Prepping Various Boxes For Aging

[RickHendeson]

Thanks for all the thoughtful and diverse responses!

[PapaDisco]

I've been experimenting with vacuum sealing for the past 6 years.

I've been thinking about trying the Ziploc hand pump 'vacuum' sealer: http://seattlefoodgeek.com/2011/06/ziploc-vacuum-bags-vs-foodsaver-for-sous-vide-at-home/

Anybody try one before? I'm not intending to suck all the air out like a true vacuum sealer, and I like that (maybe) these things are less likely to crush a dress box. Other big issue is the permeability (or not) of a ziploc versus the plastic used in a traditional vacuum sealer. I've heard that regular ziploc actually pass quite a bit of gas through the plastic. I don't know if the vacuum ziplocs are made of a different material.

[Ginseng]

I've been thinking about trying the Ziploc hand pump 'vacuum' sealer...Anybody try one before?

Yes, I've used them in food applications. The bags are sort of crackly and stiff and doesn't feel like a cryovac bag. And the seals are poor. They all fail in time.

Wilkey

[BBS]

My routine:

- I jot my humi date (the date they go into the cabinet) on the bottom of the box, been doing so for years.

- Open them up and give them a general look over.

- Give them a good sniff and dream of what's to come.

- Close them up.

- Open the Staebell and engage in master level Tetris until the new acquisition has a place in the cabinet.

- Close the Staebell.

....and party on....

[earthson]

From my very limited knowledge on aging, conditions which help the microbial community inside the cigars should be the goal of aging. I believe that the Pseudomonas genus is the main contributor to cigar aging. Studies have shown that the some genus members degrade nicotine, but improves leaf quality over time. Which seems to support anecdotal evidence that as cigars age longer, the relative strength of the cigar decreases. However, I do not know if this is the case after the cigar is made or during fermentation in bales, the studies I've read did not test tobacco in the final product, only individual leaves. Beyond this I cannot comment on how to store cigars, this is only what I've found during my research on the topic and what I believe the goals of aging are.

Can I get a reference source for these studies? I would love to read the methods and measurement... especially if this is a study that was done over a relatively short amount of time.

[reffy]

Can I get a reference source for these studies? I would love to read the methods and measurement... especially if this is a study that was done over a relatively short amount of time.

Of course! Most studies observing the general bacterial communities were done between 6-24 months (2nd and 4th). The other studies were examining how the Pseudomonas genus specifically affects the leaf. However, the only problem I can see is if you can access the journals I've read. I have free access from my college, but I'm not too sure about the availability to the general public.

1) Chen, Chunmei, Xuemei Li, Jinkui Yang, Xiaowei Gong, Bing Li, and Ke-Qin Zhang. "Isolation of Nicotine-degrading Bacterium Pseudomonas Sp. Nic22, and Its Potential Application in Tobacco Processing." International Biodeterioration & Biodegradation 62.3 (2008): 226-31. Web. 26 Mar. 2015.

2) Huang, Jingwen, Jinkui Yang, Yanqing Duan, Wen Gu, Xiaowei Gong, Wei Zhe, Can Su, and Ke-Qin Zhang. "Bacterial Diversities on Unaged and Aging Flue-cured Tobacco Leaves Estimated by 16S RRNA Sequence Analysis." Applied Microbiology & Biotechnology 88.2 (2010): 553-62. Web. 26 Mar. 2015.

3) Zhao, Lei, Chenjing Zhu, Yang Gao, Chang Wang, Xuanzhen Li, Ming Shu, Yuping Shi, and Weihong Zhong. "Nicotine Degradation Enhancement by Pseudomonas Stutzeri ZCJ during Aging Process of Tobacco Leaves." World Journal of Microbiology & Biotechnology 28.5 (2012): 2077-086. Web. 26 Mar. 2015.

4) Zhao, Mingqin, Baoxiang Wang, Fuxin Li, Liyou Qiu, Fangfang Li, Shumin Wang, and Jike Cui. "Analysis of Bacterial Communities on Aging Flue-cured Tobacco Leaves by 16S RDNA PCR–DGGE Technology." Applied Microbiology & Biotechnology 73.6 (2007): 1435-440. Web. 26 Mar. 2015.

If you find any other studies of interest feel free to contact me!

[earthson]

Of course! Most studies observing the general bacterial communities were done between 6-24 months (2nd and 4th). The other studies were examining how the Pseudomonas genus specifically affects the leaf. However, the only problem I can see is if you can access the journals I've read. I have free access from my college, but I'm not too sure about the availability to the general public.

Thank you so much! I should be able to get free access - I'm doing research with USGS right now and I know my project advisor has access to JSTOR and many of the other major journal access points.

I've dabbled a bit in growing edible and medicinal fungi as a hobby and our current research involves creating substrates for bioremediation/augmented-attenuation technology. I am perpetually in awe of the Chinese and Japanese researchers who are simply light years beyond almost any other teams working with fungi or bacterial populations.

[reffy]

I've dabbled a bit in growing edible and medicinal fungi as a hobby and our current research involves creating substrates for bioremediation/augmented-attenuation technology. I am perpetually in awe of the Chinese and Japanese researchers who are simply light years beyond almost any other teams working with fungi or bacterial populations.

I believe in one of the studies they suggested that, through inoculation of tobacco leaves with leaf enhancing bacteria, they hope to increase the rate at which the tobacco gains the more positive qualities. I haven't seen any studies submitted yet which suggest that they have completed experiments in this, but it is certainly an exciting prospect!

[earthson]

I believe in one of the studies they suggested that, through inoculation of tobacco leaves with leaf enhancing bacteria, they hope to increase the rate at which the tobacco gains the more positive qualities. I haven't seen any studies submitted yet which suggest that they have completed experiments in this, but it is certainly an exciting prospect!

It makes me think of the recent quality of CC in that some smokes are now simply amazing after less than a year.

[Rushman]

about the only way I can successfully age cigars is to let someone else keep them for me - out of my dirty little hands.....El Pres' OLH does a fine job of this.

if only he could install a webcam!

[Habana Mike]

Can I get a reference source for these studies? I would love to read the methods and measurement... especially if this is a study that was done over a relatively short amount of time.

Enjoy!

Characterization of environmentally friendly nicotine degradation by Pseudomonas putida biotype A strain S16

http://mic.sgmjournals.org/content/153/5/1556.full.pdf

Shu Ning Wang, Zhen Liu,3 Hong Zhi Tang,3 Jing Meng3 and Ping Xu

Correspondence

Ping Xu

[email protected]

State Key Laboratory of Microbial Technology, Shandong University, Jinan,

People’s Republic of China 250100

Received 13 December 2006

Accepted 9 January 2007

Nicotine and some related alkaloids in tobacco and tobacco wastes are harmful to health and the

environment, and a major environmental requirement is to remove them from tobacco and tobacco

wastes. In this study, an isolated strain, S16, identified as Pseudomonas putida biotype A, was

used to investigate nicotine degradation. Possible intermediates were identified based on the

results of NMR, Fourier-transform (FT)-IR and UV spectroscopy, GC-MS and high-resolution MS

(HR-MS) analysis. The pathway of nicotine degradation in P. putida was proposed to be from

nicotine to 2,5-dihydroxypyridine through the intermediates N-methylmyosmine,

29-hydroxynicotine, pseudooxynicotine, 3-pyridinebutanal,C-oxo, 3-succinoylpyridine and

6-hydroxy-3-succinoylpyridine. N-Methylmyosmine, 2,5-dihydroxypyridine and succinic acid were

detected and satisfactorily verified for the first time as intermediates of nicotine degradation. In

addition, an alcohol compound, 1-butanone,4-hydroxy-1-(3-pyridinyl), was found to be a novel

product of nicotine degradation. These findings provide new insights into the microbial metabolism

of nicotine and the environmentally friendly route of nicotine degradation.

INTRODUCTION

Nicotine, a major alkaloid synthesized as the L-isomer in

tobacco plants, plays a critical role in smoking addiction. In

China, 20 % of the world’s population (1.2 billion people)

consumes 30 % of the world’s cigarettes. If current smoking

patterns persist, tobacco will kill around 100 million Chinese

in the next 50 years (Holden, 2001). In 2000, about

4.9 million smoking-related premature deaths occurred

throughout the world. In the USA, tobacco use was

responsible for nearly one in five deaths, or an estimated

440 000 deaths per year, in the period 1995–1999. Smoking

accounts for at least 30 % of all cancer deaths and 87 % of

lung cancer deaths (American Cancer Society, 2005).

Currently, regulatory strategies to control the tobaccoinduced

disease epidemic are very much focused on nicotine.

Reduced-nicotine cigarette products have been advocated to

gradually lower the level of nicotine dependence (Benowitz

& Henningfield, 1994). The American Medical Association

has endorsed a public health strategy in which the nicotine

level of tobacco would be forcibly reduced (Henningfield

et al., 1998).

Nicotine is also a significant toxic waste product in tobacco

production. The tobacco-manufacturing process and all

activities that use tobacco produce solid or liquid wastes

with high concentrations of nicotine (Novotny & Zhao,

1999). A non-recyclable, powdery, nicotine-containing

waste is formed during tobacco production, which has an

average nicotine content of 18 grams per kilogram dry

weight. This waste is classified as ‘toxic and hazardous’ by

European Union regulations when the nicotine content

exceeds 500 milligrams per kilogram dry weight (Civilini

et al., 1997). Therefore, there is a major environmental

requirement to remove nicotine from tobacco wastes.

Nicotine degradation by micro-organisms has received

increasing attention in the past 50 years because microorganisms

have the potential to reduce nicotine levels in

tobacco and to detoxify tobacco wastes (Civilini et al., 1997;

Wang et al., 2004). However, the current understanding of

nicotine metabolism in micro-organisms is poor. Some

bacteria, such as Pseudomonas sp. no. 41 (Wada & Yamasaki,

1954), Pseudomonas convexa PC1 (Thacker et al., 1978),

Arthrobacter oxidans (Sguros, 1955), A. oxidans P-34

(Gherna et al., 1965; reclassified as Arthrobacter ureafaciens

by Kodama et al., 1992), A. oxidans pAO1 (Decker & Bleeg,

1965; reclassified as Arthrobacter nicotinovorans by Kodama

3These authors contributed equally to this work.

Abbreviations: BSTFA, N,O-bis(trimethylsilyl)trifluoroacetamide; DCIP,

2,6-dichlorophenolindophenol; DCW, dry cell weight; DHP, 2,5-dihydroxypyridine;

ESI, electrospray ionization; FT-IR, Fourier-transform IR;

HR-MS, high-resolution MS; HSP, 6-hydroxy-3-succinoylpyridine; SP, 3-

succinoylpyridine; TMS, trimethylsilyl.

The GenBank/EMBL/DDBJ accession number for the nucleotide

sequence of the 16S rRNA gene of strain S16 determined in this study

is AY574282.

1556 2006/005223 G 2007 SGM Printed in Great Britain

Microbiology (2007), 153, 1556–1565 DOI 10.1099/mic.0.2006/005223-0

t al., 1992) and Achromobacter nicotinophagum (Hylin,

1959), have been proposed to degrade nicotine, mainly via

two different pathways (Kaiser et al., 1996). In the genus

Arthrobacter, the pathway and related metabolic mechanism

in the molecular biology of nicotine degradation have been

thoroughly elucidated (Gherna et al., 1965; Brandsch et al.,

1982; Schenk et al., 1998; Igoli & Brandsch, 2003). However,

the nicotine-degradation mechanisms in Pseudomonas and

other genera are less well documented. In Pseudomonas

species, pseudoxynicotine, methylamine, 3-succinoylpyridine

(SP) and 6-hydroxy-3-succinoylpyridine (HSP) were

detected as the initial catabolic products of nicotine by Wada

& Yamasaki (1954) and Tabuchi (1955). HSP has been

supposed to be further metabolized by cleavage at the 3

position of HSP with the formation of 2,5-dihydroxypyridine

(DHP) and succinic acid (Kaiser et al., 1996).

However, until now, no research on the detection or

satisfactory characterization of N-methylmyosmine, DHP

and succinic acid in nicotine degradation by Pseudomonas

species has been reported, and no complete pathway could

be reliably constructed. Furthermore, although the identi-

fication of the intermediates in earlier investigations was

probably correct, the techniques used at the time did not

conform to current standards of metabolite characterization.

In our previous publication (Wang et al., 2005), a ‘green’

route to HSP from the nicotine of tobacco waste employing

whole cells of Pseudomonas sp. S16 was developed, and

HSP was easily purified with a 43.8 % (w/w) yield and

characterized. However, other metabolites from nicotine

have not yet been completely detected and identified. In this

paper, we further describe the isolation, identification and

characterization of the nicotine-degrading bacterium S16

and the possible intermediates of nicotine degradation. A

proposed pathway for nicotine degradation by strain S16 is

also postulated and discussed according to the results of

NMR, Fourier-transform IR (FT-IR) and UV spectroscopy,

GC-MS, and high-resolution MS (HR-MS) analysis, as is the

transformation reaction by the cell-free extract.

METHODS

Isolation and growth of bacteria. A soil sample (0.5 g, wet

weight) from a field under continuous tobacco cropping in

Shandong, People’s Republic of China, was incubated with liquid

medium containing 1 g L-(2)-nicotine l21 at 30 uC with shaking at

120 r.p.m. in an incubator. The liquid culture medium was a minimal

medium containing (per litre) 13.3 g K2HPO4.3H2O, 4 g

KH2PO4, 0.2 g MgSO4.7H2O and 0.5 ml trace elements solution.

The trace elements solution contained (per litre of 0.1 M HCl)

0.05 g CaCl2.2H2O, 0.05 g CuCl2.2H2O, 0.008 g MnSO4.H2O,

0.004 g FeSO4.7H2O, 0.1 g ZnSO4, 0.1 g Na2MoO4.2H2O and 0.05 g

Na2WO4.2H2O. Nicotine (¢99 % purity, purchased as the free base

from Fluka) was added to the medium after filter-sterilization. After

bacterial growth was observed, the culture was used as an inoculum

and transferred twice. The final culture (0.1 ml) was serially diluted

and spread onto agar plates containing nicotine. After 2 days, colonies

began to appear on plates incubated at 30 uC. The colonies were

picked and streaked to purity on nicotine agar plates. One isolate

with higher capacity for nicotine degradation was selected for

further study. It was routinely and optimally cultured with 3 g nicotine

l21 at 30 uC and pH 7.0.

Strain identification and characterization. Physiological characteristics

were determined according to Palleroni (1984). Cell morphology

was observed using a Hitachi S-570 scanning electron

microscope. Fatty acids were analysed using the Sherlock microbial

identification system, version 4.0 (MIDI) with the TSBA (revision

4.10) database of the Deutsche Sammlung von Mikroorganismen

und Zellkulturen (DSMZ). The 16S rRNA gene was amplified by

PCR using the oligonucleotides 27f (59-AGAGTTTGATCMTGGCTCAG-39)

and 1492r (59-TACGGYTACCTTGTTACGACTT-39)

as primers (Johnson, 1994). The fragment generated was purified by

agarose gel electrophoresis and band extraction before sequencing

analysis. Related sequences were obtained from the GenBank database

using the BLASTN search program.

Nicotine degradation by resting cells. Cells were harvested in

mid-exponential phase by centrifugation at 6000 g for 15 min at

4 uC, then washed three times with 0.05 M sodium phosphate buffer

(pH 7.0). These cells were called resting cells. The degradation

experiment was performed in a 5 l flask containing 3.37 mg dry cell

weight (DCW) ml21 of resting cells (OD620 ~6; 1.0 OD620

unit=0.56 mg DCW ml21

), 3 g nicotine l21 and 1 l sterilized

0.05 M sodium phosphate buffer or deionized water (pH adjusted

to 7.0 at the beginning of the reaction), at 30 uC with shaking at

120 r.p.m.

General analytical methods. During the course of bacterial

growth or resting cell reaction, aliquots of the culture or cell suspension

were sampled and the cells removed by centrifugation at 6000 g

for 15 min at 4 uC. The supernatant was used for GC, HPLC, UV

absorption and TLC analysis. GC and HPLC analysis was performed

as previously described (Wang et al., 2005). Identification and quantitative

data for nicotine, DHP and intermediates were obtained by

comparing the retention time and peak areas of the unknown compounds

with those of standards (DHP was from SynChem OHG) or

of the intermediates of known concentration isolated and purified

from this study. The supernatant was also diluted with 0.1 M HCl

and scanned with a UV-3100 spectrophotometer (Shimadzu) to

record the UV absorption spectra of the metabolites. TLC analysis

was carried out as previously described (Wang et al., 2005), with a

slight modification, using silica gel HSGF254 0.20 mm plates

(Huanghai) and chloroform/ethanol/methanol/0.5 M NaOH

(30 : 15 : 2 : 1.5, v/v) for development. The spots of the metabolites

were examined under UV light (254 nm).

Isolation of metabolites SP and HSP. After incubation of nicotine

and resting cells for ~3 h, several possible metabolites were

found by TLC analysis (Fig. 1b), and metabolites SP and HSP accumulated.

In order to isolate the two metabolites, the reaction was

stopped by centrifugation at 12 000 g for 10 min at 4 uC to remove

the resting cells. A 1 l volume of supernatant was evaporated at

50 uC under reduced pressure to about 100 ml. To obtain metabolite

SP, the condensate was adjusted to pH 4.0 with 1 M HCl, and

extracted with chloroform. The chloroform phase was concentrated

by evaporation and a white crystalline substance was obtained. To

obtain metabolite HSP, the mother liquor after removing metabolite

SP was evaporated again to about 50 ml, and 3 M HCl was added

until the mixture became cloudy. From this mixture, a brick-red

precipitate was obtained by filtration and drying. The two metabolites

were purified by recrystallization.

Identification of metabolites SP and HSP. The identification of

the metabolites SP and HSP was performed by UV, FT-IR, MS and

NMR analysis. The UV spectra were recorded with a UV-3100 spectrophotometer

(Shimadzu) in 0.1 M HCl and 0.1 M NaOH. FT-IR

http://mic.sgmjournals.org 1557

Nicotine degradation in Pseudomonas putida

pectra were obtained on a Nexus 470 spectrometer (Nicolet). MS

analysis was run on an API 4000 mass spectrometer with electrospray

ionization (ESI) (Applied Biosystems) using methanol as solvent.

NMR spectra were recorded for solutions in DMSO-d6 on an

Avance 600 spectrometer (Bruker) operated at 600 MHz for 1

H and

at 150 MHz for 13C. DEPT90, DEPT135, H-H COSY, HMQC and

HMBC spectra of metabolite SP were also recorded.

Isolation and HR-MS analysis of metabolite A. In order to

obtain metabolite A, the reaction was stopped by centrifugation to

remove the resting cells after incubation for ~4 h; at that time

metabolite A had accumulated to a maximum (Fig. 1b). The reaction

solution was adjusted to pH 11.0 with 1 M NaOH and

extracted with chloroform. The chloroform phase was concentrated

and applied to preparative TLC on silica gel HSGF254 0.50 mm

plates (Huanghai) employing chloroform/ethanol/methanol/0.5 M

NaOH (30 : 15 : 2 : 1.5, v/v) for development. The fluorescent spots

were detected under a UV light (254 nm). The band of metabolite A

on the plate was scraped off and eluted with methanol and ultrasound.

After concentration, the eluate was analysed by GC-MS

(Waters GCT mass spectrometer, coupled to an Agilent HP6890 gas

chromatograph) to record the high-resolution mass spectrum of

metabolite A. The system was equipped with a J&W DB-5MS

column (0.25 mm internal diameter650 m length60.25 mm film

thickness, Folsom). Chromatographic conditions were: 0.5 ml injection

volume (splitless injection, 30 : 1); carrier gas, helium at a constant

flow of 1.0 ml min21

; temperature programme 50 uC for

2 min, then to 280 uC at a rate of 10 uC min21 for 10 min. The ionization

energy was 70 eV, and the temperature was 280 uC with a

mass-to-charge ratio of 20–600.

GC-MS analysis for other metabolites. During the nicotinedegradation

experiment, the reaction mixture was sampled hourly.

After removing the cells, the samples (25 ml) were evaporated to

dryness at 50 uC under reduced pressure and dissolved in 2 ml acetonitrile.

The acetonitrile solution (0.2 ml) was transferred to a vial

and dried under a stream of nitrogen. The residue was silylated by

addition of 100 ml bis(trimethylsilyl)trifluoroacetamide (BSTFA;

Sigma-Aldrich) at 70 uC for 3 h. After drying under a stream of

nitrogen, the sample was redissolved in acetonitrile. Two controls

were silylated by the same procedure. One contained nicotine alone

without resting cells, and the other contained resting cells without

nicotine. The samples were analysed by GC-MS (GCD 1800C,

Hewlett Packard) equipped with a 50 m J&W DB-5MS column

(Folsom).

Transformation of nicotine and its metabolites by a cell-free

extract. Cells were harvested in mid-exponential phase by centrifugation

at 6000 g for 15 min at 4 uC, washed three times with 0.05 M

sodium phosphate buffer (pH 7.0), then suspended in the same

buffer and disrupted by ultrasonification in an ice/water bath. After

centrifugation at 20 000 g for 30 min at 4 uC, the clear supernatant

was used as cell-free extract for degradation of nicotine, and the

metabolites SP and HSP. All the reactions were performed in a 1 ml

total volume at 30 uC with gentle shaking. Reaction mixtures contained

0.05 M sodium phosphate buffer (pH 7.0), an appropriate

volume of cell-free extract (a final protein content of 0.3 mg ml21

)

and substrates at 0.3 mg ml21

. The changes in the substrates and

the formation of products were determined by UV spectroscopy, GC

and HPLC after 30 min (10 min for the HSP degradation reaction).

RESULTS

Characterization and identification of a

nicotine-degrading bacterium

A nicotine-degrading bacterium was isolated from soil

samples obtained from a field under continuous tobacco

cropping in Shandong, People’s Republic of China, and

designated strain S16. It could use nicotine as the sole

carbon, nitrogen and energy source. S16 grew rapidly and

optimally with 3 g nicotine l21

, and at 30 uC and pH 7.0,

and completely degraded nicotine within 10 h with a

maximum biomass of 1.4 mg DCW ml21 (Wang et al.,

2004).

Strain S16 was deposited at the China Center for Type

Culture Collection (CCTCC; accession no. M 205038). It

was a Gram-negative, mobile, rod-shaped bacterium

(0.52–0.6561.05–1.43 mm) with one or two flagella at

one pole (data not shown). Its physiological characteristics

(data not shown) were identical to those of Pseudomonas

putida (Holt et al., 1994). S16 could grow on glucose, xylose,

arabinose, citrate, galactose, rhamnose, glycerol, valine,

Fig. 1. (a) UV and ( TLC analysis of nicotine metabolites.

Nicotine (3 g l”1

) and 3.37 mg DCW ml”1 resting cells were

incubated at pH 7.0 and 30 6C in 0.05 M sodium phosphate

buffer (pH 7.0) (a) and deionized water (pH 7.0) (. In (a),

only nicotine was in the reaction mixture at 0 h (maximum

absorbance at 259 nm), and later metabolites (maximum absorbance

at ~223, 263, 275 and 310 nm) were formed and then

degraded. ( Nic, nicotine standard; DHP, 2,5-dihydroxypyridine

standard.

1558 Microbiology 153

S. N. Wang and others

rginine, alanine, fructose, mannose, creatine and ethanol.

However, it could not grow on lactose, sucrose, mannitol,

sorbitol, trehalose, raffinose or inositol. Fatty acid analysis of

S16 gave a 0.881 similarity index with P. putida biotype A in

the TSBA (revision 4.10) database. The partial 16S rRNA

gene sequence of S16 (1453 nt; GenBank accession no.

AY574282) was determined. BLASTN search analysis revealed

that the sequence showed high homology (¢99.0 %) with

those of P. putida (AB029257.1), Pseudomonas monteilii

(AF064458.1), Pseudomonas plecoglossicida (AB009457.1),

P. putida KT2440 (AE016774.1) and P. putida ATCC 12633T

(AF094736.1). The results suggested that strain S16 was

closely related to the genus Pseudomonas. Based on the

comparative 16S rRNA gene sequencing, chemotaxonomy,

and morphological and physiological data, we concluded

that strain S16 belonged to the species P. putida biotype A.

Nicotine degradation by resting cells of S16

To detect the products of nicotine degradation, we carried

out the experiments in two different media using resting

cells of S16. When the degradation experiment was

performed in 0.05 M sodium phosphate buffer (pH 7.0),

nicotine was fully degraded in 5 h (Fig. 1a), while it took

more than 8 h to completely decompose nicotine in

deionized water (pH adjusted to 7.0 at the beginning of

the reaction; see Fig. 1b). As nicotine was degraded in

deionized water, the pH of the suspension dropped, and the

low pH value of the mixture inhibited the reaction from

proceeding. This suggested that acidic metabolites were

formed by the reaction. Moreover, there were substantial

changes in both the intensity and the wavelength of the

absorption maximum of the UV absorption spectra of the

reaction mixture in the degradation experiments (Fig. 1a).

The altered UV absorption suggested the formation of

metabolites. Furthermore, several possible metabolites were

also found in the TLC analysis (Fig. 1b).

Isolation and identification of metabolites SP

and HSP

Two principal metabolites, SP and HSP, were produced

during the incubation reaction (Fig. 1b), and they were

isolated and identified according to the description in

Methods. The physical and chemical properties of SP are

summarized in Table 1. The NMR data of HSP have been

presented in a previous publication (Wang et al., 2005).

Other properties of HSP determined in this study were:

melting point, 290–293 uC; UV absorption, lmax (in 0.1 M

HCl)=276.6 (EM 10246.5), 206.6 (EM 16298.7), lmin (in

0.1 M HCl)=230.4; lmax (in 0.1 M NaOH)=304.2 (EM

20384.7), lmin (in 0.1 M NaOH)=240.8; FTIR (KBr), 3428

and 3240 cm21 (OH), 1719 (CO) cm21

. These results

confirmed that the metabolites were SP and HSP. The

structures of SP and HSP are shown in Fig. 4 (SP is VII and

HSP is VIII).

Isolation and identification of metabolite A by

TLC and GC-HR-MS analysis

Metabolite A was produced in the early phase and later

disappeared during nicotine degradation by the resting cells

of S16 (Fig. 1b). It was partially purified by preparative TLC

and analysed by GC-HR-MS. The metabolite was unstable

and its content in the methanol elution decreased several

hours later with the formation of other compounds, even

though the sample was refrigerated. Accordingly, it was

difficult to obtain a pure sample. Metabolite A had a low Rf

value (0.04) and exhibited the following mass spectrum [m/z

(relative intensity, %)]: [160.0993 (M+, 63.1), 159.0914

(100.0), 144.0686 (15.0), 130.0651 (6.2), 119.0601 (26.7),

Table 1. Physical and chemical properties of metabolite SP from nicotine degradation by S16

Property Value

Appearance White crystal

Melting point 159–161 uC

Molecular formula C9H9NO3

Molecular mass 179.2

MS (ESI), m/z 180.3 [M+H]

+

Rf by TLC (chloroform/ethanol/methanol/0.5 M

NaOH 30 : 15 : 2 : 1.5)

0.37

UV absorption lmax (0.1 M HCl)=222.4 (EM 6289.4), 263.2 (EM 5373.6),

lmin (0.1 M HCl)=211.0, 241.4; lmax (0.1 M NaOH)=229.8

(EM 9648.6), 266.6 (EM 3649.6), lmin (0.1 M NaOH)=212.8, 252.4

FTIR (KBr) 3435 and 3358 cm21 (KBr, OH), 1708 cm21

(CO) 1

H NMR, 600 MHz in DMSO-d6 (d, mult. J) 12.20 (brs, 1H, 10-COOH), 9.14 (s, 1H, H-2), 8.80 (d, J=4.9 Hz, 1H, H-6),

8.31 (d, J=7.8 Hz, 1H, H-4), 7.56 (dd, J=4.9, 7.8 Hz, 1H, H-5), 3.29

(t, J=6.2 Hz, 2H, H-8), 2.60 (t, J=6.2 Hz, 2H, H-9) 13C NMR, 150 MHz in DMSO-d6 (d) 198.22 (7-CO), 173.71 (10-COOH), 153.50 (C-6), 149.17 (C-2), 135.43 (C-4),

131.87 (C-3), 123.97 (C-5), 33.59 (C-8), 27.87 (C-9)

http://mic.sgmjournals.org 1559

Nicotine degradation in Pseudomonas putida

117.0561 (11.6), 90.0432 (3.8), 78.0350 (2.2), 63.0246 (2.7),

42.0376 (1.7)]. This mass spectrum was identical to the

results of analysis by low-resolution GC-MS (Table 2, group

B II). The molecular mass determined (160.0993) was in

good agreement with that calculated from the molecular

formula C10H12N2 (160.1000) within 4.7 p.p.m. error. With

the help of the exact mass measurement by GC-HR-MS

(within 5 p.p.m. error) and the interpretation of ion

fragments, we were able to elucidate the chemical structure

(Debrauwer, 2000; Ishikawa et al., 2004; Cai et al., 2002). In

this way, metabolite A was identified as N-methylmyosmine,

and the structure is shown as II in Fig. 3.

Formation of DHP in the degradation mixture

When the degradation experiment was performed in

deionized water (the initial pH value was adjusted to

~7.0), a spot (metabolite was found by TLC analysis

to show blue fluorescence under 300 nm UV light, and to

change to a visibly brown colour after several hours’

exposure to air (Fig. 1b). Its Rf value (0.84) was the same as

that of the DHP standard in TLC analysis. Furthermore,

HPLC analysis of the sample obtained from the reaction

performed in 0.05 M sodium phosphate buffer (pH 7.0)

showed the same retention time (4.17 min) as that of the

authentic DHP standard (Fig. 2). Further GC-MS analysis

of the reaction sample after silylation with BSTFA indicated

that mass spectra of the metabolite were identical to those of

the DHP standard (Table 2). Thus it was confirmed that

metabolite B was DHP, an important intermediate of

nicotine degradation by S16.

Identification of other metabolites by GC-MS

analysis

Unlike the two major metabolites SP and HSP, other

metabolites from nicotine degradation were difficult to

isolate and purify because either only small amounts were

produced or they were unstable. By GC-MS analysis, the

structures of some metabolites could be identified by

comparing their mass spectra with those from the standard

GC-MS spectral library (Wiley275), especially after silylation

with BSTFA (Fig. 3). The metabolites were succinic

acid (IX), lactic acid (XI) and 3-hydroxybutyric acid (XII)

(Table 2, group A, Fig. 4).

For those compounds whose mass spectra could not be well

matched in the standard GC-MS library, the structures were

identified according to both their molecular ions and their

fragment ions (Table 2, group B, Fig. 4). They were

tentatively suggested to be pseudooxynicotine (IV) and 1-

butanone,4-hydroxy-1-(3-pyridinyl) (VI) according to the

Table 2. Mass spectra of products, or their TMS derivatives, from the metabolism of nicotine by resting cells of S16

Product/TMS derivative m/z of major ion peaks (relative intensity, partially proposed composition)

Group A*

Succinic acid (IX) 247 (10.2), 172 (3.4), 147 (100), 129 (5.5), 75 (20.5), 73 (50.7), 55 (8.5), 45 (7.0)

DHP (X) 255 (12.7), 240 (100), 168 (7.6), 112 (10.9), 84 (13.8), 73 (40)

Lactic acid (XI) 219 (5.4), 191 (14.6), 190 (15.3), 147 (100), 133 (8.1), 117 (82.1), 88 (6.5), 73 (95.4),

66 (10.2), 59 (6.8), 45 (15.3)

3-Hydroxybutyric acid (XII) 233 (7.2), 191 (17.0), 147 (100), 133 (7.9), 130 (8.6), 117 (44.8), 88 (15.7), 75 (29.3),

73 (93.8), 66 (11.7), 59 (7.7), 45 (20.0)

Group BD

N-Methylmyosmine (II) 160 (68.7, M+); 159 (100, [M-H]

+); 144 (15.6, [M-H-CH3]

+); 130 (6.4); 119 (40.9);

117 (17.9); 106 (7.2); 92 (15.7); 84 (28.4); 78 (20.9, [M-H-C5H8N]

+); 65 (14.1); 51

(15.7); 42 (24.1); 39 (16.7)

Pseudooxynicotine (IV) 178 (13.9, M+); 124 (12.0); 106 (100, [M-C3H6NHCH3]

+); 78 (80.4, [M- COC3H6NHCH3]

+);

51 (34.6)

1-Butanone,4-hydroxy-1-

(3-pyridinyl) (VI)

237 (3.1, M+); 222 (45.6, [M-CH3]

+); 204 (3.5); 194 (15.6, [M-(CH2)2-CH3]

+); 148 (36.0, [MOSi(CH3)3]

+); 130 (11.5); 121 (25.5); 116 (30.2, [C3H6OSi(CH3)2]

+); 106 (39.6,

[M-C3H6OSi(CH3)3]

+); 78 (50.1, [M-COC3H6OSi(CH3)3]

+); 75 (100); 73 (54.6, [si(CH3)3]

+);

51 (14.1)

SP (VII) 251 (14.5, M+); 236 (100, [M-CH3]

+); 208 (76.7, [M-(CH2)2-CH3]

+); 162 (13.0, [M-OSi(CH3)3]

+);

134 (30.2, [M-CO-OSi(CH3)3]

+); 106 (93.0, [M-(CH2)2-CO-OSi(CH3)3]

+); 78 (54.6, [M-CO-(CH2)2-

CO-OSi(CH3)3]

+); 73 (23.8, [si(CH3)3]

+); 51 (24.8)

HSP (VIII) 339 (49.4, M+); 324 (100, [M-CH3]

+); 296 (15.9, [M-(CH2)2-CH3]

+); 242 (48.1); 194 (63.8,

[M-(CH2)2-CO-OSi(CH3)3]

+); 151 (8.1); 134 (38.4); 119 (31.3); 91 (26.7); 77 (23.9); 73 (29.0,

[si(CH3)3]

+)

*Group A products were identified according to the standard spectra from the standard library (Wiley275) or standard compounds.

DGroup B products were tentatively identified according to their molecular ions and fragment ions, whose mass spectra could not be well

matched in the standard library of GC-MS.

1560 Microbiology 153

S. N. Wang and others

ass spectra and interpretation of ion fragments. For

compound VI, the high-resolution mass spectrum [m/z

(relative intensity, %)] [237.1196 (M+, 3.7), 222.0953

(100.0), 204.0822 (7.2), 194.1000 (24.3), 148.0758 (42.1),

130.0647 (9.0), 121.0531 (21.2), 117.0717 (25.7), 116.0656

(34.1), 106.0289 (27.7), 78.0343 (18.4), 75.0265 (43.1),

73.0475 (22.2), 51.0240 (3.6)] was also determined and was

identical to the results of analysis by low-resolution GC-MS

(Table 2, group B, VI). The molecular mass determined

(237.1196) was in good agreement with that calculated from

the molecular formula C12H19NO2Si (237.1185), within

4.6 p.p.m. error. Moreover, the trimethylsilyl (TMS)

derivatives of SP and HSP were also detected in the reaction

mixture.

Transformation of nicotine and its metabolites

by cell-free extract

Nicotine and its metabolites SP and HSP could be

transformed into related products by the cell-free extract

of S16 (Table 3). By GC, HPLC and UV analysis, the

products of the enzymic reactions could be detected.

Nicotine was transformed into N-methylmyosmine (II)

and SP. SP and HSP were converted to HSP and DHP,

respectively. The results showed that the metabolic sequence

of these compounds in the presence of cell-free extract was

from nicotine to SP, HSP and DHP via N-methylmyosmine.

DISCUSSION

Earlier research has suggested that Pseudomonas species are

able to oxidize nicotine into II, IV, VII (SP), VIII (HSP), IX

and X (DHP), but II, IX and X (DHP) have either never been

detected or not been satisfactorily characterized with

confirmatory evidence in these studies (Wada &

Yamasaki, 1954; Tabuchi, 1955; Kaiser et al., 1996). In

this study, one important metabolite, SP, was purified and

characterized thoroughly by NMR, FT-IR, UV and MS

analysis. To our knowledge, this is the first time that certain

physical and chemical properties of SP have been

investigated in detail. DHP was also detected and identified

for the first time as a nicotine metabolite by TLC, HPLC and

MS analysis by comparison with the standard compound.

N-Methylmyosmine was partially purified and identified by

preparative TLC and GC-HR-MS analysis. The structures of

other metabolites (IX, XI and XII) were identified by

comparing their mass spectra with those from the standard

GC-MS spectral library (Wiley275). For the other two

compounds whose mass spectra could not be well matched

in the standard GC-MS library, the structures were

identified according to both their molecular ions and

fragment ions. They were suggested to be pseudooxynicotine

(IV) and 1-butanone,4-hydroxy-1-(3-pyridinyl) (VI).

In addition, the mechanism of the L-6-hydroxynicotine

oxidase in the nicotine degradation pathway of Arthrobacter

species (Decker & Dai, 1967; Dai et al., 1968) could be

helpful to understand the initial attack upon nicotine at the

pyrrolidine ring by S16. The flavoprotein converts L-6-

hydroxynicotine to 6-hydroxypseudooxynicotine in the

presence of oxygen. The overall process consists of the

enzyme-catalysed dehydrogenation of L-6-hydroxynicotine

to produce 6-hydroxy-N-methylmyosmine and hydrogen

peroxide, and the spontaneous hydrolysis of 6-hydroxy-Nmethylmyosmine

to form 6-hydroxypseudooxynicotine. A

similar mechanism has been described for 2,4,6-trichlorophenol

4-monooxygenase in Ralstonia eutropha JMP134,

which catalyses the sequential dechlorination of 2,4,6-

trichlorophenol to 6-chlorohydroxyquinol by oxidative and

hydrolytic reactions (Xun & Webster, 2004). Hecht et al.

(2000) have reported that nicotine can be metabolized

through 29 or 59 hydroxylation by cytochrome P450 2A6

and human liver microsomes by two different pathways:

29-hydroxynicotine is further decomposed into IV and VII

(Fig. 4), while 59-hydroxynicotine is converted to cotinine.

Because IV and VII were produced in considerable amounts

Fig. 2. HPLC profile of the metabolites of

nicotine degradation by resting cells of strain

S16 at pH 7.0 and 30 6C in 0.05 M sodium

phosphate buffer (pH 7.0). The reaction mixture

at 0 h (solid line) contained nicotine

(2.5 g l”1

, 15.43 mM) alone, and at 3.5 h

(dotted line) contained DHP (0.06 g l”1

,

0.54 mM), SP (1.31 g l”1

, 7.32 mM), HSP

(0.24 g l”1

, 1.23 mM) and other metabolites.

The dashed line represents the DHP standard.

mAU, milliabsorbance unit.

http://mic.sgmjournals.org 1561

Nicotine degradation in Pseudomonas putida

rom nicotine degradation by S16 in this study, the highly

unstable compound 29-hydroxynicotine was considered to

have a transient existence as the product of hydrolysis in the

biochemical reactions that lead to the breaking of the C–N

bond.

From the known reaction mechanism and general chemical

considerations, it can be proposed that the initial attack

upon nicotine by S16 is as follows: nicotine (I)RNmethylmyosmine

(II)R29-hydroxynicotine (III)Rpseudooxynicotine

(IV). Analogically, pseudooxynicotine (IV)

could be converted into 3-pyridinebutanal,C-oxo (V) by the

same or a similar amine oxidase, while 3-pyridinebutanal,Coxo

(V) could easily be oxidized to 3-succinoyl-pyridine

(VII) by aldehyde dehydrogenases. However, we did not

find compound V, possibly because it is a transient

intermediate in cells.

In nicotinic acid degradation by Pseudomonas fluorescens N-

9, hydroxylation at carbon 6 is the first step in the pathway,

and this is followed by an oxidative decarboxylation to yield

DHP, which is catalysed by nicotinic acid dehydrogenase

Fig. 4. Proposed pathway of nicotine degradation in P. putida

S16. Bracketed compounds were not detected.

Fig. 3. GC-MS chromatograms of the products, or their TMS

derivatives, of nicotine degradation by resting cells of strain

S16. The compounds were obtained from the residue of samples

evaporated to dryness, and derivatized by silylation. The

process of sample treatment was not fully quantitative. I, nicotine

(14.5 min); II, N-methylmyosmine (15.2 min); IV, pseudooxynicotine

(18.2 min); VI, 1-butanone,4-hydroxy-1-(3-pyridinyl)

(17.6 min); VII, SP (18.6 min); VIII, HSP (19.2 min); IX, succinic

acid (13.7 min); X, DHP (14.6 min); XI, lactic acid (10.3 min);

XII, 3-hydroxybutyric acid (11.7 min).

1562 Microbiology 153

S. N. Wang and others

nd 6-hydroxynicotinate 3-monooxygenase (Behrman

& Stainier, 1957; Hurh et al., 1994; Nakano et al.,

1999). DHP is further degraded by 2,5-dihydryoxypyridine

oxygenase into maleamic acid and formic acid (Gauthier &

Rittenberg, 1971a, , which are used as nutrients and energy

sources to synthesize new cell compounds. In this study, SP

and HSP could be transformed by the cell-free system or the

resting cells (data not shown) of S16 into HSP and DHP,

respectively, so a reaction similar to nicotinic acid metabolism

in P. fluorescens is believed to happen in nicotine

degradation by S16: SP (VII)RHSP (VIII)Rsuccinic acid

(IX)+DHP (X). Furthermore, as shown in Fig. 2, most of

the nicotine (58.9 %, molar conversion) was metabolized

into SP, HSP and DHP. However, lactic acid (XI) and 3-

hydroxybutyric acid (XII) were detected in this study, and

not maleamic acid and formic acid, which might be a result

of the rapid reaction caused by the high degradation activity

of the cells and an insufficient oxygen supply.

It is interesting that a newly found compound, 1-

butanone,4-hydroxy-1-(3-pyridinyl) (VI) was produced

during nicotine degradation by the resting cells of S16,

and then utilized completely (Fig. 3). At the same time,

lactic acid showed similar behaviour, which indicated that

oxygen supply was initially insufficient during nicotine

degradation by the resting cells of S16. It can be assumed

that a limited oxygen supply makes the degradation reaction

slower and leads to accumulation of NAD(P)H and

metabolites. However, a high level of NAD(P)H and

metabolites is toxic to cells, and it is necessary to convert

NAD(P)H and toxic metabolites into NAD(P)+ and nontoxic

compounds. In nicotine degradation, the aldehyde

compound 3-pyridinebutanal,C-oxo (V), which is usually

thought of as a toxic compound, is believed to be

transformed into the corresponding alcohol, 1-butanone,4-hydroxy-1-(3-pyridinyl)

(VI), accompanied by

oxidation of NAD(P)H. Alcohol dehydrogenases, which

catalyse the reversible conversion of aromatic and heterocyclic

aldehydes to their corresponding alcohols, have been

reported in many micro-organisms (Bradshaw et al., 1992a,

b; Hummel, 1999; Mee et al., 2005; Peng et al., 2006; Tasaki

et al., 2006). When the oxygen supply is adequate, 1-

butanone,4-hydroxy-1-(3-pyridinyl) and lactic acid can be

oxidized by corresponding dehydrogenases and further

decomposed. From another point of view, the formation of

the alcohol and the acid SP also verified that the aldehyde

compound 3-pyridinebutanal,C-oxo (V) was an important

intermediate of nicotine degradation.

Based on our investigations and the known reaction

mechanisms, we propose the pathway for nicotine degradation

in P. putida S16 shown in Fig. 4.

The pathways of nicotine metabolism vary in different

bacteria. In the Gram-positive Arthrobacter genus, nicotine

is hydroxylated at the 6 position before the pyrrolidine ring

is opened. The opposite occurs in the Gram-negative

Pseudomonas genus, and the further metabolic pathways

also differ considerably. 2,3,6-Trihydroxypyridine and DHP

are produced, respectively, in these two bacterial genera after

the removal of the side chain which is formed by opening the

pyrrolidine ring.

In summary, nicotine and some of its metabolites are harmful

to health and the environment; this study provides new

insights into the nicotine metabolism of micro-organisms and

into an environmentally friendly route of nicotine degradation

(Wang et al., 2004, 2005). It is notable that compound

IV is the direct precursor of the tobacco-specific lung

carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone

(NNK) (Hecht, 1999; Hecht et al., 2000). The present

study suggests that nicotine and its metabolites such as

compound IV might be eliminated in later reactions by the

Pseudomonas genus when the bacteria are employed to reduce

the nicotine content in tobacco and to detoxify tobacco

wastes. Moreover, nicotine can be transformed into renewable

functionalized pyridines by biocatalytic processes that are

difficult to achieve by chemical means (Schmid et al., 2001).

We believe that this is a promising strategy to convert nicotine

in tobacco and tobacco wastes into valuable compounds by

means of biotechnology. Nicotine could be transformed by P.

putida S16 (Fig. 4) into valuable compounds such as HSP and

DHP, which are precursors for the synthesis of drugs and

insecticides (Spande et al., 1992; Roduitet al., 1997; Nakano et

al., 1999). Preliminary bioconversion processes with S16 have

indicated that they are capable of transforming nicotine into

HSP with high yields (Wang et al., 2005). However, the

enzymes and genes involved in nicotine degradation by S16

and other Pseudomonas bacteria are still unclear, so future

work in our group will focus on the molecular biology of

nicotine biodegradation.

Table 3. Transformation of nicotine and its metabolites by cell-free extract of S16

Values are the means±SD of three independent tests, in mg ml21

.

Substrate

(0.3 mg ml”1

)

Addition (mM) Decrease of substrate

concentration

Product

Nicotine DCIP (0.25) 0.17±0.019 II (0.05±0.024),

SP (0.07±0.021)

SP NAD (200) 0.16±0.021 HSP (0.09±0.020)

HSP NADH (300) 0.19±0.016 DHP (0.05±0.017)

http://mic.sgmjournals.org 1563

Nicotine degradation in Pseudomonas putida

CKNOWLEDGEMENTS

The authors like to thank the grant from the National Natural Science

Foundation of China (grant no. 20607012) awarded to S. N. W. The

authors gratefully acknowledge Mr Jian Huang (Shanghai Apple Flavour

& Fragrance Co.) for GC/MS analysis, and they also wish to thank

Professor Ji Mao Lin (School of Chemistry, Shandong University), Dr

Hui Xue Ren (School of Chemistry, Shandong University) and Dr Ji

Wen Zhang (School of Chemistry, Northwest Sci-Tech University of

Agriculture and Forestry) for their warm-hearted help, useful suggestions

and valuable discussions of NMR and FT-IR analysis, and Miss

Xiao Feng Cai for assistance in preparation of the manuscript.

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Edited by: D. J. Arp

[earthson]

Habana Mike, you are a wacko! Thanks! This will get my feet wet until I get the login info to access the rest of the articles.

[Jeremy Festa]

Prepping?

I just buy really expensive pre-aged boxes 'cause I lack patience, I'm really lazy, and a cocky wanker

Sent from my iPhone

[reffy]

Nice Mike! Seems like good intro for an investigation into the chemicals which contribute to the perceived flavor of aged cigars

[fabes]

Love the smileys in that article

[Guest rob]

The only prepping I do is to freeze custom cigars for a week.

Everything else just goes straight into storage.

[Shikar]

I try to age with my cigars.

Hopefully I don't go up before them.

That's the only important point, rest are just minor details.

Regards.