Bioresource Technology 101 (2010) 6935–6941

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Bioresource Technology

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Degradation of nicotine in tobacco waste extract by newly isolated Pseudomonas sp.ZUTSKD

Weihong Zhong a,*, Chenjing Zhu a, Ming Shu b, Kedan Sun a, Lei Zhao a, Chang Wang a, Zhijuan Ye c,Jianmeng Chen a,**

a College of Biological and Environmental Engineering, Zhejiang University of Technology, Hangzhou 310032, PR Chinab Technology Center, China Tobacco Zhejiang Industrial Co. Ltd., Hangzhou 310009, PR Chinac Technology Center, Hangzhou Liqun Environment Protecting Paper Co. Ltd., Hangzhou 310018, PR China

a r t i c l e i n f o a b s t r a c t

Article history:Received 24 December 2009Received in revised form 25 March 2010Accepted 31 March 2010

Keywords:NicotineDegradationPseudomonas sp.Tobacco waste extract

0960-8524/$ - see front matter � 2010 Elsevier Ltd. Adoi:10.1016/j.biortech.2010.03.142

* Corresponding author. Address: Zhejiang UniversBiological and Environmental Engineering, 18 ChaowPR China. Tel.: +86 571 88320658; fax: +86 571 8832** Corresponding author. Tel.: +86 571 88320386; fa

E-mail addresses: whzhong@zjut.edu.cn (W. Zhong)

The newly isolated Pseudomonas sp. ZUTSKD was evaluated for its ability to degrade nicotine in tobaccowaste extract (TWE). The strain degraded nicotine completely when the concentration of reducing sugarin TWE was lower than 8 g L�1. Yeast extract and phosphate additions improved nicotine degradation in5% TWE. At 30 �C and pH 7.0, with additional 15 g L�1 Na2HPO4�6H2O and 6 g L�1 KH2PO4 in 5% TWE,Pseudomonas sp. ZUTSKD could degrade 97% of nicotine (1.6 g L�1) in 12 h. The data showed that strainZUTSKD could be useful to control the nicotine content in TWE.

� 2010 Elsevier Ltd. All rights reserved.

1. Introduction

Tobacco-related processes can produce solid or liquid wastescontaining high concentrations of water-soluble nicotine that canend up in groundwater. Since nicotine is harmful to humans andthe environment, tobacco wastes have been classified as ‘toxicand hazardous wastes’ by European Union Regulations (Novotnyand Zhao, 1999). Various technologies for tobacco waste treat-ments such as biomethanation (Meher et al., 1995), composting(Piotrowska-Cyplik et al., 2009) and sequencing batch reactor tech-nology (Wang et al., 2009a) have been explored.

Tobacco waste utilization for the production of ‘‘reconstitutedtobacco” has been gradually introduced into the tobacco industryworldwide (Ashok and Joao, 2001). In China, several special ‘‘recon-stituted tobacco” factories have been built during the past decade.These factories collected large quantities of tobacco wastes, includ-ing tobacco plant stems, leaf scraps, dry tobacco dust, adhesives,reinforcing fibers, mineral ash modifiers, and humectants, to pro-duce reconstituted tobacco of different quality for cigarettes facto-ries (Wang et al., 2005a). For example, more than 10,000 tons oftobacco wastes are processed by the Hangzhou Liqun Environment

ll rights reserved.

ity of Technology, College ofang Road, Hangzhou 310032,0057.x: +86 571 88320387., jchen@zjut.edu.cn (J. Chen).

Protecting Paper Co. Ltd., per year alone. Production of ‘‘reconsti-tuted tobacco” is a promising technology to deal with tobaccowastes, but high levels of nicotine and reducing sugars in aqueoustobacco waste extract (TWE) is a problem for manufacturers (Yuanet al., 2007). Therefore, attempts are being made to reduce the nic-otine content.

Biological treatment is a viable method for nicotine removaland numerous studies have demonstrated nicotine degradationby microbes. Microorganisms able to degrade nicotine includeArthrobacter sp. (Hochstein and Rittenberg, 1959; Kodama et al.,1992; Ruan et al., 2006), Pseudomonas sp. (Chen et al., 2008;Civilini et al., 1997; Ruan et al., 2005; Wang et al., 2004), Cellulo-monas sp. (Civilini et al., 1997), Ochrobactrum intermedium (Yuanet al., 2005), Rhodococcus sp. (Gong et al., 2009), Ensifer sp. strainN7 (Lei et al., 2009), and Agrobacterium sp. S33 (Wang et al.,2009c). These nicotine-degrading microbes play an important rolein the manufacturing process of tobacco by altering the content ofnicotine in the final product and in treating nicotine pollution(Brandsch, 2006). The molecular basis of nicotine degradation isalready well understood (Baitsch et al., 2001; Chiribau et al.,2005; Ganas et al., 2009, 2008; Igloi and Brandsch, 2003; Mihasanet al., 2009; Tang et al., 2009, 2008; Wang et al., 2009b, 2007,2005b), but information on culture conditions and factors effect-ing nicotine degradation in TWE are not available. Strains with astronger ability to degrade nicotine suitable for application inTWE treatment are also still required. Therefore, the objectivesof this study were to isolate and evaluate a strain suitable for

6936 W. Zhong et al. / Bioresource Technology 101 (2010) 6935–6941

TWE treatment and to test the effect of glucose and mineral ele-ments in TWE on nicotine degradation.

Table 1Concentration of different elements in 5% diluted TWE and BSM (g L�1).

Items K Ca Mg P Na Fe Mn

5% Diluted TWE(ICP-AES)

2.136 1.517 0.377 0.131 0.020 0.013 0.012

BSM (calculation) 1.021 0.0004 0.036 0.889 0.920 0.0002 0.0001

2. Methods

2.1. Chemicals and media

(�)-Nicotine (98%) isolated from tobacco was purchased fromWeifang Three Power Group Co. Ltd. (Shandong, China). Waste to-bacco leaves and tobacco waste extract (TWE) was supplied byHangzhou Liqun Environment Protecting Paper Co. Ltd. (Hangzhou,China). The contents of solid, nicotine and reducing sugar in TWEwere 54.04% (w/v, dry weight), 1.54% (w/v) and 12.57% (w/v)respectively.

The basic inorganic salt medium (BSM) contained: Na2HPO4�12H2O 5 g, KH2PO4 2 g, MgCl2�6H2O 0.2 g, MnCl2�4H2O 0.0004 g,FeCl3�6H2O 0.001 g, K2SO4 1 g, CaCl2 0.001 g in 1 L of distilledwater, the pH of medium was adjusted to 7.0–7.5.

2.2. Analytical method

The concentration of nicotine in aqueous samples was analyzedby high performance liquid chromatography (HPLC) with a SPD-10AVP instrument (SHIMADZU, Japan) equipped with an AgilentSB-C18 (4.6 � 150 mm). A mixture of methanol and 0.1 mol L�1

KH2PO4 (10:90 by volume, pH 3.0) was used as mobile phase witha flow rate of 1 ml/min. UV detector was carried out at 254 nm, andsampling quantities were 5 lL (Xie et al., 2000). For BSM or dilutedtobacco waste extract (DTWE), centrifugation (14,800g, 10 min,4 �C) was necessary to remove microbes or solid substances beforeHPLC.

Cells were harvested by centrifugation (14,800g, 10 min, 4 �C)from 5 ml of culture, washed three times with 5 ml of 50 mM phos-phate buffer (pH 7.0), and then re-suspended in 5 mL of phosphatebuffer. The optical density (OD) of bacterial cultures was deter-mined at 600 nm using a UV–Vis Spectrophotometer (Type 756,Shanghai Spectrum Instrument Co Ltd., China).

The dinitrosalicylic acid (DNS) (Kidby and Davidson, 1973)method and glucose oxidase (Gu et al., 2005) were used to measurethe concentration of reducing sugars and glucose using a UV–VisSpectrophotometer (Type 756) at 540 nm and a biosensor analyzer(SBA-40C, Shandong Academy of Sciences, China) respectively.

Acid digests of TWE with nitric acid were prepared to measurethe concentration of K, Ca, Mg, P, Na, Fe, and Mn by inductivelycoupled plasma-atomic emission spectrometry (ICP-AES, IRIS In-trepid, Thermo Electron Corporation, USA).

2.3. Strain identification and cultivation conditions

The nicotine-degrading strain ZUTSKD was isolated from wastetobacco leaves and deposited in the China Center for Type CultureCollection (CCTCC at Wuhan University, Wuhan, China) with theaccession number CCTCCM207083.

Cell morphology was observed by a transmission electronmicroscope (JEM-1200EX, Japan), and physiological characteristicswere determined by standard methods (Holt et al., 1994). The Bio-log microstation (GN) (Biolog Hayward, CA, USA) was used to iden-tify the carbon source utilization of the isolate. Chromosomal DNAwas extracted by a boiling procedure (Thomson and Henry, 1995).16S rRNA gene DNA was amplified using PCR with Taq polymerase(Fermentas, Canada), and the universal primer pair of 20F (50-agagtttgatggctca-30) and 1500R (50-cggctaccttgttacgacttc-30)(Weisburg et al., 1991) (PCR procedure: denaturation at 94 �C for4 min; followed by 30 cycles of 94 �C for 50 s, 52 �C for 1 min,and 72 �C for 2 min; and a final incubation at 72 �C for 10 min).

The partial sequence of the amplified DNA was determined bythe Shanghai Invitrogen Biological Technique Company and depos-ited in GenBank under the accession number EF538425. Related se-quences were obtained from the GenBank database (NationalCenter for Biotechnology Information, NCBI) using BLAST (http://www.ncbi.nlm.nih.gov/BLAST/). The 16S rDNA sequences werealigned using CLUSTALX software (Saitou and Nei, 1987). A phylo-genetic tree was constructed with MEGA4 software.

Strain ZUTSKD was cultured in BSM (Sun et al., 2008) at200 rpm and 30 �C. The medium was autoclaved at 121 �C for20 min or 115 �C for 30 min when sugar added. Filter-sterilizednicotine was added to the medium after cooling. Cells were har-vested in later exponential phase (OD600 equal to 0.7) and washedthree times with phosphate buffer (50 mM, pH 7.0) before inocula-tion into different medium. Each test was designed in triplicate.

2.4. Effect of glucose, K2SO4, yeast extract, and phosphate on nicotinedegradation by Pseudomonas sp. ZUTSKD

Since TWE contained more than 100 g L�1 of reducing sugars,the effect of glucose on nicotine degradation by Pseudomonas sp.ZUTSKD was evaluated at concentrations ranging from 1.0 to45 g L�1 in BSM. Then the diluted TWE was utilized to evaluatethe effect of nicotine degradation by the isolate. TWE was dilutedto 5%, 10%, 20%, 30%, and 40% (v/v) before sterilization by autoclav-ing and inoculation and the concentration of nicotine in TWE wasanalyzed by HPLC.

A high concentration of K+ is necessary for the final quality(flammable characteristics) of reconstituted tobacco product.Therefore, the effect of K2SO4 on nicotine degradation by Pseudo-monas sp. ZUTSKD was investigated in BSM containing 6, 12 and18 g L�1 (2.7, 5.4, and 8.1 g L�1 of K+) respectively.

To evaluate the effect of yeast extract in nicotine degradation,0.5, 1.0, and 1.5 g L�1 yeast extract was added BSM containing2 g L�1 of nicotine.

Due to the significant difference of P concentration in BSM and5% TWE (Table 1), the effect of phosphate was determined in 5%TWE containing 5 g L�1 Na2HPO4�12H2O and 2 g L�1 KH2PO4,10 g L�1 Na2HPO4�6H2O and 4 g L�1 KH2PO4, and 15 g L�1 Na2HPO4�6H2O and 6 g L�1 KH2PO4, resulting in final P concentration of0.889, 1.778, and 2.667 g L�1, respectively.

A double-factor orthogonal experiment L9 (3)3 (Table 2) wasperformed to test the reciprocal effect between yeast extract andphosphate on nicotine degradation in 5% diluted TWE.

3. Results and discussion

3.1. Identification and characteristics of strain ZUTSKD capable ofnicotine degradation

Isolate ZUTSKD is a gram-negative bacterium with the strongestability to degrade nicotine among all the strains we isolated fromwaste tobacco leaves. Based on 16S rRNA gene sequence similarity,it was identified as a member of the genus Pseudomonas (99% iden-tity with other strains of the genus Pseudomonas according to Gen-bank BLAST). The phylogenetic relationship with other reportednicotine-degrading strains is shown in Fig. 1. Pseudomonas sp.

Table 2Orthogonal test design L9 (3)3 and results.

Sample A. Yeast extract,g L�1 (level)

B. Phosphatea

g L�1 (level)A/Bb

(level)Degradationratio, %

1 0.5 (1) 0.889 (1) (1) 26.732 0.5 (1) 1.778 (2) (2) 37.213 0.5 (1) 2.667 (3) (3) 57.924 1 (2) 0.889 (1) (2) 48.515 1 (2) 1.778 (2) (3) 50.416 1 (2) 2.667 (3) (1) 56.137 1.5 (3) 0.889 (1) (3) 36.508 1.5 (3) 1.778 (2) (1) 47.029 1.5 (3) 2.667 (3) (2) 56.19K1 121.86 111.74 129.88K2 155.05 134.64 141.91K3 139.71 170.24 144.83k1 40.62 37.25 43.29k2 51.68 44.88 47.30k3 46.57 56.75 48.28R 11.06 19.50 4.98Controlc 0 0 25.27

a Calculated as P-concentration.b Reciprocal effect between yeast extract and phosphate.c The medium without additional yeast extract and phosphate; K, sum of the

degradation ratio of different factors at the same level; k, the average of K; R, thedifference between the maximum and minimum values of k value.

Fig. 1. Phylogenetic tree based on 16S rDNA sequences of Pseudomonas sp. ZUTSKD (MEG0.02 substitutions per site.

0 10 20 30 40 50

0

1

2

3

4

5

6

7A

Nic

otin

e co

ncen

tratio

n (g

L-1)

Time (h)

B

OD

600

Fig. 2. Effect of original nicotine concentration on the n

W. Zhong et al. / Bioresource Technology 101 (2010) 6935–6941 6937

ZUTSKD utilizes nicotine as sole resource of carbon, nitrogen andenergy when grown in BSM. Growth and nicotine degradationwere observed at nicotine concentrations of 2–5.8 g L�1, but wereabsent at 6.5 g L�1 (Fig. 2). Pseudomonas sp. ZUTSKD grew and de-graded nicotine at pH 5–9 in BSM containing 2 g L�1 nicotine andthe optimal pH was 7.5 (Sun et al., 2008).

3.2. Effect of glucose on nicotine degradation by Pseudomonas sp.ZUTSKD

Strain ZUTSKD grew in BSM containing glucose in the range of0–40 g L�1 (data not shown). Glucose in the range of 1–10 g L�1

significantly improved growth and the optimal concentrationwas 4–8 g L�1 (Fig. 3A). One to 8 g L�1 of glucose significantly en-hanced the degradation of nicotine (Fig. 3B), but degradation wasinhibited above 10 g/L of glucose. It appears that 4 g L�1 glucosemight be sufficient for the growth of strain ZUTSKD since only75% glucose could be utilized after 24 h of cultivation (Fig. 3C).

These results suggested that Pseudomonas sp. ZUTSKD could notbe directly applied in the degradation of nicotine in TWE, since thereducing sugar concentration was higher than 125 g L�1. There-fore, TWE was diluted prior to nicotine degradation experiments.Significant nicotine degradation occurred only when TWE was

A software). Bootstrap values (%) are indicated at the nodes. The scale bars represent

0 10 20 30 40 50

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

2.0 g L-1

4.2 g L-1

5.2 g L-1

5.8 g L-1

6.5 g L-1

Time (h)

icotine degradation (A) and cell growth in BSM (B).

0.0

0.5

1.0

1.5

2.0

302520151086421

Dry

Cel

l Mas

s (g

L-1)

Concentration of Glucose (g L-1)0

0 h 7 h 12 h 19 h 24 h

A

0.0

0.5

1.0

1.5

2.0

302520151086421

Res

idua

l Nic

otin

e (g

L-1)

Concentration of Glucose (g L-1)0

0 h 7 h 12 h 19 h 24 h

B

0 5 10 15 20 2502468

1012141618202224262830

Res

idua

l glu

cose

(g L

-1)

Time (h)

1 g L-1

2 g L-1

4 g L-1

6 g L-1

8 g L-1

10 g L-1

15 g L-1

20 g L-1

25 g L-1

30 g L-1

C

Fig. 3. Effect of additional glucose concentration on the growth (A), nicotine degradation (B), and glucose consumption (C) of ZUTSKD in BSM.

6938 W. Zhong et al. / Bioresource Technology 101 (2010) 6935–6941

diluted to 5% and 10% (v/v) (containing about 6 and 12 g L�1 reduc-ing sugars, respectively), and 100% and 78% nicotine, respectively,were degraded after 24 h (Fig. 4). When TWE was diluted to 20%

(containing about 24 g L�1 reducing sugars), only 14% and 25% nic-otine were degraded after 24 and 48 h, respectively. At a 30% and40% dilution of TWE (containing about 36 and 48 g L�1 reducing

0 10 20 30 40 500

1

2

3

4

5

6

7

8

Res

idua

l nic

otin

e co

ncen

tratio

n (g

L-1)

Time (h)

5% diluted 10% diluted 20% diluted 30% diluted 40% diluted

Fig. 4. Degradation of nicotine by Pseudomonas sp. ZUTSKD in diluted TWE.

0 3 6 9 120.0

0.4

0.8

1.2

1.6

2.0

2.4 B

Nic

otin

e co

ncen

tratio

n (g

L-1)

Time (h)

Control 0.5 g L-1

1.0 g L-1

1.5 g L-1

A

0 4 8 120.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

Nic

otin

e co

ncen

tratio

n (g

L-1)

Time (h)

5% TWE 5% TWE+1 g L-1 YE

C D

Fig. 5. Effect of yeast extract addition on nicotine degradation and cell growth in BSM (A,Pseudomonas sp. ZUTSKD.

W. Zhong et al. / Bioresource Technology 101 (2010) 6935–6941 6939

sugars, respectively), only 14% or less nicotine was degraded after24 h and 48 h cultivation, respectively.

3.3. Effect of yeast extract, K2SO4, and phosphate on nicotinedegradation by Pseudomonas sp. ZUTSKD

The addition of yeast extract improved the nicotine degradationand growth (Fig. 5A and B). Yeast extract also improved nicotinedegradation in 5% TWE (diluted with BSM) (Fig. 5C and D).

K2SO4 had no significant influence on cell growth and nicotinedegradation (data not shown). In contrast, the addition of phos-phate promoted nicotine degradation in TWE (Fig. 6).

The double-factor orthogonal experiment using L9 (3)3 demon-strated that the degradation ratio of the experimental group washigher than that of the control group (Table 2) and that yeastextract and phosphate improved nicotine degradation in TWE.Phosphate had the strongest effect on nicotine degradation (largestmargin, R = 19.50) while the reciprocal effect between yeast extractand phosphate had a lesser effect (smallest margin, R = 4.98). Thus,the optimal additional concentrations can be determined by the k

0 3 6 9 120.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7D

ry c

ell w

eigh

t (g

L-1)

Time (h)

Control 0.5 g L-1

1.0 g L-1

1.5 g L-1

0 4 8 12 160.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

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Con

cent

ratio

n of

β-D

-Glu

(g L-1

)

Time (h)

5% WTE 5% WTE+1g L-1 YE

B) and nicotine degradation and D-glucose consumption in 5% diluted TWE (C, D) by

0 3 6 9 120.0

0.3

0.6

0.9

1.2

1.5

1.8

0 3 6 9 120.0

0.4

0.8

1.2

1.6

2.0N

icot

ine

conc

entra

tion

(g L

-1)

Time (h)

Control 0.889 g L-1

1.778 g L-1

2.667 g L-1

A

Con

cent

ratio

n of

β-D

-Glu

(g L

-1)

Time (h)

B

Control 0.889 g L-1

1.778 g L-1

2.667 g L-1

Fig. 6. Effect of additional phosphate (calculated as P concentration) on nicotine degradation (A) and D-glucose consumption (B) by Pseudomonas sp. ZUTSKD in 5% dilutedTWE.

6940 W. Zhong et al. / Bioresource Technology 101 (2010) 6935–6941

values of yeast extract and phosphate, which were 1.0 and2.667 g L�1 (P concentration) respectively.

3.4. Comparison with other reported nicotine-degrading strains

A comparison between Pseudomonas sp. ZUTSKD and other nic-otine-degrading strains is presented in Table 3. Pseudomonas sp.S16 (Wang et al., 2004) and Agrobacterium sp. S33 (Wang et al.,

Table 3Nicotine biodegradation by various microorganisms with different process.

Microorganism Medium Initial nicotine(g L�1)

Inoculum P

Pseudomonas puida Tobacco extract 3.2 Unknown B

Pseudomonas sp. S16 MSMa 4.0 0.12 g L�1 BMSM 2.0 0.12 g L�1 BMSM 1.0 0.12 g L�1 BTobacco solid waste 1.2 84 mg B

Pseudomonas sp. HF-1 ISMb 1.3 Unknown B

Ochrobactrumintermedium DN2

BSMc 0.5 Unknown B

BSM with yeast extractand glucose

1.2 15%(v/v) B

DTWE 1.2 Unknown BDTWE with 1 g L�1 yeastextract

2.1 Unknown Fb

4.3

5.5

Pseudomonas sp. Nic22

ISMc 3.2 Unknown B

Rhodococcus sp. Y22 ISM 1.0 Unknown B

Agrobacterium sp. S33 Minimal medium 1.0 0.26 g L�1 BNicotine medium 3.0 2.05 g L�1 B

Pseudomonas sp.ZUTSKD

BSM with 1.5 g L�1 YE 2.1 0.01 g L�1 B5% DTWE (v/v) 1.6 13.1 mg B10% DTWE (v/v) 2.6 14.9 mg B

a MSM: mineral salts medium.b ISM: inorganic salt medium.c BSM: basci salt medium.d ‘‘–”: not mentioned.

2009c) appears to show the highest degrading activity since theydegraded 100% of nicotine (4 g L�1) in 13 h and 100% of nicotine(3 g L�1) in 1.5 h, respectively. Rhodococcus sp. Y22 (Gong et al.,2009), O. intermedium DN2 (Yuan et al., 2005) and Pseudomonassp. HF-1 (Ruan et al., 2005) could degrade 100% of nicotine(1.0 g L�1) in 52 h, 97.7% of nicotine (0.5 g L�1) in 36 h, and 99%of nicotine (1.3 g L�1) in 25 h, respectively. Pseudomonas sp.ZUTSKD could degrade 96% and 100% of nicotine (2.1 g L�1) in

rocess Workingvolume

Efficiency ofdegradation

Tolerance (g L�1) Reference

Nicotine Glucose

atch 150 ml 97%/40 h –d – Civilini et al.(1997)

atch Unknown 100%/13 h 6 – Wang et al.(2004)atch Unknown 100%/6 h 6 –

atch Unknown 100%/6 h 6 –atch 300 ml 100%/6 h 6 –atch 100 ml 99.6%/25 h 2 – Ruan et al.

(2005)atch 100 ml 97.7%/36 h 5 Yuan et al.

(2005)atch 25 L 95.6%/10 h 5 – Yuan et al.

(2006)atch 100 ml 94.4%/24 h 5.5 – Yuan et al.

(2007)ed-atch

22 L 1st fed, 98.7%/42 h

20 L 2nd fed, 99.2%/22 h

20L 3rd fed, 92.7%/22 h

atch Unknown 96.5%/24 h 3 – Chen et al.(2008)

atch Unknown 100%/52 h 3 – Gong et al.(2009)

atch 50 ml 100%/6 h 5 – Wang et al.(2009b)atch 50 ml 100%/1.5 h

atch 100 ml 96.1%/9 h 5.8 40 This studyatch 100 ml 97.4%/12 hatch 100 ml 92.7%/28 h

W. Zhong et al. / Bioresource Technology 101 (2010) 6935–6941 6941

BSM after 9 h and 12 h, respectively (Table 3, Fig. 5A). Moreover,Pseudomonas sp. ZUTSKD could tolerate 5.8 g L�1 of nicotine, whichis close to the nicotine tolerance level of S16 and DN2. Further-more, Pseudomonas sp. ZUTSKD tolerated 40 g L�1 of glucose.

Only one other study (Yuan et al., 2007) reported on nicotinedegradation in DTWE. The strain used was O. intermedium DN2and it degraded 94% of the nicotine (1.2 g L�1) in 24 h. Pseudomonassp. ZUTSKD degraded 97% of nicotine (1.6 g L�1) in 12 h (Table 3,Fig. 6). Therefore, Pseudomonas sp. ZUTSKD might be another po-tential candidate for biodegradation of nicotine in DTWE.

Since TWE is smeared back onto the reconstituted tobacco pa-pers to recover the original taste and smell of cigarette, a smallquantity of nicotine would be desirable in treated TWE and there-fore, degradation conditions will have to be adjusted to achieve afinal desirable nicotine concentration.

4. Conclusions

Pseudomonas sp. ZUTSKD utilizes nicotine as sole source of car-bon, nitrogen and energy and can potentially be used application inTWE treatment. The conditions for TWE treatment that allow con-trol over the final nicotine content and the effect of biodegradationon taste and other characteristics of reconstituted tobacco stillhave to be determined.

Acknowledgements

This study was supported by Science and Technology Depart-ment of Zhejiang Province of PR China under Grant No.2007C23035 and China Tobacco Zhejiang Industrial Co. Ltd., forwhich the authors are thankful.

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