- Email: [email protected]
Biosorption and biodegradation of triphenyltin by Brevibacillus brevis
Bioresource Technology 129 (2013) 236–241
Contents lists available at SciVerse ScienceDirect
Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Biosorption and biodegradation of triphenyltin by Brevibacillus brevis Jinshao Ye a, Hua Yin b,c,⇑, Hui Peng a, Jieqiong Bai a, Danping Xie d, Linlin Wang a a
Department of Environmental Engineering, Jinan University, Guangzhou 510632, Guangdong, China College of Environmental Science and Engineering, South China University of Technology, Guangzhou 510006, Guangdong, China c Key Lab of Pollution Control and Ecosystem Restoration in Industry Clusters, Ministry of Education, Guangzhou 510006, Guangdong, China d Key Lab of Water and Air Pollution Control of Guangdong Province, Guangzhou 510655, Guangdong, China b
h i g h l i g h t s " Biosorption and biodegradation of TPT by Brevibacillus brevis were studied. 2+
2+
" TPT and coexisted Cu , Cd , Pb
2+
and Zn2+ were adsorbed effectively by B. brevis.
" TPT was transferred into the cytoplasm of B. brevis. " TPT was transformed to DPT, MPT and inorganic tin inside cells. 2+
" H2O2, glucose, rhamnolipid, Cu
a r t i c l e
and Zn2+ obviously improved TPT degradation.
i n f o
Article history: Received 10 September 2012 Received in revised form 13 November 2012 Accepted 19 November 2012 Available online 29 November 2012 Keywords: Biosorption Biodegradation Triphenyltin Brevibacillus brevis
a b s t r a c t Triphenyltin (TPT) is an endocrine disruptor highly toxic to non-target organisms, and has contaminated the environment worldwide. To accelerate TPT elimination, the study on the behavior and mechanism of TPT biosorption and biodegradation by Brevibacillus brevis was conducted. The results revealed that TPT and coexisted Cu2+, Cd2+, Pb2+ and Zn2+ in solution could be adsorbed effectively by B. brevis, and TPT was further transformed to diphenyltin, monophenyltin and tin intracellularly. The removal efficiency of 0.5 mg L1 TPT after degradation by 0.3 g L1 biomass for 5 d was about 60%. Suitable kinds and levels of oxygen, nutrient, surfactant and metals obviously improved TPT biodegradation. When concentrations of H2O2, glucose, rhamnolipid, Cu2+ and Zn2+ varied from 1.5 to 6 mmol L1, 0.5 to 5 mg L1, 5 to 25 mg L1, 0.5 to 6 mg L1 and 0.5 to 1 mg L1, separately, TPT biodegradation efficiencies increased 15–25%. Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction Triphenyltin (TPT) is one of the endocrine disruptors highly toxic to many different non-target organisms, and has contaminated the global environment for its worldwide use as major ingredients of antifouling products and fungicides (Antes et al., 2011; Zhang et al., 2008). Recently, there are many reports that highlighted the development of sensitive and selective analytical methods for TPT determination in various kinds of samples, and explored the accumulation patterns and toxicity of TPT at environmentally relevant concentrations to different organisms (Lyssimachou et al., 2009; Rantakokko et al., 2010). Biological activity (Sakultantimetha et al., 2011a) and physicochemical processes (Zhao et al., 2011) are responsible for TPT ⇑ Corresponding author at: College of Environmental Science and Engineering, South China University of Technology, Guangzhou 510006, Guangdong, China. Tel.: +86 20 39380508; fax: +86 20 85226615. E-mail addresses: [email protected], [email protected] (H. Yin). 0960-8524/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biortech.2012.11.076
elimination to diphenyltin (DPT) and monophenyltin (MPT), and ultimately to inorganic tin under favorable conditions. Some physico-chemical methods, including thermal decomposition (Mesubi and Olatunji, 1983), photodegradation (Zhao et al., 2011), have been employed for the treatment of aqueous TPT. However, the disadvantages of these methods such as high energy or chemical requirements, and high cost have recently shifted studies to developing more efficient degradation processes for TPT control in terrestrial and aquatic environments. Moreover, long half-life of TPT varying from weeks to years has demonstrated that the transformation of TPT and its derivatives in environments that are not exposed to UV light, especially in sediments, is primarily dependent on biodegradation. Some studies attempting to identify the effects of microorganisms on TPT bio-removal determined that supplemental substrates, initial TPT concentration and certain environmental factors seemed to influence the biodegradation efficiencies (Heroult et al., 2008; Stasinakis et al., 2005). Yen et al. (2001) demonstrated that the persistence of TPT acetate in soil was affected by microbial
J. Ye et al. / Bioresource Technology 129 (2013) 236–241
degradation, soil moisture and temperature as well as adsorption. In a 231 d incubation experiment, TPT degradation was found significantly slower in the sterile soil compared to non-sterilized sample (Paton et al., 2006). Even though the biodegradation of TPT has been illustrated by these reports, it must be stressed that information is still severely limited regarding the biodegradation mechanisms, including biosorption, transfer and dearylation. Although biosorption is an emerging and innovative technology using biomass to remove heavy metals and dyes from wastewater (Bulgariu and Bulgariu, 2012), only a few studies have attempted to verify the contribution of biosorption to organotins bio-removal. Among those organotin compounds, tributyltin (TBT) was the only one that had been chosen to conduct adsorption experiment using pure microbial cultures. For example, the adsorbed amount by a single cell of Pseudoalteromonas sp. was up to about 107.5 TBT molecules (Mimura et al., 2008). After biosorption, TPT may transport across cell membrane owing to metabolism or hydrophobicity of TPT that causes intracellular accumulation and biodegradation. The present work aimed to investigate TPT biosorption by Brevibacillus brevis, and improve TPT biodegradation by adding H2O2, glucose and rhamnolipid. To determine the possible effects of heavy metals on TPT biodegradation, Cu2+, Cd2+, Pb2+ and Zn2+ were selected in the study. Moreover, the biosorption of these coexisted metals during TPT biodegradation was also evaluated. The extraand intracellular phenyltins (PTs), including TPT, DPT and MPT, were detected to reveal the mechanisms of TPT biodegradation.
2. Methods
237
0.01, 0.05, 0.1, 0.2, 0.3, 0.4 and 0.5 g L1, individually. TPT solution without biomass was used as the control. The ratios of TPT biosorption are calculated as follows:
Q ¼ ðC o C t Þ 100=C o where Q represents biosorption ratio (%), Co and Ct are initial and final concentration of TPT, individually.
2.4. Biodegradation experiments The flask with 20 mL MSM containing 0.5 mg L1 TPT and 0.3 g L1 B. brevis was inoculated in the dark at 25 °C on a rotary shaker at 100 r min1 for 5 d to elucidate the best conditions. The effects of H2O2, glucose, rhamnolipid, Cu2+, Cd2+, Pb2+ and Zn2+ on TPT biodegradation were assessed. In these experiments, glucose was added at 0.5, 1, 2, 5, 10, 15, 20, 25, 30 and 40 mg L1, H2O2 levels were set at 0, 0.5, 1, 1.5, 3, 6, 9, 12 and 15 mmol L1, individually, and rhamnolipid concentration varied from 0 to 40 mg L1. To determine the removal of both TPT and coexisted heavy metals in MSM, Cu2+, Cd2+, Pb2+ and Zn2+ with initial concentrations of 0, 0.5, 1, 2, 4, 6, 8 and 10 mg L1 were selected, respectively. MSM with 0.5 mg L1 TPT and 1 mg L1 Zn2+ was treated by 0.3 g L1 B. brevis for 5 d to ascertain the metabolites of TPT biodegradation. The controls were run in parallel in flasks with solutions that were uninoculated. All of the experiments were performed in triplicate, and the mean values were used in the calculations.
2.1. Strain and chemicals 2.5. Extraction and derivatization of extracellular and intracellular PTs B. brevis was preserved in the laboratory of the Environmental Engineering Department at Jinan University. This strain was isolated from the sediment samples collected at a town called Guiyu in Guangdong Province, China, which is infamous for its involvement in primitive e-waste processing and recycling activities. The samples contained high levels of organotins and other toxic pollutants. High performance liquid chromatography-grade TPT was obtained from Sigma Aldrich (St. Louis, MO, USA). 2.2. Microbial culture B. brevis was inoculated into the medium, which contained 3 g L1 beef extract, 10 g L1 peptone and 5 g L1 NaCl, at 30 °C on a rotary shaker at 100 r min1 for 24 h. Subsequently, the cells were separated from the medium by centrifugation at 3500g for 5 min. The harvested biomass was washed three times with sterile distilled water and used for further experiments.
After biodegradation, 10 mL hexane was added into 20 mL solution. The PTs in the mixture were sonicated for 20 min in an ultrasonic bath and allowed to set until phase separation. After the organic phase was removed, 10 mL hexane was added into the aqueous phase, and then the operation was repeated again. The organic part was collected, followed by concentrating using a rotary evaporator at 30 °C. The residues that represented the extracellular PTs were dissolved by 5 mL methanol and derivatised in pH 4.5 acetate buffer with 2 mL of 2% sodium diethyl dithiocarbonate. To obtain the intracellular fraction, the aqueous phase with cells was collected after the above extraction in a glass vessel, and subsequently kept in an ice bath during the cell disruption to prevent overheating. Cell disruption was performed at 650 W for 5 min (5 s:10 s pulse on:off basis). Then, the intracellular PTs were extracted and derivatised using the same methods as the extracellular ones.
2.6. Analytical methods of TPT and its metabolites 2.3. Biosorption studies Biosorption of 0.5 mg L1 TPT was performed at 25 °C in 20 mL mineral salts medium (MSM), which was composed of (mg L1 water) 100 Na2HPO412H2O, 50 KH2PO4, 40 NaCl, 20 NH4NO3 and 5 MgSO4, by shaking on a rotary shaker at 100 r min1. After biosorption, cells were separated from MSM by centrifugation at 3500g for 5 min. Residual TPT in resultant supernatant was detected to determine the biosorption efficacies. To verify the effect of pH on TPT biosorption, TPT solution with the initial pH ranging from 5.0 to 9.0 was adsorbed by 0.3 g L1 biomass. The effect of contact time by 0.3 g L1 biomass was investigated in the range of 5–90 min at the original pH of solution. To determine the effect of cell dosage, biomass levels were set at
The PTs were analyzed by gas chromatography–mass spectrometry (GC–MS) (QP2010, Shimadzu) equipped with a type Rxi-5MS GC column (30 m 0.25 mm 0.25 lm). Helium was used as the carrier gas with a constant flow at 1.1 mL min1. The column temperature program started at 50 °C, held for 1.5 min. Subsequently, the oven was heated to 300 °C at a rate of 10 °C min1 and held for 4 min. The solvent cut time was set to 2.6 min. The GC–MS interface temperature was maintained at 280 °C. Mass spectra were recorded at 1 scan s1 under electronic impact with electron energy of 70 eV, mass ranged 50–650 atoms to mass unit. The temperature of ion source was set at 250 °C. Sample of 2 lL was injected directly. The detection limits of TPT, DPT and MPT were 250, 110 and 110 ng L1.
238
J. Ye et al. / Bioresource Technology 129 (2013) 236–241
with the decrease of the residual TPT in MSM, the chances for this part of TPT to contact cells were reduced accordingly. Therefore, TPT biosorption consisted of an initially rapid phase and a subsequently slow one.
3. Results and discussions 3.1. TPT biosorption
100
B 100
80
80
Biosorption (%)
A Biosorption (%)
Fig. 1A suggested that pH significantly affected TPT biosorption. An increasing trend of TPT biosorption with ascending pH from 5.0 to 6.5 was observed. Because there was high level of protons that competed with TPT to form bonds with functional groups on the cell surface, the low biosorption efficiency occurred under these acidic conditions (Ye et al., 2010). Furthermore, the metabolism of B. brevis, which was attributed to TPT biosorption, was inhibited by excess H+. Within the pH range from 6.5 to 9.0, B. brevis exhibited better efficacy on TPT biosorption than in the acidic conditions. According to the data obtained from the persistence of TPT in soil, adsorption amounts of TPT appeared also pH-dependant, being higher when soil pH was high (Marcic et al., 2006). The solubility of TPT chloride in water within pH range of 6–8 is approximately 1 mg L1 at 25 °C (Yi et al., 2012). Therefore, the removal of TPT with an initial concentration of 0.5 mg L1 from MSM in the current experiments was primarily attributed to the biosorption instead of precipitation. The study on the effects of biomass dosage ranging from 0.01 to 0.50 g L1 revealed that the biosorption of TPT was quite effective, and more than 97% TPT was adsorbed by 0.30 g L1 B. brevis within 1 h (Fig. 1B). Similar to the uptake mechanism of other pollutants, this quick removal process was mainly owing to the physicochemical interactions, such as electrostatic attraction, cellular affinity and ion exchange, between TPT and cell surface (Ye et al., 2010). Because this step was independent on metabolism, and hydrophobic TPT tended to interact with lipid and protein, which were the main membrane constituents, the binding of TPT was a rapid process. This is consistent with TBT uptake by dead algal species, in which approximately 85–95% TBT with an initial concentration of 0.3 mg L1 was almost instantaneously adsorbed by Chlorella miniata, C. sorokiniana, Scenedesmus dimorphus and S. platydiscus when cells and TBT were mixed (Tam et al., 2002). As shown in Fig. 1C, the biosorption efficiency of TPT by 0.30 g L1 B. brevis increased instantly and almost reached the biosorption equilibrium within 40 min. Subsequently, the efficiency of TPT biosorption increased slowly with the elongated contact time. These results further confirmed that TPT biosorption was an efficient process primarily dependent on the physicochemical interactions between B. brevis and TPT. After then, active sites for TPT biosorption on the cell surface became less available. Moreover,
60 40 20
40 20 0
7.0 pH
8.0
9.0
0
0.1
0.2
0.3
0.4
TPT degrading strain requires a period of time to adapt to the contaminated environment, since TPT can inhibit the expression of different intracellular enzymes, including cytochrome P450 and glutathione-S-transferase, which are both involved in biotransformation of xenobiotics (Yi et al., 2012). In order to shorten this adaption phase and accelerate the degradation, different levels of glucose were added in the present experiment. Fig. 2 showed that degradation efficiency increased with rising concentrations of glucose varied from 0 to 5 mg L1. This is consistent with TBT degradation in which the amendment with suitable nutrients encouraged microbial activity, resulting in better microbial performance and higher removal of TBT (Sakultantimetha et al., 2011b). However, glucose with higher levels in the current experiment exerted suppressive effect on TPT biodegradation. This is worth further exploring why B. brevis did not perform well under such conditions. One possible explanation is that when there existed high level of exogenous organic carbon, the cells primarily metabolized it, consequently decreasing the degradation of toxic target contaminant. Furthermore, the change in pH induced by excessive utilization of exogenous glucose might pose detrimental effect on TPT biosorption and biodegradation (Hunziker et al., 2001), since the depressed effect of low pH on TPT biosorption has been determined (Fig. 1). To verify this inference, a series of degradation experiments in MSM with the same concentration of glucose were conducted. Considering that the pH maintained between 6.55 and 6.82 after degrading TPT for 5 d, the decreasing degradation efficacy of TPT with higher level of glucose coexisted in MSM was not attributed to the fluctuation of pH, but to the excessive usage of glucose. 3.3. Effect of H2O2 on TPT biodegradation Since H2O2 has been commonly employed to facilitate the degradation of chemical pollutants, especially some persistent organic pollutants (Švrcˇek et al., 2010), we added H2O2 into MSM to examine its possible effect on TPT degradation. Fig. 3 exhibited that 0.5–15 mmol L1 H2O2 oxidized approximately 5–11% TPT, and a further positive influence on aerobic biodegradation of TPT was observed when H2O2 and B. brevis simultaneously presented in MSM, with H2O2 as a supplemental oxygen source. It was seen that the joint removal rate of TPT by cells and H2O2 elevated to the maximum at 86.8% when the concentration of H2O2 was 6.0 mmol L1. This positive behavior in enhancing TPT degradation by adequate oxygen supply was also observed in TBT biodegradation. Aerobic degradation of TBT has been proven to be faster than
0.5
-1
C
100 80 60 40 20
100
8
80
7
60
6
40
5 degradation pH
20
4
0
0 0 10 20 30 40 50 60 70 80 90 Time (min) Fig. 1. Effect of pH, dosage and time on biosorption of 0.5 mg L1 TPT. (A) pH. (B) Dosage. (C) Time.
pH
Dosage (g L )
Degradation (%)
6.0
Biosorption (%)
0 5.0
60
3.2. Influence of glucose on TPT biodegradation
3 0
10
20
30
40
Glucose concentration (mg L-1) Fig. 2. Effect of glucose concentration on pH value and degradation of 0.5 mg L1 TPT by 0.3 g L1 B. brevis at 25 °C for 5 d.
239
J. Ye et al. / Bioresource Technology 129 (2013) 236–241
3.5. Influence of heavy metals on TPT biodegradation
80 60 40
H2O2 H 2O2+ B. brevis
20 0 0.0 3.0 6.0 9.0 12.0 15.0 -1 H2 O2 concentration (mmol L )
Fig. 3. Effect of H2O2 on degradation of 0.5 mg L1 TPT by 0.3 g L1 B. brevis at 25 °C for 5 d.
that under anaerobic conditions (Bernat and Długon´ski, 2006; Stasinakis et al., 2005). However, the removal efficiency reduced to 72.8% when the level of H2O2 went up to 9 mmol L1. Because of its oxidative effect, H2O2 could attack various biological macromolecules, including the catabolic enzymes that were responsible for TPT biodegradation, which induced the decrease of degradation kinetics. Thereby, high levels of H2O2 depressed TPT removal when compared to low levels of H2O2 in the current experiment, but still enhanced the degradation in comparison with the control. 3.4. Influence of surfactant on TPT biodegradation
3.6. Metabolites of TPT biodegradation Although the mechanism of TBT elimination has been revealed in some reports, information on that of TPT biodegradation is still limited. To determine the potential metabolites and transmembrane transfer of PTs, the extracted compounds after TPT biodegradation were explored. Fig. 6A–C exhibited that TPT had been transformed into DPT and MPT, since they were detected by GC– MS-SIM (selective ion monitoring), and the retention times of
100
A
80
Biosorption (%)
Degradation %
Within the range of 5–40 mg L1, rhamnolipid exhibited significant promotion of TPT removal (Fig. 4). This was due to the strong emulsification of TPT by rhamnolipid as the low solubility and high hydrophobicity of TPT limited its transport into microbial cells. Rhamnolipid is a biosurfactant that has both hydrophobic and hydrophilic domains, and is capable of lowering the surface tension of TPT. Furthermore, it can regulate the hydrophobicity of cell surface, through which rhamnolipid will enhance the affinity of the degrading strain to TPT and consequently improve biosorption and metabolism, leading to a higher bioavailability of TPT. Similar results were found in some studies regarding the degradation of other hydrophobic contaminants. For example, the solubilization of TBT increased from 13% to the maximum at 26% by the addition of Tween 80 at 1 critical micelle concentration (Sakultantimetha et al., 2011a). Sponza and Gök (2010) proved that rhamnolipid treatment caused a significant increase of 5- and 6ring PAH degradation. The presence of rhamnolipid also enhanced alkane biodegradation, resulting in up to 71% removal at 60 °C and 42% at 18 °C (Perfumo et al., 2007). Apart from emulsification, similar to the effects of suitable concentration of glucose on TPT degradation, rhamnolipid could also serve as nutrient for cell metabolism due to its bioavailability, which was partially responsible for promotion of TPT biodegradation. However, less improvement of TPT biodegradation was achieved by higher levels of rhamnolipid due to the excessive utilization of this easily metabolized chemical.
Organisms and environments are frequently exposed simultaneously or sequentially to a variety of pollutants via multiple exposure routes. Heavy metals are one of the most abundant and harmful pollutants found in polluted environments. They may inhibit the growth and metabolic activity of microorganisms and thus further depress the biodegradation of persistent organic pollutants (Hong et al., 2007). In order to determine the influence of heavy metals on TPT bio-treatment, some ubiquitous metals found in polluted environments, including Cu, Cd, Pb and Zn, were selected to add into MSM individually during the biodegradation period. The bioaccumulation of these heavy metals by B. brevis through biosorption process was detected and shown in Fig. 5A. When the initial levels were up to 10 mg L1, the removal efficacies of Cu2+, Cd2+, Pb2+ and Zn2+ were 60.5%, 59.2%, 90.9% and 33.2%, separately, illustrating that B. brevis was an effective biosorbent owing to its native tolerance to heavy metals. A similar result was presented that as an indigenous microorganism isolated from heavy metals polluted soil, B. brevis could effectively tolerate Cd and Ni, increase plant growth and promote rhizobacterium (Vivas et al., 2006). Fig. 5B confirmed that B. brevis was suitable to jointly remove TPT and these coexisted heavy metals, although some of them depressed TPT biodegradation to a certain extent. The reduction of TPT biodegradation in the presence of Cd2+ or Pb2+ implied at least one plausible reason that the metabolic activity of B. brevis was negatively affected by these heavy metals, because cadmium and lead are two of the ‘‘Big Three’’ metals that are known for their high toxicity (Volesky, 2007). Apart from the metabolism inhibition, the synchronously bio-adsorbed Cd2+ or Pb2+ competed with TPT to form a bond with functional groups, which were partially attributed to the decrease of TPT bio-elimination. In contrast, Cu2+ and lower levels of Zn2+ exhibited positive effect on TPT degradation. Cu2+ and Zn2+ are important cofactors for many biological processes and are essential for enzymatic activity. For example, B. brevis contains metalloprotease (Serkina et al., 1999) of which zinc is essential for the activity and stability (Lorenzen et al., 2011). The inhibition of the metabolic regulation caused by excessive levels of Zn2+ accordingly induced the depression of TPT degradation. These results revealed that the effects of heavy metals on TPT biodegradation depend on the concentration and species of those metals, as well as their biosorption.
60 40 20
B 100
100 80 60 Cu Cd Pb Zn
40 20
0 10 20 30 40 Rhamnolipid concentration (mg L-1) Fig. 4. Effect of rhamnolipid on degradation of 0.5 mg L1 TPT by 0.3 g L1 B. brevis at 25 °C for 5 d.
80 60 40
Cu Cd Pb Zn
20 0
0
0
Degradation (%)
Degradation (%)
100
0
2 4 6 8 10 Metal concentration (mg L-1)
0
2 4 6 8 10 Metal concentration (mg L-1)
Fig. 5. Effect of heavy metals on degradation of 0.5 mg L1 TPT by 0.3 g L1 B. brevis at 25 °C for 5 d. (A) Heavy metal biosorption. (B) TPT biodegradation.
240
J. Ye et al. / Bioresource Technology 129 (2013) 236–241
A
(x10,000) TIC
5.0 255.00 (1.53) 197.00 (1.53) 253.00 (1.92)
Abundance
4.0 227.00 (2.67)
MPT 3.0
2.0
1.0
13.7
13.8
13.9
14.0
14.1
14.2
14.3
14.4
14.5
14.6
t(min) (x10,000) TIC
B
3.0 303.00 (1.56) 301.00 (2.01) 275.00 (1.89)
Abundance
2.5 273.00 (2.49)
DPT
2.0 1.5 1.0 0.5
18.9
19.0
19.1
19.2
19.3
19.4
19.5
23.8
23.9
19.6
19.7
19.8
t/min
Abundance
C
(x1,000,000) TIC 351.00 (1.50) 349.00 (1.35) 1.00 154.00 (9.16) 197.00 (2.07)
TPT
0.75
0.50
0.25
23.3
23.4
23.5
23.6
23.7
24.0
24.1
24.2
t/min
TPT MPT DPT
60
20
40
10
20 0
TPT (ug Sn L-1)
30
80
E 100
0 1
2
3 4 Time (d)
5
6
40 TPT MPT DPT
80
30
60 20 40 10
20 0
DPT,MPT (ug Sn L-1)
40
100
DPT,MPT (ug Sn L-1)
TPT (ug Sn L-1)
D 120
0 1
2
3 4 Time (d)
5
6
Fig. 6. Metabolites of TPT biodegradation. (A) Qualitative ions of MPT. (B) Qualitative ions of DPT. (C) Qualitative ions of TPT. (D) Total concentrations of TPT, DPT and MPT. (E) Intracellular concentrations of TPT, DPT and MPT.
MPT, DPT and TPT were 14.164, 19.391 and 23.685 min, respectively. MPT appeared as the predominant metabolite (Fig. 6D), suggesting that the degradation of TPT to MPT was a fast process while the transformation of MPT to inorganic tin possessed slow degradation kinetics. This finding is in agreement with previous observations from aquatic environmental monitoring in which MPT is often the predominant species (Marcic et al., 2006). In natural freshwater and soils, TPT seemed to rapidly degrade to MPT, while DPT was rarely detected (Heroult et al., 2008). Because all benzene rings of TPT bond individually with tin atom, the benzene ringcleavage reactions may occur respectively and synchronously. Therefore, the relatively higher concentrations of MPT attained in the current experiment illustrated that a portion of MPT was formed directly from TPT biodegradation, in addition to being produced by the transformation of DPT.
The majority of residual TPT and metabolites was detected inside B. brevis (Fig. 6E), proving that TPT degradation took place intracellularly. Therefore, TPT bio-removal by B. brevis included biosorption, membrane transport and biodegradation. After the rapid biosorption period, TPT was transferred into cytoplasm by active transport and through the interaction of hydrophobic TPT with cell membrane (Ortiz et al., 2005). TBT degradation by Aeromonas veronii also illustrated that TBT and its metabolites bound to the cell membrane lipids due to their hydrophobicity (Cruz et al., 2007). During the biodegradation process, some DBT and MBT were released to extracellular circumstance likely owing to the detoxification of PTs by living cells and the apoptosis of some cells under PTs exposure. B. brevis showed great potential in TPT degradation during the first three days, since the residual TPT was only 40.8 lg Sn L1 by
J. Ye et al. / Bioresource Technology 129 (2013) 236–241
the end of the third day. With the further extension of exposure time, the degradation speed per day for TPT was evidently lower, which was related to the cause that chances for residual TPT to form bonds with enzymes were reduced. Moreover, without exogenous organic carbon besides PTs, which were decreased with the elongated degradation time, the metabolic activity of B. brevis was negatively affected. Since DBT and MBT consumed adenosine triphosphate (ATP), which has the characteristic of ‘‘high energy’’, for their bio-removal, ATP for TPT utilization would decrease. This is the other reason relevant to the slow degradation of TPT during 3–6 d. 4. Conclusions TPT and coexisted Cu2+, Cd2+, Pb2+ and Zn2+ in solution could be adsorbed effectively by B. brevis simultaneously, and TPT was transferred into cytoplasm and further transformed to DPT, MPT and inorganic tin. The removal efficiency of 0.5 mg L1 TPT after degradation by 0.3 g L1 biomass for 5 d was about 60%. When H2O2, glucose, rhamnolipid, Cu2+ and Zn2+ varied from 1.5 to 6 mmol L1, 0.5 to 5 mg L1, 5 to 25 mg L1, 0.5 to 6 mg L1 and 0.5 to 1 mg L1, separately, TPT biodegradation efficiencies were approximately increased 15–25%. Acknowledgements The authors would like to thank the National Natural Science Foundation of China (Nos. 21007020, U0933002, and 50978122), Science and Technology Star Foundation of Pearl River (No. 2012J2200056), and Key Laboratory of Water and Air Pollution Control of Guangdong Province (2011A060901002) for the financial support of this work. References Antes, F.G., Krupp, E., Flores, E.M.M., Antes, F.G., Krupp, E., Flores, E.M.M., 2011. Speciation and degradation of triphenyltin in typical paddy fields and its uptake into rice plants. Environ. Sci. Technol. 45, 10524–10530. Bernat, P., Długon´ski, J., 2006. Acceleration of tributyltin chloride (TBT) degradation in liquid cultures of the filamentous fungus Cunninghamella elegans. Chemosphere 62, 3–8. Bulgariu, D., Bulgariu, L., 2012. Equilibrium and kinetics studies of heavy metal ions biosorption on green algae waste biomass. Bioresour. Technol. 103, 489–493. Cruz, A., Caetano, T., Suzuki, S., Mendo, S., 2007. Aeromonas veronii, a tributyltin (TBT)-degrading bacterium isolated from an estuarine environment, Ria de Aveiro in Portugal. Mar. Environ. Res. 64, 639–650. Heroult, J., Nia, Y., Denaix, L., Bueno, M., Lespes, G., 2008. Kinetic degradation processes of butyl- and phenyltins in soils. Chemosphere 72, 940–946. Hong, H.B., Nam, I.H., Kim, Y.M., Chang, Y.S., Schmidt, S., 2007. Effect of heavy metals on the biodegradation of dibenzofuran in liquid medium. J. Hazard. Mater. 140, 145–148. Hunziker, R.W., Escher, B.I., Schwarzenbach, R.P., 2001. pH dependence of the partitioning of triphenyltin and tributyltin between phosphatidylcholine liposomes and water. Environ. Sci. Technol. 35, 3899–3904. Lorenzen, I., Trad, A., Grötzinger, J., 2011. Multimerisation of a disintegrin and metalloprotease protein-17 (ADAM17) is mediated by its EGF-like domain. Biochem. Biophys. Res. Commun. 415, 330–336.
241
Lyssimachou, A., Ramón, M., Porte, C., 2009. Comparative study on the metabolism of the androgen precursor androstenedione in two gastropod species: in vitro alterations by TBT and TPT. Comp. Biochem. Phys. C 149, 409–413. Marcic, C., Hecho, I.L., Denaix, L., Lespes, G., 2006. TBT and TPhT persistence in a sludged soil. Chemosphere 65, 2322–2332. Mesubi, M.A., Olatunji, G.A., 1983. A study of the thermal decomposition of some triphenyltin carboxylates. Thermochim. Acta 67, 307–313. Mimura, H., Sato, R., Furuyama, Y., Taniike, A., Yagi, M., Yoshida, K., Kitamura, A., 2008. Adsorption of tributyltin by tributyltin resistant marine Pseudoalteromonas sp. cells. Mar. Pollut. Bull. 57, 877–882. Ortiz, A., Teruel, J.A., Aranda, F.J., 2005. Effect of triorganotin compounds on membrane permeability. BBA – Biomembranes 1720, 137–142. Paton, G.T., Cheewasedtham, W., Marr, I.L., Dawson, J.J.C., 2006. Degradation and toxicity of phenyltin compounds in soil. Environ. Pollut. 144, 746–751. Perfumo, A., Banat, I.M., Marchant, R., Vezzulli, L., 2007. Thermally enhanced approaches for bioremediation of hydrocarbon-contaminated soils. Chemosphere 66, 179–184. Rantakokko, P., Hallikainen, A., Airaksinen, R., Vuorinen, P.J., Lappalainen, A., Mannio, J., Vartiainen, T., 2010. Concentrations of organotin compounds in various fish species in the Finnish lake waters and Finnish coast of the Baltic Sea. Sci. Total Environ. 408, 2474–2481. Sakultantimetha, A., Keenan, H.E., Beattie, T.K., Bangkedphol, S., Cavoura, O., 2011a. Bioremediation of tributyltin contaminated sediment: degradation enhancement and improvement of bioavailability to promote treatment processes. Chemosphere 83, 680–686. Sakultantimetha, A., Keenan, H.E., Beattie, T.K., Bangkedphol, S., Cavoura, O., 2011b. Effects of organic nutrients and growth factors on biostimulation of tributyltin removal by sediment microorganisms and Enterobacter cloacae. Appl. Microbiol. Biotechnol. 90, 353–360. Serkina, A.V., Gorozhankina, T.F., Shevelev, A.B., Chestukhina, G.G., 1999. Propeptide of the metalloprotease of Brevibacillus brevis 7882 is a strong inhibitor of the mature enzyme. FEBS Lett. 456, 215–219. Sponza, D.T., Gök, O., 2010. Effect of rhamnolipid on the aerobic removal of polyaromatic hydrocarbons (PAHs) and COD components from petrochemical wastewater. Bioresour. Technol. 101, 914–924. Stasinakis, A.S., Thomaidis, N.S., Nikolaou, A., Kantifes, A., 2005. Aerobic biodegradation of organotin compounds in activated sludge batch reactors. Environ. Pollut. 134, 431–438. ˇ erveny´, L., 2010. The Švrcˇek, J., Marhoul, A., Kacˇer, P., Kuzma, M., Pánek, L., C influence of operating conditions on the efficiency of vapor phase hydrogen peroxide in the degradation of 4-(dimethylamino)benzaldehyde. Chemosphere 81, 617–625. Tam, N.F.Y., Chong, A.M.Y., Wong, Y.S., 2002. Removal of tributyltin (TBT) by live and dead microalgal cells. Mar. Pollut. Bull. 45, 362–371. Vivas, A., Biró, B., Németh, T., Barea, J.M., Azcón, R., 2006. Nickel-tolerant Brevibacillus brevis and arbuscular mycorrhizal fungus can reduce metal acquisition and nickel toxicity effects in plant growing in nickel supplemented soil. Soil Biol. Biochem. 38, 2694–2704. Volesky, B., 2007. Biosorption and me. Water Res. 41, 4017–4029. Ye, J.S., Yin, H., Mai, B.X., Peng, H., Qin, H.M., He, B.Y., Zhang, N., 2010. Biosorption of chromium from aqueous solution and electroplating wastewater using mixture of Candida lipolytica and dewatered sewage sludge. Bioresour. Technol. 101, 3893–3902. Yen, J.H., Tsai, C.C., Su, C.C., Wang, Y.S., 2001. Environmental dissipation of fungicide triphenyltin acetate and its potential as a groundwater contaminant. Ecotoxicol. Environ. Saf. 49, 164–170. Yi, A.X.L., Leung, K.M.Y., Lam, M.H.W., Lee, J.S., Giesy, J.P., 2012. Review of measured concentrations of triphenyltin compounds in marine ecosystems and metaanalysis of their risks to humans and the environment. Chemosphere 89, 1015– 1025. Zhang, Z.B., Hu, J.Y., Zhen, H.J., Wu, X.Q., Huang, C., 2008. Reproductive inhibition and transgenerational toxicity of triphenyltin on medaka (Oryzias latipes) at environmentally relevant tip levels. Environ. Sci. Technol. 42, 8133–8139. Zhao, Y.J., He, J.C., Chen, Q., He, J., Hou, H.Q., Zheng, Z., 2011. Evaluation of 206 nm UV radiation for degrading organometallics in wastewater. Chem. Eng. J. 167, 22–27.
Contents lists available at SciVerse ScienceDirect
Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Biosorption and biodegradation of triphenyltin by Brevibacillus brevis Jinshao Ye a, Hua Yin b,c,⇑, Hui Peng a, Jieqiong Bai a, Danping Xie d, Linlin Wang a a
Department of Environmental Engineering, Jinan University, Guangzhou 510632, Guangdong, China College of Environmental Science and Engineering, South China University of Technology, Guangzhou 510006, Guangdong, China c Key Lab of Pollution Control and Ecosystem Restoration in Industry Clusters, Ministry of Education, Guangzhou 510006, Guangdong, China d Key Lab of Water and Air Pollution Control of Guangdong Province, Guangzhou 510655, Guangdong, China b
h i g h l i g h t s " Biosorption and biodegradation of TPT by Brevibacillus brevis were studied. 2+
2+
" TPT and coexisted Cu , Cd , Pb
2+
and Zn2+ were adsorbed effectively by B. brevis.
" TPT was transferred into the cytoplasm of B. brevis. " TPT was transformed to DPT, MPT and inorganic tin inside cells. 2+
" H2O2, glucose, rhamnolipid, Cu
a r t i c l e
and Zn2+ obviously improved TPT degradation.
i n f o
Article history: Received 10 September 2012 Received in revised form 13 November 2012 Accepted 19 November 2012 Available online 29 November 2012 Keywords: Biosorption Biodegradation Triphenyltin Brevibacillus brevis
a b s t r a c t Triphenyltin (TPT) is an endocrine disruptor highly toxic to non-target organisms, and has contaminated the environment worldwide. To accelerate TPT elimination, the study on the behavior and mechanism of TPT biosorption and biodegradation by Brevibacillus brevis was conducted. The results revealed that TPT and coexisted Cu2+, Cd2+, Pb2+ and Zn2+ in solution could be adsorbed effectively by B. brevis, and TPT was further transformed to diphenyltin, monophenyltin and tin intracellularly. The removal efficiency of 0.5 mg L1 TPT after degradation by 0.3 g L1 biomass for 5 d was about 60%. Suitable kinds and levels of oxygen, nutrient, surfactant and metals obviously improved TPT biodegradation. When concentrations of H2O2, glucose, rhamnolipid, Cu2+ and Zn2+ varied from 1.5 to 6 mmol L1, 0.5 to 5 mg L1, 5 to 25 mg L1, 0.5 to 6 mg L1 and 0.5 to 1 mg L1, separately, TPT biodegradation efficiencies increased 15–25%. Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction Triphenyltin (TPT) is one of the endocrine disruptors highly toxic to many different non-target organisms, and has contaminated the global environment for its worldwide use as major ingredients of antifouling products and fungicides (Antes et al., 2011; Zhang et al., 2008). Recently, there are many reports that highlighted the development of sensitive and selective analytical methods for TPT determination in various kinds of samples, and explored the accumulation patterns and toxicity of TPT at environmentally relevant concentrations to different organisms (Lyssimachou et al., 2009; Rantakokko et al., 2010). Biological activity (Sakultantimetha et al., 2011a) and physicochemical processes (Zhao et al., 2011) are responsible for TPT ⇑ Corresponding author at: College of Environmental Science and Engineering, South China University of Technology, Guangzhou 510006, Guangdong, China. Tel.: +86 20 39380508; fax: +86 20 85226615. E-mail addresses: [email protected], [email protected] (H. Yin). 0960-8524/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biortech.2012.11.076
elimination to diphenyltin (DPT) and monophenyltin (MPT), and ultimately to inorganic tin under favorable conditions. Some physico-chemical methods, including thermal decomposition (Mesubi and Olatunji, 1983), photodegradation (Zhao et al., 2011), have been employed for the treatment of aqueous TPT. However, the disadvantages of these methods such as high energy or chemical requirements, and high cost have recently shifted studies to developing more efficient degradation processes for TPT control in terrestrial and aquatic environments. Moreover, long half-life of TPT varying from weeks to years has demonstrated that the transformation of TPT and its derivatives in environments that are not exposed to UV light, especially in sediments, is primarily dependent on biodegradation. Some studies attempting to identify the effects of microorganisms on TPT bio-removal determined that supplemental substrates, initial TPT concentration and certain environmental factors seemed to influence the biodegradation efficiencies (Heroult et al., 2008; Stasinakis et al., 2005). Yen et al. (2001) demonstrated that the persistence of TPT acetate in soil was affected by microbial
J. Ye et al. / Bioresource Technology 129 (2013) 236–241
degradation, soil moisture and temperature as well as adsorption. In a 231 d incubation experiment, TPT degradation was found significantly slower in the sterile soil compared to non-sterilized sample (Paton et al., 2006). Even though the biodegradation of TPT has been illustrated by these reports, it must be stressed that information is still severely limited regarding the biodegradation mechanisms, including biosorption, transfer and dearylation. Although biosorption is an emerging and innovative technology using biomass to remove heavy metals and dyes from wastewater (Bulgariu and Bulgariu, 2012), only a few studies have attempted to verify the contribution of biosorption to organotins bio-removal. Among those organotin compounds, tributyltin (TBT) was the only one that had been chosen to conduct adsorption experiment using pure microbial cultures. For example, the adsorbed amount by a single cell of Pseudoalteromonas sp. was up to about 107.5 TBT molecules (Mimura et al., 2008). After biosorption, TPT may transport across cell membrane owing to metabolism or hydrophobicity of TPT that causes intracellular accumulation and biodegradation. The present work aimed to investigate TPT biosorption by Brevibacillus brevis, and improve TPT biodegradation by adding H2O2, glucose and rhamnolipid. To determine the possible effects of heavy metals on TPT biodegradation, Cu2+, Cd2+, Pb2+ and Zn2+ were selected in the study. Moreover, the biosorption of these coexisted metals during TPT biodegradation was also evaluated. The extraand intracellular phenyltins (PTs), including TPT, DPT and MPT, were detected to reveal the mechanisms of TPT biodegradation.
2. Methods
237
0.01, 0.05, 0.1, 0.2, 0.3, 0.4 and 0.5 g L1, individually. TPT solution without biomass was used as the control. The ratios of TPT biosorption are calculated as follows:
Q ¼ ðC o C t Þ 100=C o where Q represents biosorption ratio (%), Co and Ct are initial and final concentration of TPT, individually.
2.4. Biodegradation experiments The flask with 20 mL MSM containing 0.5 mg L1 TPT and 0.3 g L1 B. brevis was inoculated in the dark at 25 °C on a rotary shaker at 100 r min1 for 5 d to elucidate the best conditions. The effects of H2O2, glucose, rhamnolipid, Cu2+, Cd2+, Pb2+ and Zn2+ on TPT biodegradation were assessed. In these experiments, glucose was added at 0.5, 1, 2, 5, 10, 15, 20, 25, 30 and 40 mg L1, H2O2 levels were set at 0, 0.5, 1, 1.5, 3, 6, 9, 12 and 15 mmol L1, individually, and rhamnolipid concentration varied from 0 to 40 mg L1. To determine the removal of both TPT and coexisted heavy metals in MSM, Cu2+, Cd2+, Pb2+ and Zn2+ with initial concentrations of 0, 0.5, 1, 2, 4, 6, 8 and 10 mg L1 were selected, respectively. MSM with 0.5 mg L1 TPT and 1 mg L1 Zn2+ was treated by 0.3 g L1 B. brevis for 5 d to ascertain the metabolites of TPT biodegradation. The controls were run in parallel in flasks with solutions that were uninoculated. All of the experiments were performed in triplicate, and the mean values were used in the calculations.
2.1. Strain and chemicals 2.5. Extraction and derivatization of extracellular and intracellular PTs B. brevis was preserved in the laboratory of the Environmental Engineering Department at Jinan University. This strain was isolated from the sediment samples collected at a town called Guiyu in Guangdong Province, China, which is infamous for its involvement in primitive e-waste processing and recycling activities. The samples contained high levels of organotins and other toxic pollutants. High performance liquid chromatography-grade TPT was obtained from Sigma Aldrich (St. Louis, MO, USA). 2.2. Microbial culture B. brevis was inoculated into the medium, which contained 3 g L1 beef extract, 10 g L1 peptone and 5 g L1 NaCl, at 30 °C on a rotary shaker at 100 r min1 for 24 h. Subsequently, the cells were separated from the medium by centrifugation at 3500g for 5 min. The harvested biomass was washed three times with sterile distilled water and used for further experiments.
After biodegradation, 10 mL hexane was added into 20 mL solution. The PTs in the mixture were sonicated for 20 min in an ultrasonic bath and allowed to set until phase separation. After the organic phase was removed, 10 mL hexane was added into the aqueous phase, and then the operation was repeated again. The organic part was collected, followed by concentrating using a rotary evaporator at 30 °C. The residues that represented the extracellular PTs were dissolved by 5 mL methanol and derivatised in pH 4.5 acetate buffer with 2 mL of 2% sodium diethyl dithiocarbonate. To obtain the intracellular fraction, the aqueous phase with cells was collected after the above extraction in a glass vessel, and subsequently kept in an ice bath during the cell disruption to prevent overheating. Cell disruption was performed at 650 W for 5 min (5 s:10 s pulse on:off basis). Then, the intracellular PTs were extracted and derivatised using the same methods as the extracellular ones.
2.6. Analytical methods of TPT and its metabolites 2.3. Biosorption studies Biosorption of 0.5 mg L1 TPT was performed at 25 °C in 20 mL mineral salts medium (MSM), which was composed of (mg L1 water) 100 Na2HPO412H2O, 50 KH2PO4, 40 NaCl, 20 NH4NO3 and 5 MgSO4, by shaking on a rotary shaker at 100 r min1. After biosorption, cells were separated from MSM by centrifugation at 3500g for 5 min. Residual TPT in resultant supernatant was detected to determine the biosorption efficacies. To verify the effect of pH on TPT biosorption, TPT solution with the initial pH ranging from 5.0 to 9.0 was adsorbed by 0.3 g L1 biomass. The effect of contact time by 0.3 g L1 biomass was investigated in the range of 5–90 min at the original pH of solution. To determine the effect of cell dosage, biomass levels were set at
The PTs were analyzed by gas chromatography–mass spectrometry (GC–MS) (QP2010, Shimadzu) equipped with a type Rxi-5MS GC column (30 m 0.25 mm 0.25 lm). Helium was used as the carrier gas with a constant flow at 1.1 mL min1. The column temperature program started at 50 °C, held for 1.5 min. Subsequently, the oven was heated to 300 °C at a rate of 10 °C min1 and held for 4 min. The solvent cut time was set to 2.6 min. The GC–MS interface temperature was maintained at 280 °C. Mass spectra were recorded at 1 scan s1 under electronic impact with electron energy of 70 eV, mass ranged 50–650 atoms to mass unit. The temperature of ion source was set at 250 °C. Sample of 2 lL was injected directly. The detection limits of TPT, DPT and MPT were 250, 110 and 110 ng L1.
238
J. Ye et al. / Bioresource Technology 129 (2013) 236–241
with the decrease of the residual TPT in MSM, the chances for this part of TPT to contact cells were reduced accordingly. Therefore, TPT biosorption consisted of an initially rapid phase and a subsequently slow one.
3. Results and discussions 3.1. TPT biosorption
100
B 100
80
80
Biosorption (%)
A Biosorption (%)
Fig. 1A suggested that pH significantly affected TPT biosorption. An increasing trend of TPT biosorption with ascending pH from 5.0 to 6.5 was observed. Because there was high level of protons that competed with TPT to form bonds with functional groups on the cell surface, the low biosorption efficiency occurred under these acidic conditions (Ye et al., 2010). Furthermore, the metabolism of B. brevis, which was attributed to TPT biosorption, was inhibited by excess H+. Within the pH range from 6.5 to 9.0, B. brevis exhibited better efficacy on TPT biosorption than in the acidic conditions. According to the data obtained from the persistence of TPT in soil, adsorption amounts of TPT appeared also pH-dependant, being higher when soil pH was high (Marcic et al., 2006). The solubility of TPT chloride in water within pH range of 6–8 is approximately 1 mg L1 at 25 °C (Yi et al., 2012). Therefore, the removal of TPT with an initial concentration of 0.5 mg L1 from MSM in the current experiments was primarily attributed to the biosorption instead of precipitation. The study on the effects of biomass dosage ranging from 0.01 to 0.50 g L1 revealed that the biosorption of TPT was quite effective, and more than 97% TPT was adsorbed by 0.30 g L1 B. brevis within 1 h (Fig. 1B). Similar to the uptake mechanism of other pollutants, this quick removal process was mainly owing to the physicochemical interactions, such as electrostatic attraction, cellular affinity and ion exchange, between TPT and cell surface (Ye et al., 2010). Because this step was independent on metabolism, and hydrophobic TPT tended to interact with lipid and protein, which were the main membrane constituents, the binding of TPT was a rapid process. This is consistent with TBT uptake by dead algal species, in which approximately 85–95% TBT with an initial concentration of 0.3 mg L1 was almost instantaneously adsorbed by Chlorella miniata, C. sorokiniana, Scenedesmus dimorphus and S. platydiscus when cells and TBT were mixed (Tam et al., 2002). As shown in Fig. 1C, the biosorption efficiency of TPT by 0.30 g L1 B. brevis increased instantly and almost reached the biosorption equilibrium within 40 min. Subsequently, the efficiency of TPT biosorption increased slowly with the elongated contact time. These results further confirmed that TPT biosorption was an efficient process primarily dependent on the physicochemical interactions between B. brevis and TPT. After then, active sites for TPT biosorption on the cell surface became less available. Moreover,
60 40 20
40 20 0
7.0 pH
8.0
9.0
0
0.1
0.2
0.3
0.4
TPT degrading strain requires a period of time to adapt to the contaminated environment, since TPT can inhibit the expression of different intracellular enzymes, including cytochrome P450 and glutathione-S-transferase, which are both involved in biotransformation of xenobiotics (Yi et al., 2012). In order to shorten this adaption phase and accelerate the degradation, different levels of glucose were added in the present experiment. Fig. 2 showed that degradation efficiency increased with rising concentrations of glucose varied from 0 to 5 mg L1. This is consistent with TBT degradation in which the amendment with suitable nutrients encouraged microbial activity, resulting in better microbial performance and higher removal of TBT (Sakultantimetha et al., 2011b). However, glucose with higher levels in the current experiment exerted suppressive effect on TPT biodegradation. This is worth further exploring why B. brevis did not perform well under such conditions. One possible explanation is that when there existed high level of exogenous organic carbon, the cells primarily metabolized it, consequently decreasing the degradation of toxic target contaminant. Furthermore, the change in pH induced by excessive utilization of exogenous glucose might pose detrimental effect on TPT biosorption and biodegradation (Hunziker et al., 2001), since the depressed effect of low pH on TPT biosorption has been determined (Fig. 1). To verify this inference, a series of degradation experiments in MSM with the same concentration of glucose were conducted. Considering that the pH maintained between 6.55 and 6.82 after degrading TPT for 5 d, the decreasing degradation efficacy of TPT with higher level of glucose coexisted in MSM was not attributed to the fluctuation of pH, but to the excessive usage of glucose. 3.3. Effect of H2O2 on TPT biodegradation Since H2O2 has been commonly employed to facilitate the degradation of chemical pollutants, especially some persistent organic pollutants (Švrcˇek et al., 2010), we added H2O2 into MSM to examine its possible effect on TPT degradation. Fig. 3 exhibited that 0.5–15 mmol L1 H2O2 oxidized approximately 5–11% TPT, and a further positive influence on aerobic biodegradation of TPT was observed when H2O2 and B. brevis simultaneously presented in MSM, with H2O2 as a supplemental oxygen source. It was seen that the joint removal rate of TPT by cells and H2O2 elevated to the maximum at 86.8% when the concentration of H2O2 was 6.0 mmol L1. This positive behavior in enhancing TPT degradation by adequate oxygen supply was also observed in TBT biodegradation. Aerobic degradation of TBT has been proven to be faster than
0.5
-1
C
100 80 60 40 20
100
8
80
7
60
6
40
5 degradation pH
20
4
0
0 0 10 20 30 40 50 60 70 80 90 Time (min) Fig. 1. Effect of pH, dosage and time on biosorption of 0.5 mg L1 TPT. (A) pH. (B) Dosage. (C) Time.
pH
Dosage (g L )
Degradation (%)
6.0
Biosorption (%)
0 5.0
60
3.2. Influence of glucose on TPT biodegradation
3 0
10
20
30
40
Glucose concentration (mg L-1) Fig. 2. Effect of glucose concentration on pH value and degradation of 0.5 mg L1 TPT by 0.3 g L1 B. brevis at 25 °C for 5 d.
239
J. Ye et al. / Bioresource Technology 129 (2013) 236–241
3.5. Influence of heavy metals on TPT biodegradation
80 60 40
H2O2 H 2O2+ B. brevis
20 0 0.0 3.0 6.0 9.0 12.0 15.0 -1 H2 O2 concentration (mmol L )
Fig. 3. Effect of H2O2 on degradation of 0.5 mg L1 TPT by 0.3 g L1 B. brevis at 25 °C for 5 d.
that under anaerobic conditions (Bernat and Długon´ski, 2006; Stasinakis et al., 2005). However, the removal efficiency reduced to 72.8% when the level of H2O2 went up to 9 mmol L1. Because of its oxidative effect, H2O2 could attack various biological macromolecules, including the catabolic enzymes that were responsible for TPT biodegradation, which induced the decrease of degradation kinetics. Thereby, high levels of H2O2 depressed TPT removal when compared to low levels of H2O2 in the current experiment, but still enhanced the degradation in comparison with the control. 3.4. Influence of surfactant on TPT biodegradation
3.6. Metabolites of TPT biodegradation Although the mechanism of TBT elimination has been revealed in some reports, information on that of TPT biodegradation is still limited. To determine the potential metabolites and transmembrane transfer of PTs, the extracted compounds after TPT biodegradation were explored. Fig. 6A–C exhibited that TPT had been transformed into DPT and MPT, since they were detected by GC– MS-SIM (selective ion monitoring), and the retention times of
100
A
80
Biosorption (%)
Degradation %
Within the range of 5–40 mg L1, rhamnolipid exhibited significant promotion of TPT removal (Fig. 4). This was due to the strong emulsification of TPT by rhamnolipid as the low solubility and high hydrophobicity of TPT limited its transport into microbial cells. Rhamnolipid is a biosurfactant that has both hydrophobic and hydrophilic domains, and is capable of lowering the surface tension of TPT. Furthermore, it can regulate the hydrophobicity of cell surface, through which rhamnolipid will enhance the affinity of the degrading strain to TPT and consequently improve biosorption and metabolism, leading to a higher bioavailability of TPT. Similar results were found in some studies regarding the degradation of other hydrophobic contaminants. For example, the solubilization of TBT increased from 13% to the maximum at 26% by the addition of Tween 80 at 1 critical micelle concentration (Sakultantimetha et al., 2011a). Sponza and Gök (2010) proved that rhamnolipid treatment caused a significant increase of 5- and 6ring PAH degradation. The presence of rhamnolipid also enhanced alkane biodegradation, resulting in up to 71% removal at 60 °C and 42% at 18 °C (Perfumo et al., 2007). Apart from emulsification, similar to the effects of suitable concentration of glucose on TPT degradation, rhamnolipid could also serve as nutrient for cell metabolism due to its bioavailability, which was partially responsible for promotion of TPT biodegradation. However, less improvement of TPT biodegradation was achieved by higher levels of rhamnolipid due to the excessive utilization of this easily metabolized chemical.
Organisms and environments are frequently exposed simultaneously or sequentially to a variety of pollutants via multiple exposure routes. Heavy metals are one of the most abundant and harmful pollutants found in polluted environments. They may inhibit the growth and metabolic activity of microorganisms and thus further depress the biodegradation of persistent organic pollutants (Hong et al., 2007). In order to determine the influence of heavy metals on TPT bio-treatment, some ubiquitous metals found in polluted environments, including Cu, Cd, Pb and Zn, were selected to add into MSM individually during the biodegradation period. The bioaccumulation of these heavy metals by B. brevis through biosorption process was detected and shown in Fig. 5A. When the initial levels were up to 10 mg L1, the removal efficacies of Cu2+, Cd2+, Pb2+ and Zn2+ were 60.5%, 59.2%, 90.9% and 33.2%, separately, illustrating that B. brevis was an effective biosorbent owing to its native tolerance to heavy metals. A similar result was presented that as an indigenous microorganism isolated from heavy metals polluted soil, B. brevis could effectively tolerate Cd and Ni, increase plant growth and promote rhizobacterium (Vivas et al., 2006). Fig. 5B confirmed that B. brevis was suitable to jointly remove TPT and these coexisted heavy metals, although some of them depressed TPT biodegradation to a certain extent. The reduction of TPT biodegradation in the presence of Cd2+ or Pb2+ implied at least one plausible reason that the metabolic activity of B. brevis was negatively affected by these heavy metals, because cadmium and lead are two of the ‘‘Big Three’’ metals that are known for their high toxicity (Volesky, 2007). Apart from the metabolism inhibition, the synchronously bio-adsorbed Cd2+ or Pb2+ competed with TPT to form a bond with functional groups, which were partially attributed to the decrease of TPT bio-elimination. In contrast, Cu2+ and lower levels of Zn2+ exhibited positive effect on TPT degradation. Cu2+ and Zn2+ are important cofactors for many biological processes and are essential for enzymatic activity. For example, B. brevis contains metalloprotease (Serkina et al., 1999) of which zinc is essential for the activity and stability (Lorenzen et al., 2011). The inhibition of the metabolic regulation caused by excessive levels of Zn2+ accordingly induced the depression of TPT degradation. These results revealed that the effects of heavy metals on TPT biodegradation depend on the concentration and species of those metals, as well as their biosorption.
60 40 20
B 100
100 80 60 Cu Cd Pb Zn
40 20
0 10 20 30 40 Rhamnolipid concentration (mg L-1) Fig. 4. Effect of rhamnolipid on degradation of 0.5 mg L1 TPT by 0.3 g L1 B. brevis at 25 °C for 5 d.
80 60 40
Cu Cd Pb Zn
20 0
0
0
Degradation (%)
Degradation (%)
100
0
2 4 6 8 10 Metal concentration (mg L-1)
0
2 4 6 8 10 Metal concentration (mg L-1)
Fig. 5. Effect of heavy metals on degradation of 0.5 mg L1 TPT by 0.3 g L1 B. brevis at 25 °C for 5 d. (A) Heavy metal biosorption. (B) TPT biodegradation.
240
J. Ye et al. / Bioresource Technology 129 (2013) 236–241
A
(x10,000) TIC
5.0 255.00 (1.53) 197.00 (1.53) 253.00 (1.92)
Abundance
4.0 227.00 (2.67)
MPT 3.0
2.0
1.0
13.7
13.8
13.9
14.0
14.1
14.2
14.3
14.4
14.5
14.6
t(min) (x10,000) TIC
B
3.0 303.00 (1.56) 301.00 (2.01) 275.00 (1.89)
Abundance
2.5 273.00 (2.49)
DPT
2.0 1.5 1.0 0.5
18.9
19.0
19.1
19.2
19.3
19.4
19.5
23.8
23.9
19.6
19.7
19.8
t/min
Abundance
C
(x1,000,000) TIC 351.00 (1.50) 349.00 (1.35) 1.00 154.00 (9.16) 197.00 (2.07)
TPT
0.75
0.50
0.25
23.3
23.4
23.5
23.6
23.7
24.0
24.1
24.2
t/min
TPT MPT DPT
60
20
40
10
20 0
TPT (ug Sn L-1)
30
80
E 100
0 1
2
3 4 Time (d)
5
6
40 TPT MPT DPT
80
30
60 20 40 10
20 0
DPT,MPT (ug Sn L-1)
40
100
DPT,MPT (ug Sn L-1)
TPT (ug Sn L-1)
D 120
0 1
2
3 4 Time (d)
5
6
Fig. 6. Metabolites of TPT biodegradation. (A) Qualitative ions of MPT. (B) Qualitative ions of DPT. (C) Qualitative ions of TPT. (D) Total concentrations of TPT, DPT and MPT. (E) Intracellular concentrations of TPT, DPT and MPT.
MPT, DPT and TPT were 14.164, 19.391 and 23.685 min, respectively. MPT appeared as the predominant metabolite (Fig. 6D), suggesting that the degradation of TPT to MPT was a fast process while the transformation of MPT to inorganic tin possessed slow degradation kinetics. This finding is in agreement with previous observations from aquatic environmental monitoring in which MPT is often the predominant species (Marcic et al., 2006). In natural freshwater and soils, TPT seemed to rapidly degrade to MPT, while DPT was rarely detected (Heroult et al., 2008). Because all benzene rings of TPT bond individually with tin atom, the benzene ringcleavage reactions may occur respectively and synchronously. Therefore, the relatively higher concentrations of MPT attained in the current experiment illustrated that a portion of MPT was formed directly from TPT biodegradation, in addition to being produced by the transformation of DPT.
The majority of residual TPT and metabolites was detected inside B. brevis (Fig. 6E), proving that TPT degradation took place intracellularly. Therefore, TPT bio-removal by B. brevis included biosorption, membrane transport and biodegradation. After the rapid biosorption period, TPT was transferred into cytoplasm by active transport and through the interaction of hydrophobic TPT with cell membrane (Ortiz et al., 2005). TBT degradation by Aeromonas veronii also illustrated that TBT and its metabolites bound to the cell membrane lipids due to their hydrophobicity (Cruz et al., 2007). During the biodegradation process, some DBT and MBT were released to extracellular circumstance likely owing to the detoxification of PTs by living cells and the apoptosis of some cells under PTs exposure. B. brevis showed great potential in TPT degradation during the first three days, since the residual TPT was only 40.8 lg Sn L1 by
J. Ye et al. / Bioresource Technology 129 (2013) 236–241
the end of the third day. With the further extension of exposure time, the degradation speed per day for TPT was evidently lower, which was related to the cause that chances for residual TPT to form bonds with enzymes were reduced. Moreover, without exogenous organic carbon besides PTs, which were decreased with the elongated degradation time, the metabolic activity of B. brevis was negatively affected. Since DBT and MBT consumed adenosine triphosphate (ATP), which has the characteristic of ‘‘high energy’’, for their bio-removal, ATP for TPT utilization would decrease. This is the other reason relevant to the slow degradation of TPT during 3–6 d. 4. Conclusions TPT and coexisted Cu2+, Cd2+, Pb2+ and Zn2+ in solution could be adsorbed effectively by B. brevis simultaneously, and TPT was transferred into cytoplasm and further transformed to DPT, MPT and inorganic tin. The removal efficiency of 0.5 mg L1 TPT after degradation by 0.3 g L1 biomass for 5 d was about 60%. When H2O2, glucose, rhamnolipid, Cu2+ and Zn2+ varied from 1.5 to 6 mmol L1, 0.5 to 5 mg L1, 5 to 25 mg L1, 0.5 to 6 mg L1 and 0.5 to 1 mg L1, separately, TPT biodegradation efficiencies were approximately increased 15–25%. Acknowledgements The authors would like to thank the National Natural Science Foundation of China (Nos. 21007020, U0933002, and 50978122), Science and Technology Star Foundation of Pearl River (No. 2012J2200056), and Key Laboratory of Water and Air Pollution Control of Guangdong Province (2011A060901002) for the financial support of this work. References Antes, F.G., Krupp, E., Flores, E.M.M., Antes, F.G., Krupp, E., Flores, E.M.M., 2011. Speciation and degradation of triphenyltin in typical paddy fields and its uptake into rice plants. Environ. Sci. Technol. 45, 10524–10530. Bernat, P., Długon´ski, J., 2006. Acceleration of tributyltin chloride (TBT) degradation in liquid cultures of the filamentous fungus Cunninghamella elegans. Chemosphere 62, 3–8. Bulgariu, D., Bulgariu, L., 2012. Equilibrium and kinetics studies of heavy metal ions biosorption on green algae waste biomass. Bioresour. Technol. 103, 489–493. Cruz, A., Caetano, T., Suzuki, S., Mendo, S., 2007. Aeromonas veronii, a tributyltin (TBT)-degrading bacterium isolated from an estuarine environment, Ria de Aveiro in Portugal. Mar. Environ. Res. 64, 639–650. Heroult, J., Nia, Y., Denaix, L., Bueno, M., Lespes, G., 2008. Kinetic degradation processes of butyl- and phenyltins in soils. Chemosphere 72, 940–946. Hong, H.B., Nam, I.H., Kim, Y.M., Chang, Y.S., Schmidt, S., 2007. Effect of heavy metals on the biodegradation of dibenzofuran in liquid medium. J. Hazard. Mater. 140, 145–148. Hunziker, R.W., Escher, B.I., Schwarzenbach, R.P., 2001. pH dependence of the partitioning of triphenyltin and tributyltin between phosphatidylcholine liposomes and water. Environ. Sci. Technol. 35, 3899–3904. Lorenzen, I., Trad, A., Grötzinger, J., 2011. Multimerisation of a disintegrin and metalloprotease protein-17 (ADAM17) is mediated by its EGF-like domain. Biochem. Biophys. Res. Commun. 415, 330–336.
241
Lyssimachou, A., Ramón, M., Porte, C., 2009. Comparative study on the metabolism of the androgen precursor androstenedione in two gastropod species: in vitro alterations by TBT and TPT. Comp. Biochem. Phys. C 149, 409–413. Marcic, C., Hecho, I.L., Denaix, L., Lespes, G., 2006. TBT and TPhT persistence in a sludged soil. Chemosphere 65, 2322–2332. Mesubi, M.A., Olatunji, G.A., 1983. A study of the thermal decomposition of some triphenyltin carboxylates. Thermochim. Acta 67, 307–313. Mimura, H., Sato, R., Furuyama, Y., Taniike, A., Yagi, M., Yoshida, K., Kitamura, A., 2008. Adsorption of tributyltin by tributyltin resistant marine Pseudoalteromonas sp. cells. Mar. Pollut. Bull. 57, 877–882. Ortiz, A., Teruel, J.A., Aranda, F.J., 2005. Effect of triorganotin compounds on membrane permeability. BBA – Biomembranes 1720, 137–142. Paton, G.T., Cheewasedtham, W., Marr, I.L., Dawson, J.J.C., 2006. Degradation and toxicity of phenyltin compounds in soil. Environ. Pollut. 144, 746–751. Perfumo, A., Banat, I.M., Marchant, R., Vezzulli, L., 2007. Thermally enhanced approaches for bioremediation of hydrocarbon-contaminated soils. Chemosphere 66, 179–184. Rantakokko, P., Hallikainen, A., Airaksinen, R., Vuorinen, P.J., Lappalainen, A., Mannio, J., Vartiainen, T., 2010. Concentrations of organotin compounds in various fish species in the Finnish lake waters and Finnish coast of the Baltic Sea. Sci. Total Environ. 408, 2474–2481. Sakultantimetha, A., Keenan, H.E., Beattie, T.K., Bangkedphol, S., Cavoura, O., 2011a. Bioremediation of tributyltin contaminated sediment: degradation enhancement and improvement of bioavailability to promote treatment processes. Chemosphere 83, 680–686. Sakultantimetha, A., Keenan, H.E., Beattie, T.K., Bangkedphol, S., Cavoura, O., 2011b. Effects of organic nutrients and growth factors on biostimulation of tributyltin removal by sediment microorganisms and Enterobacter cloacae. Appl. Microbiol. Biotechnol. 90, 353–360. Serkina, A.V., Gorozhankina, T.F., Shevelev, A.B., Chestukhina, G.G., 1999. Propeptide of the metalloprotease of Brevibacillus brevis 7882 is a strong inhibitor of the mature enzyme. FEBS Lett. 456, 215–219. Sponza, D.T., Gök, O., 2010. Effect of rhamnolipid on the aerobic removal of polyaromatic hydrocarbons (PAHs) and COD components from petrochemical wastewater. Bioresour. Technol. 101, 914–924. Stasinakis, A.S., Thomaidis, N.S., Nikolaou, A., Kantifes, A., 2005. Aerobic biodegradation of organotin compounds in activated sludge batch reactors. Environ. Pollut. 134, 431–438. ˇ erveny´, L., 2010. The Švrcˇek, J., Marhoul, A., Kacˇer, P., Kuzma, M., Pánek, L., C influence of operating conditions on the efficiency of vapor phase hydrogen peroxide in the degradation of 4-(dimethylamino)benzaldehyde. Chemosphere 81, 617–625. Tam, N.F.Y., Chong, A.M.Y., Wong, Y.S., 2002. Removal of tributyltin (TBT) by live and dead microalgal cells. Mar. Pollut. Bull. 45, 362–371. Vivas, A., Biró, B., Németh, T., Barea, J.M., Azcón, R., 2006. Nickel-tolerant Brevibacillus brevis and arbuscular mycorrhizal fungus can reduce metal acquisition and nickel toxicity effects in plant growing in nickel supplemented soil. Soil Biol. Biochem. 38, 2694–2704. Volesky, B., 2007. Biosorption and me. Water Res. 41, 4017–4029. Ye, J.S., Yin, H., Mai, B.X., Peng, H., Qin, H.M., He, B.Y., Zhang, N., 2010. Biosorption of chromium from aqueous solution and electroplating wastewater using mixture of Candida lipolytica and dewatered sewage sludge. Bioresour. Technol. 101, 3893–3902. Yen, J.H., Tsai, C.C., Su, C.C., Wang, Y.S., 2001. Environmental dissipation of fungicide triphenyltin acetate and its potential as a groundwater contaminant. Ecotoxicol. Environ. Saf. 49, 164–170. Yi, A.X.L., Leung, K.M.Y., Lam, M.H.W., Lee, J.S., Giesy, J.P., 2012. Review of measured concentrations of triphenyltin compounds in marine ecosystems and metaanalysis of their risks to humans and the environment. Chemosphere 89, 1015– 1025. Zhang, Z.B., Hu, J.Y., Zhen, H.J., Wu, X.Q., Huang, C., 2008. Reproductive inhibition and transgenerational toxicity of triphenyltin on medaka (Oryzias latipes) at environmentally relevant tip levels. Environ. Sci. Technol. 42, 8133–8139. Zhao, Y.J., He, J.C., Chen, Q., He, J., Hou, H.Q., Zheng, Z., 2011. Evaluation of 206 nm UV radiation for degrading organometallics in wastewater. Chem. Eng. J. 167, 22–27.