when the OLR increased to 3.0 kg/m3 d, Firmicutes, rather than the Proteobacteria, became the most predominant group in the sludge. The predominance of the Firmicutes was understandable for the reason that Firmicutes are well-known fermenters and syntrophic bacteria to degrade volatile fatty acids (37), which could increase with the concomitant increase of OLR (38). Another dominant phylum Bacteroidetes are proteolytic bacteria (39), which may be involved in the degradation of the proteins. The increased concentration of ammonia nitrogen in the effluent, which derived from the degradation of proteins, was a good indicator of the active role of the Bacteroidetes in the reactor.
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FIG. 5. Bacterial community shift (left) and archeal community shift (right) in different periods.
With the enrichment of Firmicutes and Bacteroidetes, the performance of the bioreactor at the end of the second stage was much better than that at the beginning of the periodⅠ. When the OLR increased to 7.2 kg/m3 d, the relative abundance of the Firmicutes increased to 48.4% and the Bacteroidetes declined to 9.5%. Firmicutes and Bacteroidetes are known for being extremely resistant and versatile microorganisms (40). Previous studies have found that these microorganisms were able to degrade complex organic compounds like vegetable and fruit residues, and abundant in reactors treating such organic compounds (37,40). Hence, Firmicutes and Bacteroidetes can better adapt to the higher OLR and dominated in the reactor. All this together could partly explain the abundance of Firmicutes and Bacteroidetes, and the good COD removal efficiency (nearly 91%, Fig. 1) under higher OLR. For archaeon, the main groups in the original sludge were Thermoplasmata, Thermoprotei and Methanobacteria. When the OLR was approximately 3.0 kg/m3 d, Methanomicrobia was the most abundant group. All the clones belonging to Methanomicrobia were affiliated with the genus Methanosaeta, which is an important acetoclastic methanogen in many high-strength organic wastewater treatment bioreactors (41). The genus Methanosaeta also plays an important role in the onset of granulation and maintenance of stable granules during system perturbations (42). The increased populations of genus Methanosaeta partly account for the good performance and increased biogas yield in the second period. However, at the end of the operation, Thermoplasmata, substituting the Methanomicrobia, became the most dominant archaeon. A similar shift had also been observed in the expanded granular sludge bed (EGSB) reactor treating wastewater from used industrial oils (43). The Thermoplasmata was also found in the anaerobic treatment of complex organic waste and wastewater, such as
polycyclic aromatic hydrocarbons (44) and food waste, wastepaper (40). The clone AP5-44 (KF564607) belonging to the Thermoplasmata was closely related to the uncultured archaeon (AF050616) found in methanogenic core (45). Due to the intolerance to high ammonia for members of the family Methanosaetaceae (41), which was produced in the degradation of protein through anaerobic digestion (46), the Methanomicrobia may gradually diminish in the reactor. Previous work also found out that fewer methanogens dominated in the population when the anaerobic reactors were used to treat protein-rich wastewater (47). Thermoplasmata is grouped into the sulfur reducers and a facultative anaerobe, which respires using sulfur plus organic carbon (48). Early study has found that the Thermoplasmata may be involved in the reduction of sulfate (49), which could be produced in the degradation of protein. It usually appears in extremophilic environments (43) and may outcompete the methanogenic archaeon in the anaerobic reactor. In conclusion, the study indicated that the UASB bioreactor could efficiently treat diluted pharmaceutical fermentation wastewater under the OLR ranging from 2.7 kg COD/m3 d to 7.2 COD/ m3 d. Firmicutes, Bacteroidetes, Thermoplasmata and Methanobacteria could better adapt to higher OLR and gradually played a leading role in the improvement of the reactor performance. Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.jbiosc.2014.02.027.
ACKNOWLEDGMENTS The authors are very grateful to the support provided by Hunan Flag Bio-tech Co., Ltd.
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Please cite this article in press as: Chen, Z., et al., Effects of increasing organic loading rate on performance and microbial community shift of an up-flow anaerobic sludge blanket reactor treating diluted..., J. Biosci. Bioeng., (2014), http://dx.doi.org/10.1016/j.jbiosc.2014.02.027