Process Biochemistry 41 (2006) 1391–1400 www.elsevier.


BTEX and ethanol removal in horizontal-flow anaerobic immobilized biomass reactor, under denitrifying condition
´ ˜ Valquiria Ribeiro Gusmao a,*, Tiago Henrique Martins a, Fabio Alexandre Chinalia a, a ´ vio HenriqueThiemann b, Isabel Kimiko Sakamoto , Ota ˆ Maria Bernadete Amancio Varesche a

´ ´ ´ ˜ Laboratorio de Processos Biologicos, Departamento de Hidraulica e Saneamento, Escola de Engenharia de Sao Carlos, ˜ ˜ ˜ Universidade de Sao Paulo, Av. Trabalhador Sao-carlense, 400, CEP 13566-590 Sao Carlos, SP, Brazil b ´ ˜ ˜ ˜ Instituto de Fısica de Sao Carlos, Universidade de Sao Paulo, Av. Trabalhador Sao-carlense, ˜ 400, CEP 13566-590 Sao Carlos, SP, Brazil Received 15 September 2005; received in revised form 1 February 2006; accepted 1 February 2006

Abstract A denitrifying consortium was used as inoculum to form a biofilm in horizontal-flow anaerobic immobilized biomass reactor. The reactor was fed with hydrocarbons, separately (benzene at 13.8, 15.4 and 26.5 mg/L; toluene 30.8 mg/L; ethylbenzene 33.3 mg/L; xylene 32.1 mg/L), and also with a benzene, toluene, ethylbenzene and xylene (BTEX) mix solution of approximately 5.0 mg/L of each hydrocarbon. The hydrocarbons were dissolved in a solution containing ethanol. Organic matter removal efficiencies were of 95% with benzene and toluene amendments and about 76% with ethylbenzene, m-xylene and the BTEX-mix amendments. Hydrocarbons removal efficiencies were of 99% at an initial concentration of benzene 26.5 mg/L, toluene 30.8 mg/L, m-xylene 32.1 mg/L, ethylbenzene 33.3 mg/L and BTEX 26.5 mg/L. Microbial diversity assessed by a small portion of 16S DNA suggested the predominance of species related to the phylotypes Pseudomonas, Paracoccus and Bacteroides. This system showed to be an alternative to treating wastewater contaminated with nitrate, ethanol and hydrocarbons. # 2006 Elsevier Ltd. All rights reserved.
Keywords: BTEX; Ethanol; Denitrifying bacteria; HAIB reactor; Paracoccus; Pseudomonas

1. Introduction Benzene, toluene, ethylbenzene and xylene (BTEX) are natural constituents of crude oil and gasoline. These compounds are often found in groundwater as a result of leaks from underground storage tanks or pipelines, improper disposal practices, inadvertent spills and leaching from landfills areas [1]. In the last decades, as a result of crude oil shortage and excess carbon monoxide in the atmosphere of large cities, some countries began to use as fuel alternative a mixture of alcohol and gasoline. According to Corseuil et al. [2], the main effect of ethanol on gasoline is the co-solvency, which increases solubility of aromatic hydrocarbons in groundwater. Therefore, contamination of groundwater supplies by gasoline amended

* Corresponding author. Tel.: +55 16 33739523; fax: +55 16 33739550. ˜ E-mail address: (V.R. Gusmao). 1359-5113/$ – see front matter # 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.procbio.2006.02.001

with ethanol and other petroleum-derived hydrocarbons is a serious and widespread environmental problem. Besides hydrocarbons, groundwater can contain high nitrate concentrations and this compound in the water supply system can be harmful to human health. On the other hand, nitrate in contaminated waters with BTEX has favorable effects, since this ion can act as potential final acceptor for the biodegradation of such hydrocarbons. The risk that nitrate, ethanol and BTEX pose to water supplies favors the development of different technologies aiming to reach new solutions to the problem of contamination. In regards to this, biological denitrification is an interesting option, as long as denitrifying cultures are used, which is also capable of removing hydrocarbons. As reported throughout the last decade of microbial research, particular microorganisms are capable to degrade hydrocarbon compounds under denitrifying conditions. Aromatic hydrocarbons, such as toluene, have been studied most intensively as a substrate in anaerobic pure cultures, including

benzene. it has been documented that benzene can be anaerobically oxidized [6] and. 2:5C2 H6 O þ 2NO3 À þ 2Hþ ! 2:5C2 H4 O2 þ N2 þ 3:5H2 O The operational feeding procedure was carried out first with benzene amendments from 13.4 mg/L. 12. 1. Ethylbenzene degradation has been reported in three denitrifying bacteria. Gusmao et al. Biomass immobilization was promoted by influent feeding-circulation for 3 days. Such a system favors the retention and growth of specially adapted cells that are very efficient in the degradation of toxic compounds such as pentaclorophenol. maintaining headspace of 50% (v/v). phenol (940 mg/L). and supplemented with sodium nitrate (350 mg/L N-NO3À). PbN1 [4] and EB1 [5]. adding stoichiometrically nitrate or sulfate as final electron acceptors. for 48 h.2. methanol (320 mg/L). Cubic particles of polyurethane foam (5 mm in size and density of 23 kg/m3) were used as immobilization support for microbial biomass with a bed porosity of 40%. 16 and 20). at final concentrations of about 30 mg/L.1. formaldehyde and BTEX [8–12]. and submitted to a N2 (100%) atmosphere. in order to evaluate cellular growth. After this period. 75. during a total of 189 days with a spatial characterization-profiling at days 24. The reactors were incubated at 30 Æ 2 8C.8 times superior to the concentrations of electron donors. in pure culture.3. 1 shows a schematic representation of the HAIB reactor. Materials and methods 2. and using lactate (900 mg/L). ethanol final concentration was of 566 mg/L. One colony. 174 and 189. benzoate (1220 mg/L) and some hydrocarbons (benzene. The microbial consortium was evaluated in the relation capacity to grow under denitrifying conditions. length/diameter ratio (L/D) of 20. denitrifying bacteria [3]. benzene (10 mg/L) and ethanol 377 mg/L (82% purity). Schematic representation of the HAIB reactor. Nitrate final amendments were carried out considering Eqs. total volume of 1995 mL and a liquid volume capacity of 800 mL. / Process Biochemistry 41 (2006) 1391–1400 The capacity for denitrification of the consortium was determined in a batch reactor by measuring the accumulation of nitrous oxide after 10% (v/v) acetylene had been added to the headspace of the reactor. (1)–(5). 2. by a HAIB reactor containing an immobilized denitrifying biofilm. Ethanol was used to enhance the hydrocarbons dissolution at final concentrations of 377 mg/L against the benzene concentrations of 13. phenol. Benzene dissolved in ethanol was added using a sterile glass syringe aiming to reach a final concentration of 10 and 377 mg/L of benzene and ethanol. which later demonstrated to be a microbial consortium. Tables 1 and 2 compare these different values. Therefore. The reactor consisted of five intermediated sampling ports along its length (L/D of 4. strains EbN1. HAIB reactor was fed with fresh substrate. The denitrifying culture was purified in anoxic liquid medium as described by Dolfing et al. The purification of the inoculum was carried out by serial dilutions. These studies have all shown the potential for developing bioremediation technologies for treating BTEX under the denitrifying conditions. C6 H6 þ 6NO3 À þ 6Hþ ! 6CO2 À þ 3N2 þ 6H2 O C7 H8 þ 7:2NO3 À þ 7:2Hþ ! 7CO2 þ 3:6N2 þ 7:6H2 O C8 H10 þ 8:4NO3 À þ 8:4Hþ ! 8CO2 þ 4:2N2 þ 9:2H2 O (1) (2) (3) (4) (5) 2. the equivalent redox potential for the electron acceptor was 5. The present study was undertaken in order to investigate nitrate. in Gas Pack Jar (BBLTM Anaerobic Systems).1392 ˜ V. In experiments using a BTEX mix of about 26. 5. The highest dilution of benzene amendment showing growth was again serially diluted. 2. The horizontal reactor was made using borosilicate glass with 100 cm length. The flasks were incubated at 30 Æ 2 8C and cellular growth was verified by optical density (OD600 nm) reading in spectrophotometer. All three isolates are closely related to each other. after 30 h of growth. The tests were performed in previously sterilized flasks that were filled with culture medium and substrate. Feeding substrate A feeding substrate was made using the microbial consortium medium [13] amended with the specific hydrocarbon. acetate (600 mg/L). . 102. ethanol. was transferred to fresh liquid medium. was also reported [7].1 (OD600 nm) was adjusted as initiative. toluene ethylbenzene and p-xylene). butyrate (880 mg/L). 144. which were dissolved in ethanol (377 mg/L). and are affiliated with the genus Azoarcus in the b-subclass of Proteobacteria. ethylbenzene and xylene removal. Although many studies have indicated that benzene persists under anaerobic conditions in petroleumcontaminated environments. The reactor was installed in a temperature-controlled chamber (30 Æ 2 8C) and influent feeding procedure was accomplished with a peristaltic pump. Microbial consortium characterization C2 H6 O þ 2:4NO3 À þ 2:4Hþ ! 2CO2 þ 1:2N2 þ 4:2H2 O Generation time tests with the microbial consortium were performed in duplicate sterile reactors.5–33. toluene. sealed with Teflon lid. the anaerobic benzene oxidation coupled to nitrate reduction. The final values for ethanol concentrations were estimated theoretically using a chemical product with 82% purity. Fig.8 and 15.3 mg/L.4. fermentative and sulfate-reducing conditions.R. 2. within the continuous horizontal reactor. glucose (1800 mg/L). Horizontal-flow anaerobic immobilized biomass (HAIB) reactor has been investigated for its applied use in wastewaters treatments. A microbial consortium at the exponential growth phase was used as the source of cells to be immobilized in the polyurethane foam. [13]. respectively. The reactors were filled with culture medium [13] and nitrate (350 mg/L N-NO3À). The culture was repeatedly cultivated by superficial streaking in Petri plates incubated at 30 Æ 2 8C. The reactor was operated with hydraulic detention time of 12 h.0 cm diameter. Nitrous oxide was determined by gas chromatograph according to Yoshinari and Knowles [14]. Purification and growth conditions The inoculum source was obtained from an upflow anaerobic sludge blanket reactor (UASB) treating poultry slaughterhouse wastewater. It was used as inoculum 1% (v/v) of the microbial consortium biomass at the exponential phase. 159. 8. toluene. ethylbenzene and xylene. Horizontal-flow anaerobic immobilized biomass reactor A horizontal-flow anaerobic immobilized biomass reactor was used aiming to assess the removal efficiency of benzene. pyruvate (880 mg/L).4 mg/L during 75 days aiming gradual metabolic Fig. for 48 h. apart from the reducing solution. Turbidity at around 0. A N2 (100%) atmosphere was sustained in the substrate feeding flasks through a gas-balloon device. propionate (740 m mg/L).8 to 15.

15.0 b 0.3 mg/L.7a 47.5 mg/L). [16].1 2.9 43.5 566. a second step of 15 cycles of denaturing temperature of 94 8C for 30 s.0 b 30.6 1. nitrate and nitrite analyses were performed according to approaches described by the Standard Methods for the Examination of Water and Wastewater [15].0 99. For this purpose a denaturing gradient of 30–70% (40% of acrylamide/bis.˜ V.0 – 351. coli it refers to the forward primer 341f (50 -CCT ACG GGA GGA GGC AGC AG-30 ) and the reverse primer 534r (50 -ATT ACC GCG GCT GCT GG-30 ). Norwalk.0 – 15.0 – 554.0 4. ethylbenzene 33.5 mg/L.4 and b – 0.9 – 0.01 mg/L for nitrite).5 mg/L of m-xylene. targeting the universal bacterial 16S rDNA. 40 or 60% of formamide and urea) was used.0 31. [18]. Hydrocarbon removal was assessed throughout the reactor’s length at the end of each applied operating regime defined according to hydrocarbon concentrations such as benzene at 13.9a 0 Removal (%) Effluent – 0. and toluene 30.0 – – 99.8mg/L. toluene 30. The extraction of rDNA was accomplished following methodology described by Griffiths et al. PCR products were used for diversity assessments by denaturing gradient gel electrophoresis.8 377.4 377.0 mg/L for acetic acid and 0.0a – – – – 849.9 – – 75 102 144 30.0 93. E.1 127.8.7 mg/L of benzene.25 mm) according to Moraes et al. Volatile acids concentrations were determined by Gas Chromatograph HP 6890 (HP Innovax column À30 m  0. [20].8a – 0. Gusmao et al.2 13.53 mm with internal diameter  2.5a – 0. Chemical and chromatographic analysis Chemical oxygen demand (COD).0 – – 75. Gel electrophoresis was carried out at a constant 2.9 mg/L of toluene. annealing at 40 8C for 40 s. 4.0 – 348. CT) with amplification reactions of an initial denaturing step of 94 8C for 5 min followed by two steps: first 20 cycles of denaturing temperature of 94 8C for 30 s. (Olympus BX60-FLA with software Image Pro-Plus).65 mm). for 42 days. In E.7 mg/L of o-xylene and 7. alkalinity. for 27 days. 4.7a – – – 839.0a 31.1 – 0. and a negative control using Methanosarcina sp). the reactor was fed with benzene 26. It was used a ‘‘Gene Amp.0 – – 99. and the spatial characterization-profiling of the organics consumption was carried out after 15 days along the horizontal reactor. / Process Biochemistry 41 (2006) 1391–1400 Table 1 Operating regime used for the HAIB reactor’s feeding procedure with benzene and toluene at HTD of 12 h Reactor feeding characteristics Operational time (days) Concentration (mg/L) Influent Total organic matter Benzene Ethanol Acetic acid Nitrate (N-NO3À) Nitrite (N-NO2À) Unidentified excreted organic matter Total organic matter Benzene Ethanol Acetic acid Nitrate (N-NO3À) Nitrite (N-NO2À) Unidentified excreted organic matter Total organic matter Benzene Ethanol Acetic acid Nitrate (N-NO3À) Nitrite (N-NO2À) Unidentified excreted organic matter Total organic matter Toluene Ethanol Acetic acid Nitrate (N-NO3À) Nitrite (N-NO2À) Unidentified excreted organic matter a b 1393 COD (mg/L) Influent 823. PCR System 2400’’ (Perkin-Elmer Cetus. m-xylene 32.3 and 26.R. Hydrocarbons concentrations were determined by a static headspace gas chromatographic method [17] on a 6890 HP gas chromatograph (HP-1 column À30 m  0.8 – – 26.5 – – – 15.2 81. 2.9 – – 0. PCR positive controls comprised of three genera of bacteria (Pseudomonas sp.5 mg/L and BTEX 26. extension at 72 8C for 1 min.0 – – 94.9a – – – 1220.8 0. solution 50 TAE.1. and also by scanning electron microscopy (Zeiss digital scanning microscope DSM-960).5 – 0. In order to assess the reactor’s response to short periods of operational regime.9 96. Microscopic analysis Morphological characteristics of microorganisms immobilized in polyurethane foam matrices were monitored using phase contrast microscopy . 5.7 – – Theoretical values based on COD analysis. Polymerase chain reaction (PCR) was performed using bacterial primers described by Muyzer et al.0 – – 96.5 42.0 99.4 0.0 99. which is a portion of about 193 bp.0 5.6 – – 88. Molecular analysis At the end of the profiling-procedure (benzene 26.5 mg/L.7a 7.4a 743. Below limit of detection method (5.3a – 5.5.0 0. microbial biomass was sampled for molecular characterization.1 – 5..3 47.0 – 96.5 mg/L (4. 33.3a 82.0 – – 83.3a – 0.25 mm with internal diameter  0.5 – 24 – 13.4 – 0. [19]. annealing at 45 8C for 40 s.4a 801.0 mg/L of ethylbenzene. respectively. extension at 72 8C for 1 min and a final extension at 72 8C for 7 min.0 b 57. the reactor was fed using hydrocarbons at concentration of 32.1a 53. the DGGE-profiling [20]. coli and Desulfococcus sp.5a 781.2a – 0.0 – 526. adaptation of the biofilm.1 mg/ L and BTEX mix of 26. ethylbenzene and BTEX-mix. 2. After this period. Samples for scanning electron microscopy were subjected to the technique described by Araujo et al.2 mg/L of m-p-xylene).0 – Effluent 100.8 566.5a 1138.4a 1.6 0. pH.8 mg/L.

4 – 261.3 a 228. 4 shows that as a result of such operating regime acetic acid concentrations increased in the effluent diminishing COD removal efficiency as shown in Table 2. 3. using Eagle Sight software. The highest concentration of acetic acid (5.0 582.8 – – 189 – 26. ethylbenzene and BTEX-mixture at HTD of 12 h Reactor feeding characteristics Operational time (days) Concentration (mg/L) Influent Total organic matter m-Xylene Ethanol Acetic acid Nitrate (N-NO3À) Nitrite (N-NO2À) Unidentified excreted organic matter Total organic matter Ethylbenzene Ethanol Acetic acid Nitrate (N-NO3À) Nitrite (N-NO2À) Unidentified excreted organic matter Total organic matter BTEX Ethanol Acetic acid Nitrate (N-NO3À) Nitrite (N-NO2À) Unidentified excreted organic matter a b COD (mg/L) Influent 1252.3 – 550. 2a). Sequences were aligned using the DNASTAR package (Lasergene Sequence Analysis Software) and checked against the NCBI-data base. during the different operating regime phases. On the other hand.1 0. suggested that ethanol consumption generated the assessed acetic acid.3a 1809.0 – 507. Fig.0 – – 99. Tables 1 and 2 show acquired and theoretical values for several monitored variables of the influent and the effluent. The increasing concentrations of produced acetic acid (214–550 mg/L) showed the lack of a proper steadysate phase probably resultant of the short period for metabolic . Results and discussion In the current study a denitrifying consortium was used for generating an immobilized microbial biofilm onto polyurethane foam within a HAIB reactor. 2a and d). Organic matter removal assessed as COD showed values up to 95% removal efficiency during the operating regime with benzene and toluene amendments.0 – 80. When the operational regime was carried out using m-xylene. The reactor performance was constantly monitored to assess the system stability as a COD removal in a steady-state. The highest steady-state stability in organic matter removal was observed in benzene and toluene experiments (Fig.1 83.R. 3d).0 a – 0. Gels were stained with ethidium bromide.0 – – 99.0 mg/L for acetic acid and 0. DGGE bands were excised from the gel.5a 1304.5 566.5 0.9 2.4a – 0.8 0.0 – – 69.0 0. On the other hand. temperature of 65 8C. Nitrate removal was observed in accordance with organic matter removal and nitrite was detected as the intermediate (Fig. acetic acid. eluted in distilled water. NNO2À. In general terms. ethanol final concentrations were increased to facilitate hydrocarbon dissolution and the experiment was carried out during 15 days.1 mg/ L) was observed in toluene amendment (30. ethylbenzene and BTEX-mix amendments the COD removal efficiency averaged 76%. 2b and c).2a 0.2 99. the organic matter removal efficiency was lower and acetic acid was detected in the effluent (Fig.0b – 159 32.0 99.1 0.0 a – 1893.1a 280. N-NO3À.5 – – 79.7a – 0.8a 0.3 566.1 – 214.0 a 0.0 99. 2e and f). 2 shows variation of temporal concentration for the monitored variables (COD.2a – – – 1409. ethylbenzene and BTEX-mix.4 101. and transformed into E. re-amplified by PCR. Fig. PCR of each band were separately cloned into a plasmid pCR 2.2 a – 279.0 a – 0. / Process Biochemistry 41 (2006) 1391–1400 Table 2 Operating regime used for the HAIB reactor’s feeding procedure with m-xylene.7 105. alkalinity and pH) which were acquired throughout the entire period of operating regime.0 99.0 a – Effluent 262. coli DHa5 according to the manufacture instructions.0 – – 174 33. separately).0 – 439. A steady-state instability tendency was observed during the operating regime with m-xylene.988a – 586.0 a 33.01 mg/L for nitrite).0 5.0 a 0.7a Removal (%) Effluent 0. 175 V for 16 h.1 566. acetic acid production and its almost total consumption were clearly observed in experiments with benzene and toluene amendments.1394 ˜ V.0b – 0.0b – – 0. showing their horizontal profiling within the reactor under the operational regime with different hydrocarbons. ethylbenzene and BTEX-mix amendments.8a 1150. Below limit of detection method (5.6 1.8 mg/L) with an ethanol concentration of 566 mg/L (Fig.0 3.0 0. DGGE-profiling documentation was accomplished in an Eagle Eye TM III (Stratagene) under excitation of 254 nm UV. the reactor showed a short period of start-up exhibiting low organic matter concentrations in the effluent. cloned and subjected to sequencing.1 TOPO-TA easy vector system (Invitrogen). The spatial characterization-profiling of volatile acids of all samples obtained throughout the reactor’s length.0 – – Theoretical values based on COD analysis. Clones were sequenced in ABI 377 DNA Sequencer (Perkin-Elmer) using M13 primers (forward and reverse.0 – 417. the acidification of the effluent was not observed as shown by pH and alkalinity values (Fig. Gusmao et al. In this period. In the operating regime with m-xylene.

9.˜ V. Short experiments with mxylene. but it was also consumed by the biofilm’s metabolism.R.2 and 127.9 mg/L. ethylbenzene and BTEX-mix showed not to have resulted in a proper steady-state phase with efficient COD removal. In the present work. Nitrite was also detected in the first operating phase (Tables 1 and 2) at concentrations up to 43.0 mg/L) was associated with an excess of electron acceptor against the concentrations of the organic matter sources (Fig. 2. the highest rates of organic mater removal as COD took place at the first portion of the horizontal reactor (L/ D = 4) as also reported by the former author This reactor portion is coincident with the highest biomass concentration values present in the system (Figs. 30. Ribeiro [21] showed that nitrates and nitrites (N-NO3À and N-NO2À) were almost totally consumed in the first portions of the horizontal reactor and that the resultant COD values in the reactor’s effluent corresponded mostly to acetic acid which were not completely degraded. although it showed a significant reduction on hydrocarbon concentrations in the effluent. Temporal variations of COD (a). pH (e) and alkalinity (f). adaptation of the biofilm within the 15 days of operation with the referred feeding substrate (Table 2). A balanced steady-state phase was obtained with benzene and toluene amendments during the operating regime of 144 days. acetic acid (d). Nitrate removal was a direct result of organic matter consumption and the reminiscent nitrate concentrations observed in the effluent (57. 4). obtained in the influent and effluent. Gusmao et al. however. These results suggested the development of a specially evolved hydrocarbon degrading biofilm which responded to significant changes in the . N-NO2À (c). N-NO3À (b). The alkalinity production within the reactor. The reactor showed high removal efficiencies of hydrocarbons (Tables 1 and 2). 3 and 4). / Process Biochemistry 41 (2006) 1391–1400 1395 Fig. avoided the total collapse of the system which exhibited a COD removal (69– 80%).

0. This author reported efficiencies up to 96% removal for compounds such as benzene (4.01. 0. 5. 3.8 mg/L (a). denitrifying microorganisms are highly versatile and widely distributed in the environment. inocula sources used in microbial studies related to the isolation of microorganisms responsible for degradation of hydrocarbons are obtained from places already contaminated by such compounds since. BTEX removal efficiency was of 99. in a concentration of 26.5 mg/L (c) and toluene at 30.5 mg/L of benzene. Results obtained at the end of the operating regime with benzene at 13.7. the highest permitted concentrations of benzene. However.5 mg/L (about 5. For the different experiments.8 mg/L (d). the investigation of alternative sources of inoculum must be considered. Xylenes were not detected. and 0. Concentration profiles for organic matter (COD) and acetic acid.3 mg/L observed in the effluent showed to be of 0. High values of hydrocarbons removal efficiencies were also observed in the study carried out by Ribeiro [21].5 mg/L. Benzene amendments experiments showed high values for hydrocarbon removal efficiencies. Characterization of culture used to generate the biofilm Usually. which is . It is for such a reason that in this study a granular sludge of UASB reactor treating residual wastewater of a poultry slaughterhouse was used.9 mg/L). 15. the possibility of isolating microorganisms adapted to these toxics is higher. toluene. Therefore. and the residual of 0.4 mg/L (b). This sludge has been used as inoculum source in this research. respectively. so that the study on metabolic diversity and versatility of microorganisms capable of carrying out BTEX removal is not restricted to contaminated areas only. benzene has been reported as difficult to biodegrade in the absence of oxygen. this work aimed to develop an alternative treatment for wastewater whose final use is not for drinking-water. 5). However.1. operating regime applied to the reactor. / Process Biochemistry 41 (2006) 1391–1400 Fig. 3. Especially during profile with 26.0 mg of each hydrocarbon). This response pattern is an indicative of possible constraint of the biomass to promote benzene removal in high concentrations.9 mg/L). The biofilm was also assessed regarding its capacity to remove toxics when they were together in a single solution (BTEX).3 and 0. 26. while biodegradation of alkylbenzenes occurs readily under a variety of anaerobic conditions [7.8 mg/L. it was observed that removal was slightly more distributed throughout the reactor (Fig. Gusmao et al. In accordance to the guideline for drinking-water quality of World Health Organization (WHO) [22].05 mg/L of toluene and ethylbenzene. The lowest toxic removal efficiency was of 93% and it was obtained during the operating regime with benzene amendments at 13.25 mg/L of benzene. It has also been suggested by several other variables shown in Fig.0%. the resultant reductions reported for hydrocarbons concentrations close adjust such treated influents to the former directives (Tables 1 and 2).R. m-xylene (3.2 mg/L).1396 ˜ V. in these areas. toluene (7.7 mg/L) and a BTEX-mix (15.23]. ethylbenzene and xylene which may be found in water are of up to 0. However.

the confirmation of denitrifying activity Fig.17. targeting BTEX contaminated wastewater. Results obtained at the end of the operating regime with m-xylene 32. and the authors used a horizontal-flow anaerobic immobilized reactor to performer their experiments. acetic acid and N-NO3À. 5. [24].5 mg/L (c). .12]. Concentration profiles of organic matter (COD).24]. a resultant of several contaminating activities [10. 4. although very little is known about its microbial diversity. / Process Biochemistry 41 (2006) 1391–1400 1397 Fig. In this work. ethylbenzene 33. m-xylene. The influence of different inocula sources in the anaerobic degradation of BTEX was carried out by Fernandes et al. The authors reported that the origin of the inoculum was significant for the process of anaerobic degradation once each distinct reactor showed a different period of adaptation and percentages of hydrocarbon removal.1 mg/L (a). A wide adaptability for metabolic adaptation towards hydrocarbons has been associated with this particular granular UASB reactor sludge treating poultry slaughterhouse [12.21.˜ V. ethylbenzene and a BTEX-mix (b). Gusmao et al. Hydrocarbons concentration profiles carried out at the end of the operating regime with benzene (a) and toluene.R. the inocula sources which contained microbial biomass originated from a reactor treating poultry slaughterhouse wastewater showed the higher BTEX removal efficiency (90%) in a shorter period of time against the efficiencies obtained with previously adapted biofilm to BTEX degradation (57%). According to the former authors.3 mg/L (b) and BTEX-mix of 26.

/ Process Biochemistry 41 (2006) 1391–1400 Fig. 15.046 hÀ1. which was different from the normal short-rod shape found for suspended cells. the band patterns in the DGGE-profiling remained the same (Fig. 6a). Therefore. Variation of absorbance. Growth tests curve and N2O accumulation after acetylene was injected in the flask containing the consortium (a). benzoate and. These cells were observed forming lumps in the first sequences of the reactor (L/D 4 and 8) with some morphology in chains (Fig. during the optical microscopic examination suspended cells morphologically associated to the pseudomonad group were observed which was later confirmed by 16S rDNA. Pseudomonas and Bacteroides (accession Fig. Growth on ethanol. Gusmao et al.5 mg/L) and the BTEX mix (26. ethanol and NNO3À concentrations (b). propionate. in the presence of benzene (10 mg/L). The biomass showed generation time of 15. This result indicated that changes were not significant in the microbial community genetic diversity after the different operating regime to which the reactor was submitted.4 and 26. After 144 days and with the modifications of the hydrocarbons amendments a steady-state regime was not achieved. 7. The knowledge of microbial diversity of evolved biofilm within HAIB denitrifying reactor is still largely unknown. similarities with the phylotypes Paracoccus. samples of the culture used as inoculum source for the HAIB and samples obtained from the reactor biofilm were analyzed by DGGEprofiling and 16S rDNA sequencing (Fig. singly. 8). Morphological changes were also observed by Shim and Yang [26] and the authors reported that Pseudomonas sp. have produced large cell clumps and had a long.0 mg/L). In order to assess microbial community diversity. The consortium showed metabolic versatility growing on different substrates under denitrifying condition.1 h and growth velocity (m) of 0.8. Some anomalies were also observed in the samples. The production of biomass was associated with the consumption of ethanol and nitrate (Fig.8 mg/L. reports of microscopic observations are important data for allowing microbial inference characterizing diversity and potential physiology of such biofilms.8 mg/L. 7b).5 mg/L and toluene at 30. In the present study. 6b). according to the NCBI-data base. ethylbenzene and pxylene dissolved in ethanol was observed. butyrate. ethanol (300 mg/L) and nitrate (393. The metabolic adaptation of the biofilm showed to provide a steadystate regime over a period of 144 days treating benzene at 13. toluene. a rod-shaped morphology predominated in the biofilm (Fig. No growth was also observed in fermentative or sulfate-reducing conditions. 7a). After the reactor inoculation with the denitrifying consortium. DGGE-profiling revealed similar band patterns for the inoculum and the biofilm samples. . 6. since N2O accumulation in the presence of acetylene is considered strong evidence that denitrification is occurring [25] (Fig. The consortium was not able to grow using methanol and phenol. lactate.1398 ˜ V. however.5 mg/L). pyruvate. (a and b) Scanning electron microscopy (5000Â) of microorganisms observed in the reactor’s biofilm under the operating-regime with benzene at 30. benzene. was suggested after the detection of N2O in the headspace of the flask where acetylene was injected. glucose. acetate. Four bands of 16S rDNA of about 193 base pairs (A–D) were sequenced and the result of such sequencing revealed. in pairs or in relatively short chains [27]. slim morphology. This similarity between the band patterns was also observed when the reactor was fed with benzene (26. 8).R.

Cole KA. [6] Lovley DR. although the possibility of this strain to be harboring two distinct 16S rDNA should be also considered. however. References [1] Phelps CD. which might have occurred during the PCR procedure. Anaerobic bacterial metabolism of hydrocarbons. desulfurizing and denitrifying effluent-treatment plant system [30]. has already been established. Line 2: sample of biofilm obtained at end of operating regime with benzene at 26. xylene or a BTEX mixture. such as BTEX. Hunt CS. Pseudomonas strains were also used in studies that undertook growth kinetics when these microorganisms were in the presence of toxics [36]. [2] Corseuil HX. 8 shows two distinct bands associated with the genus Paracoccus which produced different similarities results to the strain AM084107 (98–100%) according to the NCBI-data base. toluene. denitrifying sand filters and activated sludge systems [30. bioreactor [26]. Foresti E.32:2065–72.R. DGGE-profiling of the inoculum and the reactor’s biofilm samples. Water Res 1998. / Process Biochemistry 41 (2006) 1391–1400 1399 Fig. Bender KS. Gusmao et al. 2004. In: The first international meeting on environmental biotechnology and engineering (I IMEBE).32]. Santos RCF. Target compounds include methanol. Anaerobic benzene degradation. 8 pp. et al. Initial reactions in anaerobic ethylbenzene oxidation by a denitrifying bacterium. methylamines. Anaerobic degradation of ethylbenzene and other aromatic hydrocarbons by new denitrifying bacteria. Biodegradation of BTEX under anaerobic conditions: a review. which was potentially the most numerous microorganism present in the system. despite the fact that the former authors attribute a negligible role of this organism in studied petroleum land treatment unit.163:96–103.411:1039–43. Anaerobic benzene oxidation coupled to nitrate reduction in pure culture by two strains of Dechloromonas. biofilters treating effluent gases and from contaminated soils [31. BTEX substrate interactions and bacterial community dynamics [37]. BS2201 and BS2203) isolated from petroleum-contaminated soil were capable of degrading petroleum hydrocarbons [35]. which was isolated from chicken cecum [38]. Fig.˜ V. toluene. Ribeiro R. Chakraborty R. The Paracoccus genus has been found in different environments such as groundwater contaminated with dichloromethane [29].5 mg/L. including specie designated Bacteroides ovatuslike. [7] Coates JD. Anaerobic biodegradation of PCP via reductive dechlorination in horizontal-flow anaerobic immobilized biomass (HAIB) reactor. substituted formamides. The ability of different species to degrade unusual and several polluting compounds indicates their potential role in natural or contrived bioremediation systems. Arch Microbiol 1995. numbers and similarities are: AM084107 (98–100%). Adv Agron 2001. Young LY. Nature 2001. which is the main source of the inoculum used in this work.34]. Spormann AM. particularly for benzene and toluene. Gusmao et al.. Conclusions The current research using HAIB reactors as an improved simple system for treating contaminated wastewater has shown high values of COD and BTEX removal efficiency. [8] Damianovic MHRZ. [28] working with 880 base pairs of 16S rDNA confirmed the suggested identity of Paracoccus sp. Therefore. showed to be related to 2 base pairs replacement by the substitution of thiamine by cytosine. FEMS Microbiol Rev 1999. [3] Heider J. The association of Pseudomonas species with biodegradation of aromatic hydrocarbons. all of which are waste products of diverse commercial processes. Johnson HA. Widdel F. Two nitrate-reducing bacteria (Pseudomonas sp. strain EB1. Beller HR. [CD ROM]. sulfides and organic sulfur compounds such as carbon disulfide. J Bacteriol 1996. The influence of the gasoline oxygenate ethanol on aerobic and anaerobic BTX biodegradation. [5] Ball HA. Spormann AM. One of the sequenced bands was associated to the Bacteroides group. On the other hand. ethylbenzene and o-xylene by a co-culture of Pseudomonas putida and Pseudomonas fluorescens was confirmed in a fibrous-bed . DQ224384 (98%) and AY554420 (98%). On ˜ the other hand.178:5755–61. Lack JG.5 mg/L.70:329–57. the presence of such group of microorganisms supports the hydrocarbon removal results through means of biodegradation. dichloromethane and other solvents. Pseudomonas and Bacteroides) all associated with pollutant degrading metabolic activities closed connected with this work. DGGE-profiling and DNA-sequencing revealed a diversified and resourceful consortium composed of microorganisms from different phylotypes (Paracoccus. This difference. acetone. Biodegradation of benzene. Biodegradation 2000. respectively). tetramethylammonium compounds. The results reported in this work allowed the conclusion that anaerobic bed-packed reactor when inoculated with specially selected denitrifying consortia is effective for treating wastewaters contaminated with ethanol.33. Zaiat M. ethylbenzene.22:459–73. Line 1: inoculum. carbonyl sulfide and methanethiol. thiocyanate. Widdel F. industrial wastewater. [4] Rabus R. Reinhard M. Alvarez PJJ. 4. 8. 11:107–16. Line 3: sample obtained at end of operating regime with BTEX-mix at 26. O’Connor SM. nitrate and benzene. Bacteroides species have been isolated from the intestinal tract of some animals. Phylotypes associated with this taxa have been reported as petroleum hydrocarbons degraders [37].

Campos JR. Formaldehyde degradation in an anaerobic packed-bed bioreactor. toluene. Townsend RT. Microbiology 1995. ¨ Lipski A.40:292–6. Krausowa VI. thesis. Isolation and characterization of a bacterium that mineralizes toluene in the absence of molecular oxygen. In: The first international meeting on environmental biotechnology and engineering (I IMEBE). [16] Moraes EM. [11] Oliveira SVWB. Bioeng Biotechnol 2005.54:1255–65. p. Lemmer H. Passig FH. 2005. Paracoccus kocurii sp.91:244–53. Forest E. Int J Syst Bacteriol 1998. Anaerobic benzene biodegradation linked to nitrate reduction. Trotsenko YA. Degradation of petroleum hydrocarbons by facultative anaerobic bacteria under aerobic and anaerobic conditions. Thiemann OH. Ribeiro R. Isolation and characterization of a new Gram-negative. [12] Cattony EBM. Kitts CL. Appl Environ Microbiol 1993. Comparison of hexamethyldisilazane and critical point drying treatments for SEM analysis of anaerobic biofilms and granular sludge. 2000. Zaiat M. p.67:99–112. Bacterial succession in a petroleum land treatment unit.69:705– 10. Adorno MAT. Reuter B. Biodegradation of benzene. Appl Environ Microbiol 2002.21:230–6. Foresti E. Zhong T.141:1469–77. Richmond MH. Rapid method for coextraction of DNA and RNA from natural environments for analysis of ribosomal DNA and rRNA-based microbial community composition. 2004. a tetramethylammonium-assimilating bacterium. Anaerobic degradation of BTEX in a packed-bed reactor. 235–8. [13] Dolfing J. McDonald TJ. Boronim AM.35:889–96. Ribeiro R.44:167–74. [CD ROM]. 186 pp. Zeyer P. Ohara M. 8 pp.40:587–92.62:4329–39. Pandya Y. / Process Biochemistry 41 (2006) 1391–1400 a packed-bed reactor. Rainey FA. Palleroni NJ. Katayama Y. Foresti E. Foresti E. Int J Syst Bacteriol 1996. Zaiat M. 1975. Zaiat M. nitrate-reducing bacterium from soil.52:429–33. Kuraishi H. editors. Chinalia FA. nov. Arch Microbiol 1990. Neef A. Reichert K. 16S rRNA-based analysis of microbiota from the cecum of broiler chickens. Doronina NV. Removal of benzene. Schleifer KH. Grishchenkov VG.65:529–33. The influence of different inoculum sources on anaerobic BTX degradation in [25] [26] [27] [28] [29] [30] [31] [32] [33] [34] [35] [36] [37] [38] . 1998. Foresti E. Moraes EM. Appl Environ Microbiol 2000. Shim H. Altendorf K. Acetylene inhibition of nitrous oxide reduction by denitrifying bacteria. Waal EC.154:336–41. Cowan RM. nov. Water Res 2004. [21] Ribeiro R. Meier H. Recuperation of gasoline-contaminated water in anaerobic ˜ ˜ packed-bed reactor.—a new aerobic facultatively methylotrophic bacterium utilizing dichloromethane. et al. DC. 1988. in methanol-fed biofilms. Biology of anaerobic microorganisms New York: John Wiley & Sons. In: Proceedings of the VI Latin American workshop and symposium on anaerobic digestion. Varesche MBA. Gusmao et al. toluene. [23] Burland SM. [14] Yoshinari T. Ribeiro R. a new species of thiocyanate-utilizing facultative chemolithotroph. A gas chromatographic determination approach for total volatile acids in effluents of anaerobic reactors treating liquid and solid wastes. Yang ST. 1. and o-xylene by a coculture of Pseudomonas putida and Pseudomomas fluorescens immobilized in a fibrous-bed bioreactor. Siller H. Winter J. Whiteley AS. Kaplan CW. Universidade de Sao Paulo. Anaerobic packed-bed reactor for bioremediation of gasoline-contaminated aquifers. 2005. Int J Syst Bacteriol 1990. vol. Water Sci Technol 2002. Genetics and biochemistry of Pseudomonas.. 2003.D. Washington. Process Biochem 2005.48:529–36. [17] Nardi IR. nov. [10] Nardi IR. Paracoccus solventivorans sp. ´ [18] Araujo JC. Biochem Biophys Res Commun 1976. nov. Sao Carlos: Escola de Engenharia de Sao ˜ Carlos. Zaiat M. Ph. Inc. Syst Appl Microbiol 1998. Uitterlinden AG. Teran FC. [22] World Health Organization (WHO).59:695–700.38:1685–94. Identification of bacterial isolates from biofilters as Paracoccus alkenifer sp. [19] Griffiths RI. with emendation of the genus. Tsuzaki M.68:124–37. Schwarzenbach RP. Hiraishi A. Edwards EA. Zaiat M. Process Biochem 2000.. Water Sci Technol 2001. Appl Environ Microbiol 1996. Binder-Eicher P. Kuraishi H. Varesche MBA. Oliveira RA. UK: John Wiley.R. Chinalia FA. 1–36. Nour EAA. Foresti E. Joerger RD. In: VII Latin American workshop and symposium on anaerobic digestion (DAAL). Varesche MBA. Adorno MAT.p. Autenrieth RL. Alagappan G. Ethanol and toluene removal in a horizontal-flow anaerobic immobilized biomass reactor in the presence of sulfate. Chemosphere 2004. Zaglauer A. In: Clarke PH. Effect of temperature and dissolved oxygen on the growth kinetics of Pseudomonas putida F1 growing on benzene and toluene.1400 ˜ V. Katayama Y.70:1777–86. [24] Fernandes BS. Profiling of complex microbial populations by denaturing gradient gel electrophoresis analysis of polymerase chain reaction amplified genes coding for 16s rRNA. J Electron Microsc 2003. ˜ Gusmao VR. J Biotechnol 1999. Zaiat M. Suzina NE. [20] Muyzer G. nov. General properties and taxonomy of the genus Pseudomonas.45:175–80. Phenol degradation in horizontal-flow anaerobic immobilized biomass (HAIB) reactor under mesophilic conditions. ethylbenzene and xylene in horizontal-flow anaerobic immobilized biomass reactor with denitrifying culture. Paracoccus methylutens sp. USA: American Public Health Association/American Water Works Association/Water Environment Federation.66:5488–91. Background document for preparation of WHO guidelines for drinking-water quality Geneva: World Health Organization. Montenegro MAP. Knowles R. Bailey MJ. ˜ [9] Bolanos ML. Zehnder AJB.46:1125–30. Nakamoto S. Amann R. Population analysis in a denitrifying sand filter: conventional and in situ identification of Paracoccus spp. Paracoccus thiocyanatus sp. Sakamoto IK. [15] Standard methods for the examination of water and wastewater. Sproer C. ethylbenzene. 227–32. and Paracoccus solventivorans with emended description of Paracoccus solventivorans. and transfer of Thiobacillus versutus to the genus Paracoccus as Paracoccus versutus comb. acetone-degrading. nov. Appl Environ Microbiol 1999. Varesche MBA. Varesche MBA. Appl Environ Microbiol 2004. 19th ed. O’Donnell AG. Zhu X. Stackebrandt E. Chinalia FA. Bonner JS. Foresti E. London. Zaiat M.