Skip to main content
  • ASM
    • Antimicrobial Agents and Chemotherapy
    • Applied and Environmental Microbiology
    • Clinical Microbiology Reviews
    • Clinical and Vaccine Immunology
    • EcoSal Plus
    • Eukaryotic Cell
    • Infection and Immunity
    • Journal of Bacteriology
    • Journal of Clinical Microbiology
    • Journal of Microbiology & Biology Education
    • Journal of Virology
    • mBio
    • Microbiology and Molecular Biology Reviews
    • Microbiology Resource Announcements
    • Microbiology Spectrum
    • Molecular and Cellular Biology
    • mSphere
    • mSystems
  • Log in
  • My alerts
  • My Cart

Main menu

  • Home
  • Articles
    • Current Issue
    • Accepted Manuscripts
    • COVID-19 Special Collection
    • Archive
    • Minireviews
  • For Authors
    • Submit a Manuscript
    • Scope
    • Editorial Policy
    • Submission, Review, & Publication Processes
    • Organization and Format
    • Errata, Author Corrections, Retractions
    • Illustrations and Tables
    • Nomenclature
    • Abbreviations and Conventions
    • Publication Fees
    • Ethics Resources and Policies
  • About the Journal
    • About AEM
    • Editor in Chief
    • Editorial Board
    • For Reviewers
    • For the Media
    • For Librarians
    • For Advertisers
    • Alerts
    • RSS
    • FAQ
  • Subscribe
    • Members
    • Institutions
  • ASM
    • Antimicrobial Agents and Chemotherapy
    • Applied and Environmental Microbiology
    • Clinical Microbiology Reviews
    • Clinical and Vaccine Immunology
    • EcoSal Plus
    • Eukaryotic Cell
    • Infection and Immunity
    • Journal of Bacteriology
    • Journal of Clinical Microbiology
    • Journal of Microbiology & Biology Education
    • Journal of Virology
    • mBio
    • Microbiology and Molecular Biology Reviews
    • Microbiology Resource Announcements
    • Microbiology Spectrum
    • Molecular and Cellular Biology
    • mSphere
    • mSystems

User menu

  • Log in
  • My alerts
  • My Cart

Search

  • Advanced search
Applied and Environmental Microbiology
publisher-logosite-logo

Advanced Search

  • Home
  • Articles
    • Current Issue
    • Accepted Manuscripts
    • COVID-19 Special Collection
    • Archive
    • Minireviews
  • For Authors
    • Submit a Manuscript
    • Scope
    • Editorial Policy
    • Submission, Review, & Publication Processes
    • Organization and Format
    • Errata, Author Corrections, Retractions
    • Illustrations and Tables
    • Nomenclature
    • Abbreviations and Conventions
    • Publication Fees
    • Ethics Resources and Policies
  • About the Journal
    • About AEM
    • Editor in Chief
    • Editorial Board
    • For Reviewers
    • For the Media
    • For Librarians
    • For Advertisers
    • Alerts
    • RSS
    • FAQ
  • Subscribe
    • Members
    • Institutions
PHYSIOLOGY AND BIOTECHNOLOGY

Plastic Encapsulation of Stabilized Escherichia coli and Pseudomonas putida

M. Manzanera, S. Vilchez, A. Tunnacliffe
M. Manzanera
1Institute of Biotechnology, University of Cambridge, Cambridge CB2 1QT
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
S. Vilchez
2Department of Biochemistry, University of Cambridge, Cambridge CB2 1QW, United Kingdom
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
A. Tunnacliffe
1Institute of Biotechnology, University of Cambridge, Cambridge CB2 1QT
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
  • For correspondence: a.tunnacliffe@biotech.cam.ac.uk
DOI: 10.1128/AEM.70.5.3143-3145.2004
  • Article
  • Figures & Data
  • Info & Metrics
  • PDF
Loading

ABSTRACT

Escherichia coli and Pseudomonas putida dried in hydroxyectoine or trehalose are shown to be highly resistant to the organic solvents chloroform and acetone, and consequently, they can be encapsulated in a viable form in solid plastic materials. Bacteria are recovered by rehydration after physical disruption of the plastic. P. putida incorporated into a plastic coating of maize seeds was shown to colonize roots efficiently after germination.

Previously, we have shown how osmotic preconditioning of bacteria, followed by drying in the presence of glass-forming protectant molecules, such as trehalose or hydroxyectoine, results in a high level of desiccation tolerance, where viability is maintained throughout extended storage periods at above-ambient temperatures (8, 12). This has been termed anhydrobiotic engineering (9), in reference to anhydrobiotic organisms which naturally exhibit extreme desiccation tolerance (4, 6, 14). In this report, we describe a new approach to preserving bacteria by drying and then encapsulating the bacteria in plastic, and we demonstrate a potential application as a seed coating.

Glasses, including those derived from organic materials, are high-viscosity liquids that slow molecular diffusion and the rates of chemical reactions, including degradative processes, dramatically (7). Consequently, biological molecules embedded in some organic glasses exhibit a high degree of stability (2, 6). For example, proteins dried in a trehalose glass are protected from denaturation by organic solvents (5, 10). Whether anhydrobiotically engineered bacteria are similarly tolerant of chemical stress, as is observed for some bacterial spores (1, 13), is not known, however. We therefore tested the resistance of dried samples of Escherichia coli and Pseudomonas putida to different pure organic solvents. Growth of E. coli MC4100 and P. putida KT2440 (strains cited in reference 12) in hypersaline minimal medium (HMM), harvesting, and vacuum drying in 1 M trehalose plus 1.5% (wt/vol) polyvinylpyrrolidone (PVP; viscosity enhancer), or 1 M hydroxyectoine plus 1.5% (wt/vol) PVP, were performed as previously described (8, 12). Dried samples of E. coli and P. putida containing approximately 108 cells were mixed with 200 μl of pure acetone, chloroform, or ethanol and incubated for 5 min. Solvents were then removed under vacuum for 25 to 135 min, depending on the solvent. Control experiments were done using fresh E. coli and P. putida, and survival of solvent-treated bacteria was compared to that of untreated cells, measured by plating a dilution series and colony counting. As expected, survival of nondried E. coli and P. putida was below detection levels. Remarkably, dried bacteria tolerated acetone and chloroform treatment to a high degree, with survival rates above 90% in some instances (Table 1). The experiment was repeated three times, giving similar rates of survival. However, ethanol treatment of dried cells of both species resulted in very low or undetectable survival. This is consistent with the partial solubility of trehalose and hydroxyectoine in ethanol, which would be expected to degrade the glass matrix and attack the bacteria within.

Further control experiments were performed to test whether the protection of dried E. coli and P. putida against organic solvents was simply due to the lack of water or due to the presence of trehalose or hydroxyectoine. Bacteria suspended in 1.5% PVP alone suffered a marked drop in viability during drying, since trehalose and hydroxyectoine are required for a high degree of protection against desiccation damage, but sufficient cells survived to assess the effects of organic solvents. Dried samples containing ∼105 cells were treated as described above and compared with survival of nontreated bacteria. Table 1 shows that cells dried without trehalose or hydroxyectoine did not survive treatment with organic solvents, confirming the requirement for these excipients. Clearly, PVP alone either does not encapsulate bacteria efficiently or is soluble in organic solvents.

The fact that dried cells tolerate treatment with pure solvents suggests the possibility of incorporating microorganisms in plastic without affecting viability. To test this, ∼107 dried cells of E. coli or P. putida were crumbled to powder, which was mixed with 1 ml of chloroform and 50 mg of polystyrene, in this order. The mixture was spread on a glass plate and allowed to air dry for 15 min, when a solid plastic layer formed. When plastic layers were shredded using a sterile razor blade and incubated in Luria-Bertani medium at an appropriate temperature, growth was detected in less than 12 h. The cultures of either E. coli or P. putida were demonstrated to be pure and to consist of the same strains included in the initial plastic layer.

As an initial test of the applicability of this approach, a seed coating experiment was performed using P. putida, which is an efficient root colonizer and is beneficial to plants (3, 11). To monitor root colonization during the germination process, a bioluminescent strain, named P. putida MAX10, was first constructed. The promoterless luxCDABE operon from Photorhabdus luminescens was inserted into P. putida chromosomal DNA after conjugation between an E. coli strain carrying plasmid pUT mini-Tn5 ′luxCDABE Tcr and a spontaneous rifampin-resistant mutant of P. putida KT2440, as described previously (15). Maize seeds were sterilized in 70% (vol/vol) ethanol for 5 min, incubated a second time in 20% (vol/vol) bleach for 20 min, and washed five times in sterile distilled water. The seeds were air dried in a microbiological flow cabinet and then coated with bacteria by immersion in a mixture of dried, powdered P. putida MAX10, chloroform, and polystyrene and prepared as described above. After 2 min, the seeds were withdrawn from the mixture and allowed to dry in a sterile airflow for 10 min. Seeds were stored at 30°C in dark and dry conditions for 1, 30, or 90 days after coating and then sowed onto agar plates to allow germination. Plates were incubated at 30°C in the dark; germination was first detected after 4 days, and root colonization was monitored over the next week by detection of bacterial luminescence. Before germination, no light emission was observed (data not shown). However, after germination, luminescence of root processes was seen to develop, consistent with colonization of the rhizosphere by P. putida MAX10 (Fig. 1).

The ability to encapsulate viable cells in plastic layers should have a number of applications in biotechnology. One factor which can have a detrimental effect on dried microorganisms over the long term is humidity in the environment; increasing moisture content of the dried sample compromises viability. Storage under vacuum or in an inert atmosphere can prevent this (8, 12) but is costly and unwieldy. Long-term storage of culture collections and libraries should be facilitated by the plastic encapsulation procedure we describe, since the dried bacteria are isolated from atmospheric conditions but can be recovered easily. The use of plastic for encapsulation also allows shaping or molding into layers, sheets, films, blocks, pellets, or pills, for example, so that the procedure could be applied in many different industries.

FIG. 1.
  • Open in new tab
  • Download powerpoint
FIG. 1.

Two different stages of germination of maize seeds coated with luminescent P. putida MAX10 in a polystyrene layer 4 days (A and B) and 9 days (C and D) after the seeds were coated with bacteria. Pictures were taken under artificial light (A and C) or in the dark (B and D) with 30-min exposure, using a charge-coupled device camera. The seeds shown had been stored for 30 days after the plastic coating was applied, although identical results were also obtained with seeds stored for 1 or 90 days after coating (not shown).

View this table:
  • View inline
  • View popup
TABLE 1.

Survival of dried E. coli and P. putida after treatment with organic solvents

ACKNOWLEDGMENTS

We thank Arcadio García de Castro for useful discussions and Simon Swift for providing lux reagents.

A.T. is the Anglian Water Fellow in Biotechnology of Pembroke College, University of Cambridge. This work was partially supported by EC grant BIO4-CT98-0283 and by Merck Chemicals, Ltd.

FOOTNOTES

    • Received 31 October 2003.
    • Accepted 15 January 2004.
  • Copyright © 2004 American Society for Microbiology

REFERENCES

  1. 1.↵
    Bloomfield, S. F., and M. Arthur. 1994. Mechanisms of inactivation and resistance of spores to chemical biocides. Soc. Appl. Bacteriol. Symp. Ser.23:91S-104S.
    OpenUrlPubMed
  2. 2.↵
    Burke, M. J. 1986. The vitreous state and survival of anhydrous biological systems, p. 358-364. In A. C. Leopold (ed.), Membranes, metabolism and dry organisms. Cornell University Press, New York, N.Y.
  3. 3.↵
    Campbell, R., and M. P. Greaves. 1990. Anatomy and community structure of the rhizosphere, p. 11-34. In J. M. Lynch (ed.), The rhizosphere. Wiley & Sons, Chichester, United Kingdom.
  4. 4.↵
    Clegg, J. S. 2001. Cryptobiosis—a peculiar state of biological organization. Comp. Biochem. Physiol. B128:613-624.
    OpenUrlCrossRefPubMedWeb of Science
  5. 5.↵
    Cleland, J. L., and A. J. S. Jones. November 1994. Excipient stabilization of polypeptides treated with organic solvents. European patent EP 0 686 045 B1.
  6. 6.↵
    Crowe, J. H., J. F. Carpenter, and L. M. Crowe. 1998. The role of vitrification in anhydrobiosis. Annu. Rev. Physiol.60:73-103.
    OpenUrlCrossRefPubMedWeb of Science
  7. 7.↵
    Franks, F. 1985. Biophysics and biochemistry at low temperatures. Cambridge University Press, Cambridge, United Kingdom.
  8. 8.↵
    García de Castro, A., H. Bredholt, A. Strøom, and A. Tunnacliffe. 2000. Anhydrobiotic engineering of gram-negative bacteria. Appl. Environ. Microbiol.66:4142-4144.
    OpenUrlAbstract/FREE Full Text
  9. 9.↵
    García de Castro, A., J. Lapinski, and A. Tunnacliffe. 2000. Anhydrobiotic engineering. Nat. Biotechnol.18:473.
    OpenUrlCrossRefPubMed
  10. 10.↵
    Gribbon, E. M., S. D. Sen, B. J. Roser, and J. Kampinga. 1996. Stabilisation of vaccines using trehalose (Q-T4) technology. Dev. Biol. Stand.87:193-199.
    OpenUrlPubMed
  11. 11.↵
    Lugtenberg, B. J. J., and L. Dekkers. 1999. What makes Pseudomonas bacteria rhizosphere competent? Environ. Microbiol.1:9-13.
    OpenUrlCrossRefPubMedWeb of Science
  12. 12.↵
    Manzanera, M., A. García de Castro, A. Tøonderik, M. Rayner-Brandes, A. R. Strøom, and A. Tunnacliffe. 2002. Hydroxyectoine is superior to trehalose for anhydrobiotic engineering of Pseudomonas putida KT2440. Appl. Environ. Microbiol.68:4328-4333.
    OpenUrlAbstract/FREE Full Text
  13. 13.↵
    McDonnell, G., and A. D. Russell. 1999. Antiseptics and disinfectants: activity, action and resistance. Clin. Microbiol. Rev.12:147-179.
    OpenUrlAbstract/FREE Full Text
  14. 14.↵
    Tunnacliffe, A., and J. Lapinski. 2003. Resurrecting van Leeuwenhoek's rotifers: a reappraisal of the role of disaccharides in anhydrobiosis. Philos. Trans. R. Soc. Lond. Ser. B358:1755-1771.
    OpenUrlCrossRefPubMedWeb of Science
  15. 15.↵
    Winson, M. K., S. Swift, P. J. Hill, C. M. Sims, G. Griesmayr, B. W. Bycroft, P. Williams, and G. S. A. B. Stewart. 1998. Engineering the luxCDABE genes from Photorhabdus luminescens to provide a bioluminescent reporter for constitutive and promoter probe plasmids and mini-Tn5 constructs. FEMS Microbiol. Lett.163:193-202.
    OpenUrlCrossRefPubMedWeb of Science
PreviousNext
Back to top
Download PDF
Citation Tools
Plastic Encapsulation of Stabilized Escherichia coli and Pseudomonas putida
M. Manzanera, S. Vilchez, A. Tunnacliffe
Applied and Environmental Microbiology May 2004, 70 (5) 3143-3145; DOI: 10.1128/AEM.70.5.3143-3145.2004

Citation Manager Formats

  • BibTeX
  • Bookends
  • EasyBib
  • EndNote (tagged)
  • EndNote 8 (xml)
  • Medlars
  • Mendeley
  • Papers
  • RefWorks Tagged
  • Ref Manager
  • RIS
  • Zotero
Print

Alerts
Sign In to Email Alerts with your Email Address
Email

Thank you for sharing this Applied and Environmental Microbiology article.

NOTE: We request your email address only to inform the recipient that it was you who recommended this article, and that it is not junk mail. We do not retain these email addresses.

Enter multiple addresses on separate lines or separate them with commas.
Plastic Encapsulation of Stabilized Escherichia coli and Pseudomonas putida
(Your Name) has forwarded a page to you from Applied and Environmental Microbiology
(Your Name) thought you would be interested in this article in Applied and Environmental Microbiology.
CAPTCHA
This question is for testing whether or not you are a human visitor and to prevent automated spam submissions.
Share
Plastic Encapsulation of Stabilized Escherichia coli and Pseudomonas putida
M. Manzanera, S. Vilchez, A. Tunnacliffe
Applied and Environmental Microbiology May 2004, 70 (5) 3143-3145; DOI: 10.1128/AEM.70.5.3143-3145.2004
del.icio.us logo Digg logo Reddit logo Twitter logo CiteULike logo Facebook logo Google logo Mendeley logo
  • Top
  • Article
    • ABSTRACT
    • ACKNOWLEDGMENTS
    • FOOTNOTES
    • REFERENCES
  • Figures & Data
  • Info & Metrics
  • PDF

KEYWORDS

biotechnology
Escherichia coli
Plastics
Pseudomonas putida

Related Articles

Cited By...

About

  • About AEM
  • Editor in Chief
  • Editorial Board
  • Policies
  • For Reviewers
  • For the Media
  • For Librarians
  • For Advertisers
  • Alerts
  • RSS
  • FAQ
  • Permissions
  • Journal Announcements

Authors

  • ASM Author Center
  • Submit a Manuscript
  • Article Types
  • Ethics
  • Contact Us

Follow #AppEnvMicro

@ASMicrobiology

       

ASM Journals

ASM journals are the most prominent publications in the field, delivering up-to-date and authoritative coverage of both basic and clinical microbiology.

About ASM | Contact Us | Press Room

 

ASM is a member of

Scientific Society Publisher Alliance

 

American Society for Microbiology
1752 N St. NW
Washington, DC 20036
Phone: (202) 737-3600

Copyright © 2021 American Society for Microbiology | Privacy Policy | Website feedback

 

Print ISSN: 0099-2240; Online ISSN: 1098-5336