Anaerobic degradation of anionic surfactants by indigenous microorganisms from sediments of a tropical polluted river in Brazil
Degradación anaeróbica de tensioactivos aniónicos en microorganismos autóctonos de los sedimentos de un río tropical contaminado en Brasil
Iolanda Cristina Silveira Duarte1*, Paula de França1, Dagoberto Yukio Okada2*, Pierre Ferreira do Prado1 & Maria Bernadete Amancio Varesche2
Abstract
Linear alkylbenzene sulfonate (LAS) is widely used in the formulation of domestic and industrial cleaning products, the most synthetic surfactants used worldwide. These products can reach water bodies through the discharge of untreated sewage or ]]>
13 to 1.0x108MPN/gTVS), and consequently, LAS degradation (60.1 to 55.4%). The sediment ]]>
El alquilbenceno sulfonato lineal (LAS) es el tensoactivo sintético más usado en todo el mundo en los produtos de limpeza domestica e industrial y puede llegar a las masas de agua a través de la descarga de aguas residuales sin ]]>
13 a 1.0x108MNP/gTVS) y por lo tanto la degradación de LAS (60.1-55.4%). Los microorganismos en el sedimento del río Tietê se pueden usar como inóculo alternativo para el ]]>
Nowadays, despite these discharges little is known about the potential of water and metabolism of sediment microorganisms in those rivers (Rocha et al., 2009). As for now, the linear alkylbenzene sulfonate (LAS) concentrations of 1.6mg/L have been reported in the town of Pirapora do Bom Jesus along the Tietê ]]>
Microbial degradation of organic ]]>
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Materials and Methods
Inoculum: Sediment of Tietê River was used as inoculum for the degradation of the anionic surfactant in a bioreactor. An amount of 1.22kg of sediment was collected in the city of Salto-SP (Brazil) (23º 00’ 44.8” S - 47º 00’17.2” W) in July (dry season) using a Van Veen dredge. The ]]>
Table 1).
Anionic surfactant: The linear alkylbenzene sulfonate used in the present study was a commercial mixture of C10-C13 homologues provided by Aldrich (CAS no. 25155-30-0, technical grade).
Bioreactor: The schema of this bioreactor is shown in figure 1. The reactor was made of borosilicate glass, with a total volume of 1 500mL. Stirring was done with an impeller-type turbine, with three 14cm blades and agitation at 150rpm.
The substrate on the feed line was kept under refrigeration (4°C) for conservation and was heated in a water bath (30°C) before being discharged into the reactor. Peristaltic pumps were used to feed and discharge the effluent. Thus, we used 430mL of Tietê river sediment (inoculum) and 1 070mL synthetic substrate.
The bioreactor was monitored for 144 days with defined 24-hour cycles. Each cycle was divided into four phases: 1) Feed without stirring (15min), 2) Reaction with stirring (23h), 3) Sedimentation without stirring (30min) and 4) Output of treated effluent (drain of reactor) without stirring (15min).
The synthetic substrate in the feed line consisted of yeast extract, sucrose, sodium bicarbonate (NaHCO3), starch, potassium nitrate (KNO3) and liquid household detergent (quantified as LAS concentration) (Table 2). During the 144 cycles, the bioreactor showed different stages: biomass adaptation to synthetic substrates - 39 days; (A) LAS addition (15mg/L) – 31 days; (B) decrease in co-substrates (50%) – 61 days; (C) increase in LAS concentration (30mg/L) – 43 days. Chemical oxygen demand (COD) (raw and filtered), nitrate and solids were determined according to APHA (2005). The pH of the suspension was determined with a pH meter.
The LAS concentration was periodically measured in the liquid phase (influent and effluent) using high-performance liquid chromatography (HPLC) (Duarte, Oliveira, Buzzini, Adorno, & Varesche, 2006). The adsorbed LAS was extracted with methanol in an ultrasound bath for 30 minutes and analyzed by HLPC HPLC in triplicate (Duarte, Oliveira, Saavedra, Fantinatti-Garboggini, Oliveira, & Varesche, 2008). This extraction protocol had an efficiency of 85% (Duarte et al., 2008). The mass balance for LAS considered the surfactant in the feed (influent), in the effluent and adsorbed on the biomass in the reactor.
The most probable number (MPN) technique was used to estimate the denitrifying bacteria (Tiedje, 1982) in the biomass reactor. The biomass reactor samples were homogenized and diluted in flasks with the feeding solution used in each operational stage. The detection of bacteria was performed after 30 days of incubation at 30oC. The results were interpreted as detailed by APHA (2005).
Results
The sediment used as inoculum for surfactant degradation showed high density of denitrifying bacteria – 7.6x1012 MNP/gTVS (Table 1).
In the adaptation stage, the bioreactor showed stability with influent pH of 6.3 and effluent of 7.3. At this stage, the COD removal was 69% for COD influent and effluent of 382mg/L and 129mg/L, respectively. However, the nitrate removal achieved was 98% with an estimation of denitrifying bacteria of 7.6x1010MNP/gTVS. Solids loss was observed when compared to the initial bioreactor operation conditions (13.6 to 6.9gTVS).
With the addition of 15mg/L of LAS (stage A), the nitrate, COD removal and pH were not affected (Table 3). In this stage, 675mg of LAS were added in the reactor. After this stage, mass balance indicated that the addition of 10% LAS was adsorbed on the biomass and LAS degradation was 60% (Table 4). Denitrifying bacteria population was improved by adding LAS and the population was estimated at 2.2x1013MPNg/TVS, and the amount of solids in the reactor also increased from 6.9 to 7.3gTVS. Stage B lasted 61 days and was characterized by a 50% decrease of organic sources (co-substrates) and 1 440mg addition of LAS. The effluent pH remained stable; the nitrate removal was reduced to 84% of efficiency; also there was a decrease of COD removal efficiency (37%), LAS degradation (55.4%), estimation of denitrifying bacteria (1.0x108MNP/gTVS) and solids (4.9gTVS) (Table 3).
Due to the decrease in the population of denitrifying bacteria, and in the efficiency of LAS degradation, concentrations of co-substrates were re-established, and the concentration of LAS increased to 30mg/L. Stage C had the highest mass of LAS applied (1 944mg) and showed the highest specific LAS-load rate (9.7mgLAS/gTVS/d). The effluent pH and the most probable number of denitrifying bacteria remained similar to the previous stage, the COD removal efficiency increased to 57%. Even resuming to the previous nutritional conditions, the total volatile solids (3.2g) and nitrate removal (Table 3) decreased, and the degradation efficiency of LAS was the lowest observed during the experiment, reaching 47% (Table 4). It is probable that the addition of 30mg/L LAS and removal of co-substrates were negative concerning LAS removal in the system (Fig. 2). ]]>
Discussion
Due to their high consumption and applications, significant amounts of surfactants are released into the environment, and this release causes serious problems in rivers and oceans. High concentrations of surfactants can be found in river sediments receiving untreated effluents due to inefficient degradation of LAS. Eniola and Olayemi (2008) found surfactant concentrations ranging from 45 to 132mg/g in sediments from the Asa River in Nigeria and heterotrophic bacteria 2.9x105 and 1.2x107CFU/g.
The sediment analyzed in this study showed concentrations of LAS lower than 0.30mg/g of sediment, however, the population of denitrifying bacteria was 7.6x1012MPN/gTVS, showing that denitrifying bacteria are responsible for nitrate reduction processes in river sediments (Berna et al., 2007).
Also, biological systems using pure cultures or microbial consortia in different fermentation conditions have been used to promote the degradation of surfactants (Cserháti, Forgács, Oros, 2002). This bioavailability increase was remarkable at the beginning of LAS addition, because of the organic compounds previously adsorbed on the biomass. According to Elsgaard (2010), the presence of LAS (105mg/L) in wastewater does not inhibit nitrate removal, but inhibits iron sulfate removal.
In our study, it is possible that the nutritional conditions enriched some bacteria from the inoculum, increasing microorganisms which use LAS as carbon source. Decreased co-substrates affected the degradation efficiency of LAS (55.4%) possibly due to a lower denitrifying bacteria population estimated (1.0x108MPNg/TVS). Furthermore, biomass concentration decreased from 7.3gTVS/L (stage A) to 4.9gTVS/L (stage B). Also, mean nitrate removal decreased from 87% (stage A) to 78% (stage B).
LAS degradation under denitrifying conditions observed in this study was higher than any other reported at the same LAS concentration. An anaerobic sequence batch reactor achieved LAS degradation efficiency of 37-53%, related to a LAS concentration in the influent of 22mg/L and specific-LAS load rate ranging from 6.8 to 9.8mgLAS g/TVS, the highest LAS degradation was 53% in the stage without co-substrates (Duarte, Oliveira, Mayor, Okada, & Varesche, 2010). The LAS presence did not inhibit bacteria in an acidogenic Upflow Anaerobic Sludge Blanket (UASB) reactor. After 250 days, LAS degradation was 41%. At hydraulic retention time of 6h, this reactor used lactose (1g/L) as co-substrate. Potassium nitrate was the electron acceptor at 1:1 ratio (LAS: NO3-) and this acceptor was completely consumed (Almendariz, Meráz, Soberón, & Monroy, 2001).
A UASB reactor fed with isotonic solution and LAS (5mg/L) degraded 85% of the surfactant. In this study, another UASB reactor fed with co-substrates showed LAS degradation of 64% (Sanz, Culubret, de Ferrer, Moreno, & Berna, 2003). Moreover, UASB reactors under mesophilic and thermophilic conditions obtained removal rates ranging from 40 to 80% (Lobner, Torang, Batstone, Schmidt, & Angelidaki, 2005). The test batches lasting 165 days, which used marine sediments as inoculum for LAS degradation (concentration ranging from 10 to 50mg/L) resulted in LAS degradation of 79% and identified high phylogenetic variety in the process. Clone library showed the classes Alphaproteobacteria, Gammaproteobacteria (genus Pseudomonas), and Sedimentibacter (family Clostridiales) (Lara-Martin, Gomez-Parra, Kochling, Sanz, Amils, & Gonzalez-Mazo, 2007).
Tietê river sediments could be used as inoculum in reactors for the treatment of anionic detergents in denitrifying (anaerobic redox potential) conditions. Denitrifying bacteria are potential candidates for effective anaerobic degradation of LAS. This bacteria group was able to degrade LAS molecule independently of additional carbon sources, while removing chemical oxygen demand and nitrate in anaerobic wastewater treatment plants.
Acknowledgments
We gratefully acknowledge the Conselho Nacional de Pesquisa (CNPq) for funding this study, (Process 479900/2008-6).
References
Almendariz, F. J., Meráz, M., Soberón, G., & Monroy, O. (2001). Degradation of linear alkylbenzene sulphonate (LAS) in an acidogenic reactor bioaugment UIT a Pseudomonas aeroginousa (M113) strain. Water Science Technology, 44, 183-188. [ Links ]
American Public Health Association (APHA), American Water Works Association (AWWA), Water Environment Federation (WEF). (2005). Standard Methods for the Examination of water and wastewater. Washington DC: American Public Health Association. [ Links ]
Berna, J. L., Cassini, G., Hager, C. D., Rehman, N., López, I., Schowanek, K. D., Sterber, J., Taeger, K., & Wind, T. (2007). Anaerobic biodegradation of surfactants-Scientific Review. Tenside Surfactants Detergents, 44, 312-346. [ Links ]
Cavalli, L., Cassani, G., Vigaró, L., Pravettoni, S., Nucci, G., Lazzarin, M., & Zatta, A. (2000). Surfactants in sediments. Tenside Surfactactants Detergents, 37, 282-288. [ Links ]
CETESB - Companhia Ambiental do Estado de São Paulo. (1992). Água do mar – Teste de toxicidade crônica de curta duração com Lytechinus variegatus, Lamarck, 1816. Norma Técnica L5.250. São Paulo: CETESB. [ Links ]
Cserháti, T., Forgács, E., & Oros, G. (2002). Biological activity and environmental impact of anionic surfactants. Environmental International, 28, 337-348. [ Links ]
Duarte, I. C. S., Oliveira, L. L., Buzzini, A. P., Adorno, M. A. T., & Varesche, M. B. A. (2006). Development of a method by HPLC to determine LAS and its application in anaerobic reactors. Journal of the Brazilian Chemical Society, 17, 1360-1367. [ Links ]
Duarte, I. C. S., Oliveira, L. L., Saavedra, N. K. D., Fantinatti-Garboggini, F., Oliveira, V. M., & Varesche , M. B. A. (2008). Evaluation of the microbial diversity in a horizontal-flow anaerobic immobilized biomass reactor treating linear alkylbenzene sulfonate. Biodegradation, 19, 375-385. [ Links ]
Duarte, I. C. S., Oliveira, L. L., Mayor, M. S., Okada, D. Y., & Varesche, M. B. A. (2010). Degradation of detergent (linear alkylbenzene sulfonate) in an anaerobic stirred sequencing-batch reactor containing granular biomass. International Biodeterioration and Biodegradation, 64, 129-134. [ Links ]
Elsgaard, L. (2010). Toxicity of xenobiotics during sulfate, iron, and nitrate reduction in primary sewage sludge suspension. Chemosphere, 79, 1003-1009. [ Links ]
Eichhorn, P., Rodríguez, S. V., Baumann, W., & Knepper, T. P. (2002). Incomplete degradation of linear alkylbenzene sulfonate surfactants in Brazilian surface waters and pursuit of their metabolites in drinking waters. Science of Total Environmental, 284, 123-134. [ Links ]
Eniola, K. I. T., & Olayemi, A. B. (2008). Linear alkylbenzene sulfonate tolerance in bacteria isolated from sedimento f tropical water bodies polluted with detergents. Revista Biologia Tropical, 56, 1595-1601. [ Links ]
Garcia, M. T., Campos, E., Sanchez-Leal, J., & Ribosa, I. (2006). Effect of linear alkylbenzene sulphonate (LAS) on the anaerobic digestion of sewage sludge. Water Research, 40, 2958-2964. [ Links ]
Hatamura, E., Eysink, G. J., Bevilacqua, J. E., & Moraes, R. P. (1993). Enriquecimento das espumas por substâncias químicas como agente de exportação de poluentes no Rio Tietê. (Relatório Técnico). CETESB. [ Links ]
Lara-Martin, P. A., Gomez-Parra, A., Kochling, T., Sanz, J. L., Amils, R., & Gonzalez-Mazo, E. (2007). Presence, biotransformation and effects of sulfophenylcarboxylic acids in the benthic fish Solea senegalensis. Environment International, 33, 565-570. [ Links ]
Lobner, T., Torang, L., Batstone, D. J., Schmidt, J. E., & Angelidaki, I. (2005). Effects of process stability on anaerobic biodegradation of LAS in UASB reactors. Biotechnology Bioengineering, 89, 759-765. [ Links ]
Mortatti, J., Moraes, G. M., & Kiang, C. H. (2012). Distribuição e possível origem de metais pesados nos sedimentos de fundo ao longo da Bacia do alto Tietê: aplicação da normalização geoquímica sucessiva. Geociências, 31, 175-184. [ Links ]
Olayemi, A. B, Eniola, K. I. T., Awe, S., & Kayoe-Isola, M. T. (2003). Distribution of Bacteria in three detergent effluent-polluted water bodies in Ilorin, Nigeria. Nigeria Society for Experimental Journal, 3, 79-86. [ Links ]
Olkowska, E., Ruman, M., & Polkowska, Z. (2014). Occurrence of surface active agents in the environment. Journal of Analytical Methods in Chemistry, 2014, 1-15. [ Links ]
Rocha, P. S., Luvizotto, G. L., Kosmehl, T., Bottcher, M., Storch, V., Braunbeck, T., & Hollert, H. (2009). Sediment genotoxicity in the Tietê River (São Paulo, Brazil): in vitro comet assay versus in situ micronucleus assay studies. Ecotoxicolology and Environmental Safety, 72, 1842-1848. [ Links ]
Sanz, J. L., Culubret, E., De Ferrer, J., Moreno, A., & Berna, J. L. (2003). Anaerobic biodegradation of linear alkylbenzene sulfonate (LAS) in up-flow anaerobic sludge blanket (UASB) reactors. Biodegradation, 14, 57-64. [ Links ]
Tiedje, J. M. (1982). Denitrification. In A. Page, R. H. Miller, & D. R. Keeney (Eds.), Methods of soil analysis (pp. 1011-1026). Winsconsin: American Society of Agronomy. [ Links ]
1. Federal University of São Carlos – Campus Sorocaba, Department of Biology, João Leme dos Santos Highway (SP 264), 110, 18052-780 Sorocaba, SP, Brazil; iolanda.duarte@gmail.com, paula_defranca@yahoo.com.br, pierreprado@hotmail.com
2. University of São Paulo, Department of Hydraulics and Sanitation, Trabalhador São-carlense Avenue 400, 13566-590 São Carlos, SP, Brazil; dagokada@gmail.com, varesche@sc.usp.br
Received 25-II-2014. Corrected 21-VII-2014. Accepted 21-VIII-2014.
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