International Biodeterioration & Biodegradation xxx (2013) 1e4

Contents lists available at SciVerse ScienceDirect

International Biodeterioration & Biodegradation

journal homepage: www.elsevier.com/locate/ibiod

Methylamine removal using mixed bacterial strains in a continuous stirred tank reactor (CSTR) system
Chu-Fang Yang a, Chun-Chin Wang b, *, Chun-Hsien Tseng b
a Department of Safety Health and Environmental Engineering, National Yunlin University of Science and Technology, No. 123, University Road, Section 3, Douliou, Yunlin 64002, Taiwan b Department of Safety, Health and Environmental Engineering, Hungkuang University, No. 1018, Sec. 6, Taiwan Boulevard, Shalu District, Taichung City 43302, Taiwan

a r t i c l e i n f o
Article history: Received 22 December 2012 Received in revised form 27 March 2013 Accepted 31 March 2013 Available online xxx Keywords: Biodegradation Continuous stirred-tank reactor (CSTR) Methylamine (MA)

a b s t r a c t
Methylamine (MA) is an organic compound that can be found in many commercial applications (agricultural chemicals, including herbicides, fungicides, insecticides, biocides, and miticides; catalysts; pharmaceuticals; resin manufacturing). It has a strong odor similar to sh and can cause chemical burns to the eyes and skin. The vapor from this chemical is extremely irritating to the nose, throat, lungs, skin, and eyes. The indiscriminate discharge of improperly treated chemical contaminated wastewater or wastes into a natural water body will cause serious environmental pollution problems and severe adverse impacts to the aquatic ecology. The goal of this investigation is to remove MA using a continuous stirred-tank reactor (CSTR) and to understand the ability of mixed bacteria strains to treat different initial MA concentrations in synthetic and actual industrial wastewaters in the CSTR. The following results were obtained: When the hydraulic retention time (HRT) was 1.136 day, the MA removed efciency in the CSTR was the best. When the HRT was 1.136 day, the mixed strains could utilize 1000 mg l1 MA both from synthetic wastewater and actual industrial wastewater for growth in the CSTR. The MA removal efciency reached 100% in the CSTR. 2013 Elsevier Ltd. All rights reserved.

1. Introduction Methylamine (MA) is mainly used as a building block or chemical intermediate in the production of pharmaceuticals, pesticides, surfactants, photographic developers, dyestuffs, emulsiers, rubber products and corrosion inhibitors (Deng et al., 2009). It is found extensively in human urine (Mitchell and Zhang, 2001), and may access the brain and interfere with neurological function if present in large amounts (Helali et al., 2011). MA has a strong malodor at concentrations of 20e100 ppm (Notice of Intended Change, 1992), and is the major nitrogen-containing waste gas emitted from the composting process (Ho et al., 2008). MA is present in wastewater with decaying foodstuffs such as sh and the fermentation broths used for ethanol production (Dryahina et al., 2009). Therefore, MA removal is an important environmental contamination issue. Many researchers have made efforts in trimethylamine (TMA) removal. Chang et al. (2004) applied an aerobic bioltration system containing entrapped mixed microbial cells to treat TMA

-containing waste gas. TMA degradation in two three-stage biolters packed with compost or sludge was investigated by Ding et al. (2007). Some valuable bacterial strains capable of utilizing TMA were also isolated from various environments, such as Paracoccus sp. T231 (Kim et al., 2001), halophilic bacterial strains PH32, PH34 and GRP21 (Kim et al., 2003). However, relatively little literature on MA biodegradation has been published in recent decades. Thus, the aim of this research is to enrich potential consortia with the ability to degrade MA in activated sludge and soil in paddy elds. A continuous stirred tank reactor was applied to investigate the optimum HRT for MA removal in synthetic and actual wastewaters containing various initial MA concentrations.

2. Materials and methods 2.1. The sources of the mixed culture The mixed microbial cultures were obtained from activated sludge and agricultural soil. The former culture was obtained from an aeration tank at an industrial wastewater treatment plant located in Central Taiwan and the latter culture was obtained from rice paddy elds.

* Corresponding author. Tel.: 886 4 26318652 4110; fax: 886 4 26525245. E-mail address: chunchin@sunrise.hk.edu.tw (C.-C. Wang). 0964-8305/$ e see front matter 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.ibiod.2013.03.031

Please cite this article in press as: Yang, C.-F., et al., Methylamine removal using mixed bacterial strains in a continuous stirred tank reactor (CSTR) system, International Biodeterioration & Biodegradation (2013), http://dx.doi.org/10.1016/j.ibiod.2013.03.031

C.-F. Yang et al. / International Biodeterioration & Biodegradation xxx (2013) 1e4

2.2. Mixed culture acclimation The sludge (20 ml) and the soil (5 g) were mixed with 300 ml phosphate buffered medium (PBM) in 1-l ask. PBM contained the following inorganic compounds (in grams per liter): MgSO4$7H2O, 0.2; CaCl2$2H2O, 0.02; K2HPO4, 1.5; KH2PO4, 1.5; trace element solution, 10 ml l1. The composition of the trace element solution included (in mg per liter): FeSO4$7H2O, 300; MgCl2$4H2O, 180; CoCl2$6H2O, 106; Na2MoO4$2H2O, 34. The medium pH was adjusted to 7.5 with 5 N NaOH. Then, 200 mg l1 MA was added as the solo carbon and nitrogen sources. The ask was incubated on a shaker (180 rpm) at 30  C for 1e3 days. During this period, MA concentration and biomass growth were periodically measured. As MA was completely consumed, the mixed culture was transferred into another ask containing fresh PBM and 200 mg l1 MA. The enriched process was repeated for several times until MA degradation and cellular growth reached a stable level. 2.3. Actual wastewater pretreatment Owing to the variation of industrial wastewater loading, sometimes we might receive high-turbidity wastewater. While dealing with the actual wastewater containing massive suspended solid, the wastewater was centrifugated at 8000 rpm under 4  C for 30 min. The centrifugated wastewater was then ltered using 80mesh sieve. Then, the ltered wastewater passed through 0.45 mm Nylon membrane and the following 0.20 mm Nylon membrane and the VacuCap 0.1 mm lter (Gelman Laboratory, Ann Arbor, Michigan, USA), respectively. When the obtained wastewater contained no visible particle, the procedures of centrifugation and sieve ltration were omitted. 2.4. Reactor conguration and continuous operation

2.5. The continuous reactor experiment 2.5.1. The optimum HRT The appropriate enriched mixed culture was collected using centrifugation at 12,000 rpm under 4  C for 4 min (Kubota 3700, Kubota, Tokyo, Japan). After washing the biomass with PBM 1 time, the cell pellet was resuspended in the PBM/articial wastewater/ actual wastewater according to the experimental design, and adjusted the OD600 value to 0.1. The cell suspension was then transferred into the CSTR. The articial wastewater/actual wastewater containing 600 mg l1 MA was allowed to ow into the reactor. The bioreactor HRT was adjusted between 0.06 and 1.136 days to obtain the optimum operating conditions. Daily reactor system monitoring included MA concentration, pH and OD600 values. 2.5.2. The removal of various MA concentrations in the articial and the actual wastewaters The experimental procedure was the same as the protocol mentioned in Section 2.5.1, except for the inuent containing various MA concentrations (200e1000 mg l1) and operation under the optimum HRT. 2.6. Analytical methods After ltering with 0.22 mm lter, the MA concentration in the supernatant was analyzed using a HewlettePackard 6890 gas chromatograph (GC) system equipped with a ame ionization detector (FID) and a DB-1 column (15 m 0.25 mm ID, 0.25 mm lm thickness, Shimadzu, Kyoto, Japan). The ow rate was 10 ml N2/min, and the FID and injection port was operated at 150  C. The oven temperature was maintained at 130  C for 3 min. 3. Results and discussion

The CSTR is illustrated in Fig. 1. This system consists of three parts, the inuent tank, bioreactor and settling tank. The inuent tank volume is 4 l and exposed directly to the atmosphere. The total bioreactor volume is 4.5 l. The working volume is 4 l. The reactor temperature is maintained at 30  C using a circulating water bath system. The agitation speed is 180 rpm. The wastewater stored in the inuent tank is pumped into the reactor using a peristaltic pump (07520-40, COLE, USA). The settling tank has an overow added after the bioreactor.

3.1. The optimum HRT Fig. 2 shows the removal of 600 mg l1 MA in articial wastewater under different HRTs. The results indicated that when the HRTs were 0.06, 0.278, 0.55 and 1.136 days, the times for complete MA removal were 90, 75, 64 and 27 h, respectively. Therefore, the optimum HRT should be 1.136 day. The following CSTR experiments were performed at HRT of 1.136 day. The removal of 600 mg l1 MA using the mixed culture in the actual wastewater under various HRTs is shown in Fig. 3. The MA was not degraded by the enriched mixed culture under HRT of 0.06 day. The reason might be the HRT setting was too short to cause

Circulating water
Thermometer

700 600 500 400 300 200 100 0

Circulating water bath

Influent
Peristaltic pump Hot plate/ Magnetic stirrer

Effluent

Wastewater containing MA

Methylamine conc. (mg l-1)

HRT: 0.060 day HRT: 0.278 day HRT: 0.550 day HRT: 1.136 day

20

40

60

80

100

Time (hrs)
Settling tank

Influent tank

Fig. 1. Schematic diagram of the experimental reactor and process.

Fig. 2. Removal of 600 mg l1 methylamine using the mixed culture in synthetic wastewater under various CSTR HRTs.

Please cite this article in press as: Yang, C.-F., et al., Methylamine removal using mixed bacterial strains in a continuous stirred tank reactor (CSTR) system, International Biodeterioration & Biodegradation (2013), http://dx.doi.org/10.1016/j.ibiod.2013.03.031

C.-F. Yang et al. / International Biodeterioration & Biodegradation xxx (2013) 1e4
700 600 500 400 300 200 100 0 0 10 20 30 40 50 60 70

Methylamine conc. (mg l-1)

HRT: 0.060 day HRT: 0.278 day HRT: 0.550 day HRT: 1.136 day

As the initial MA concentration was 1000 mg l1. MA degradation accompanied pH decease (about 0.6) at each set MA concentration (Fig. 4B). The microbial consortia utilized MA to produce ammonium and formaldehyde (Rappert and Mller, 2005). Ammonium theoretically caused the increase in pH value in the solution. However, part of the ammonium was used for biomass synthesis, with formaldehyde further metabolized into formic acid and the following carbon dioxide. Carbon dioxide in the closed bioreactor easily dissolved in the wastewater to form carbonate. Therefore, the sum of total effects contributed the decline in pH value. 3.3. The removal of various MA concentrations in the actual wastewater in CSTR Removal of various initial MA concentrations (200e 1000 mg l1) and pH variation in the actual wastewater at HRT of 1.136 days is shown in Fig. 5. As with results the articial wastewater results, higher MA concentration produced longer removal period. As the initial MA was 200 mg l1, 28 h was needed to achieve complete MA removal. When the MA concentration was 400 mg l1, 37.2%, 65.7% and 87% MA removal were obtained at 10, 20 and 26 h, respectively. It needed 26 and 30 h to achieve 90.9% and 100% MA removal when the initial MA was increased concentration to 800 mg l1. With 1000 mg l1 MA as the substrate, the removal efciencies were 11%, 52.1% and 77.3% at 10, 21 and 27 h, respectively. Complete removal of 1000 mg l1 MA was at 33 h. The periods for various MA concentrations to approach complete MA removal in the actual wastewater were all superior to that in the articial wastewater. Other carbon sources present in the actual wastewater beneted MA degradation is the reason. Moreover, pH decline was also observed and the decrease at each set concentration was about 1.1 (Fig. 5B). The reason was the same as that mentioned in Section 3.2. However, owing to the lack of phosphate buffer system, the decreased pH value in the actual wastewater was larger than that in the articial wastewater. Table 1 presents MA removal using the acclimated mixed culture in the articial and actual wastewaters under batch (Tseng et al., 2011) and continuous experiments (this study). Under the same operation strategy (batch or continuous experiment), there

Time (hrs)
Fig. 3. Removal of 600 mg l1 methylamine using the mixed culture in actual wastewater under various CSTR HRTs.

washout, and this could be supported by the decrease in OD600 value (0.073 dropped to 0.017). When the HRT was prolonged to 0.278, 0.55 and 1.136 days, complete MA removal was achieved at 64, 60 and 28 h, respectively. Thus, in the following experiments, HRT of 1.136 day was applied for investigating MA degradation in the actual wastewater. 3.2. The removal of various MA concentrations in the articial wastewater in CSTR Fig. 4 presents MA removal and pH variation at the HRT of 1.136 day using articial wastewater containing various MA concentrations as the inuent. The higher substrate concentrations resulted in longer MA removal times. As 200 mg l-1 MA as the substrate, the removal efciencies were 26.9% and 81% at 9 and 22 h, respectively. It needed 28 h to completely remove 200 mg l1 MA. Increasing the MA concentration to 400 mg l1, the MA removal efciencies were 23.8%, 48.5% and 70.9% at 6, 22 and 28 h, respectively. Total biodegradation of 600 mg l1 MA was achieved at 32 h. When the MA concentration was 800 mg l1, the removal efciencies were 20%, 38.3% and 83.5% at 6, 22 and 33 h, respectively. It needed 7, 25 and 31 h to achieve 5.4%, 47.8% and 86.9% MA removal, respectively.

1200

1200

Methylamine conc. (mg l-1)

Methylamine conc. (mg l-1)

1000 800

200 mg l-1 400 mg l-1 800 mg l-1 1000 mg l-1

1000
800 600

200 mg l-1 400 mg l-1 800 mg l-1 1000mg l-1

600
400

(A)

400
200

(A)

200
0 0 5 10 15 20 25 30 35 40

0 0
8.5 8
7.5

10

15

20

25

30

35

7.8
7.6

7.4

pH

7.2

(B)

pH

6.5
6

7
5.5

(B)
0 5 10 15 20 25 30 35

6.8 0 5 10 15 20 25 30 35 40

Time (hrs)
Fig. 4. MA removal (A) and pH variation (B) in CSTR at a HRT of 1.136 day using articial wastewater as the inuent.

Time (hrs)
Fig. 5. MA removal (A) and pH variation (B) in CSTR at a HRT of 1.136 day using actual wastewater as the inuent.

Please cite this article in press as: Yang, C.-F., et al., Methylamine removal using mixed bacterial strains in a continuous stirred tank reactor (CSTR) system, International Biodeterioration & Biodegradation (2013), http://dx.doi.org/10.1016/j.ibiod.2013.03.031

C.-F. Yang et al. / International Biodeterioration & Biodegradation xxx (2013) 1e4

Table 1 MA removal using acclimated mixed culture in articial and actual wastewaters under batch and continuous experiments. Experiment Wastewater MA concentration (mg l-1) 200 400 800 1000 200 400 800 1000 200 400 800 1000 200 400 800 1000 Time for 100% MA removal (hrs) 25.5 27.5 71.5 81.5 25.5 29.5 71.5 81.5 28 32 35 33 28 28 30 33 Reference

4. Conclusions This study applied enriched mixed culture to degrade various MA concentrations in articial and actual wastewaters using the CSTR strategy. The optimum HRT for complete MA removal in both wastewaters was 1.136 day. Operating under this HRT, MA concentrations lower than 1000 mg l1 could be utilized for biomass growth and the removal efciency was 100% in both articial and actual wastewater. Acknowledgment The authors would like to thank the National Science Council of the Republic of China, Taiwan for nancially supporting this research under Contract No. NSC 98-2221-E-241-005-MY2. References
Chang, C.T., Chen, B.Y., Shiu, I.S., Jeng, F.T., 2004. Bioltration of trimethylaminecontaining waste gas by entrapped mixed microbial cells. Chemosphere 55, 751e756. Deng, Y.H., Wang, H., Zhong, L., Zhang, H.S., 2009. Trace determination of shortchain aliphatic amines in biological samples by micellar electrokinetic capillary chromatography with laser-induced uorescence detection. Talanta 77, 1337e1342. Ding, Y., Shi, J.Y., Wu, W.X., Yin, J., Chen, Y.X., 2007. Trimethylamine (TMA) bioltration and transformation in biolters. Journal of Hazardous Materials 143, 341e348.  l, P., 2009. Quantication of methylamine in Dryahina, K., Pehal, F., Smith, D., Span e the headspace of ethanol of agricultural origin by selected ion ow tube mass spectrometry. International Journal of Mass Spectrometry 286, 1e6. Helali, S., Puzenat, E., Perol, N., Sa, M.-J., Guillard, C., 2011. Methylamine and dimethylamine photocatalytic degradation- adsorption isotherms and kinetics. Applied Catalysis A: General 402, 201e207. Ho, K.L., Chung, Y.C., Lin, Y.H., Tseng, C.P., 2008. Bioltration of trimethylamine, dimethylamine, and methylamine by immobilized Paracoccus sp. CP2 and Arthrobacter sp. CP1. Chemosphere 72, 250e256. Huang, J., Zhang, H., Wang, J., 2009. Hydrolysis acidication/EGSB/biological contact oxidation/sand ltration process for treatment of methylamine production wastewater. China Water & Wastewater 25, 60e62. Kim, S.G., Bae, H.S., Oh, H.M., Lee, S.T., 2003. Isolation and characterization of novel halotolerant and/or halophilic denitrifying bacteria with versatile metabolic pathways for the degradation of trimethylamine. FEMS Microbiology Letters 225, 263e269. Kim, S.G., Bae, H.S., Lee, S.T., 2001. A novel denitrifying bacterial isolate that degrades trimethylamine both aerobically and anaerobically via two different pathways. Archives of Microbiology 176, 271e277. Mitchell, S.C., Zhang, A.Q., 2001. Methylamine in human urine. Clinica Chimica Acta 312, 107e117. Notice of Intended ChangedMethylamine, 1992. Applied Occupational and Environmental Hygiene 7, 198e199. Rappert, S., Mller, R., 2005. Microbial degradation of selected odorous substances. Waste Management 25, 940e954. Tseng, C.H., Wang, C.C., Chen, C.H., 2011. Methylamine Removal by Mixed Strains in the Batch Reactors. Paper Presented at 2011 Wastewater Treatment Technical Conference on the Chinese Institute of Environmental Engineering, November4e5, Tainan City, Taiwan. The Chinese Institute of Environmental Engineering, Taipei city, Taiwan, R.O.C Vich, D.V., Garcia, M.L., Varesche, M.B.A., 2011. Methanogenic potential and microbial community of anaerobic batch reactors at different ethylamine/sulfate ratios. Brazilian Journal of Chemical Engineering 28, 1e8.

Batch experiment

Articial wastewater

Tseng et al., 2011

Actual wastewater

Tseng et al., 2011

Continuous experiment

Articial wastewater

This study

Actual wastewater

This study

was no signicant difference between the articial and actual wastewaters. However, comparing the two operation strategies, the results indicated that the time needed for complete MA removal in the batch experiment was about 2.5 fold the time needed for complete MA removal in the continuous experiment when the initial MA concentration was higher than 800 mg l1. Owing to the lack of other experimental data, further experiments are needed to make conrmation. To our knowledge, researches about MA biological treatment are quite rare. A biolter using granular activated carbon with immobilized Paracoccus sp. CP2 was applied to the elimination of 10e 250 ppm of trimethylamine (TMA), dimethylamine (DMA), and MA. The results indicated that the system effectively treated MA (>93%), DMA (>90%), and TMA (>85%) under high load conditions, and the maximum degradation rates were 1.4, 1.2, and 0.9 g-N/kg GAC/day (Ho et al., 2008). Vich et al. controlled different MA/sulfate ratios (0.71, 1.26 and 2.18) to investigate methanogenic potential using anaerobic reactors. Their results indicated that the addition of sulfate inhibited the methane production only in the control reactors without MA addition (2011). A hydrolysis acidication/EGSB/ biological contact oxidation/sand ltration process was applied to treat MA production wastewater. Using this system, the discharge could well ll efuent standards (Huang et al., 2009). In the present research, the CSTR was used to deal with various MA concentrations using mixed cultures. Microbial consortia could utilize 1000 mg l-1 MA both from synthetic wastewater and actual industrial wastewater for growth in the CSTR, when the HRT was 1.136 day. MA was totally consumed and the removal efciency was 100%. It is believed that our mixed strains will benet the recovery of environments contaminated by MA.

Please cite this article in press as: Yang, C.-F., et al., Methylamine removal using mixed bacterial strains in a continuous stirred tank reactor (CSTR) system, International Biodeterioration & Biodegradation (2013), http://dx.doi.org/10.1016/j.ibiod.2013.03.031