ArticlePDF Available

Fundamental things apply: The case of Dehalococcoides ethenogenes

Authors:
PERSPECTIVES
Fundamental things apply:
the case of
Dehalococcoides
ethenogenes
You must remember this, /a kiss is still a kiss, /a sigh is just a sigh; /the
fundamental things apply, /as time goes by.
Herman Hupfeld (1894-1951), As time goes by (1931; made famous in the
1942 film Casablanca)
L’essentiel est invisible pour les yeux (Chapter 21).
Antoine de Saint-Exupéry (1900-1944), Le petit prince (1943)
Searching for words
Most successful applications of science, at least at some stage
in their development, are the products of basic research, much
of which takes place behind the scenes but is nonetheless
essential for the final result. Of course, there may be examples
to the contrary. George Porter (1920-2002), Nobel Laureate in
Chemistry 1967, said: “Thermodynamics owes more to the
steam engine than the steam engine owes to science.” But the
reality is that applicable results of science are generally
achieved from many fragments of information. This involves a
series of gradual scientific successes, which at the time the
work was completed their degree of recognition by the scien-
tific community may not have been comparable to the effort
invested. Despite this lack of recognition, the research may later
become fundamental to a specific application. How many pub-
lished results or techniques have been forgotten for years only
to be rediscovered and used later by the private sector? While
purely practical, applied science may be commercially attrac-
tive, financially rewarding, and possibly essential, in the long
run this approach alone will not guarantee success. It is basic
science, leading to fundamental results, that makes applied
research feasible and which therefore must be nurtured.
What are exactly “fundamental results”? Merriam
Webster’s Collegiate Dictionary defines “fundamental” as
something “of central importance”, “serving as an original or
generating source”, and “dealing with general principles rather
than practical application”.
This is, in fact, the job (or the aspiration) of basic science.
The importance of a given “fundamental” contribution will
depend, among other factors, on the priorities set by society
and by its view of life. The value assigned to a particular field
of study or the scope extended to a specific project derive
from global concerns, which, in turn, determine the potential
worth of basic research findings. While this may be true, it is
also true that present-day priorities cannot predict either the
future importance of such findings or their relevance to
researchers in other fields of study, who may already be able
to appreciate the potential applications of a seemingly trivial
result. An early example of this was described by J.J.
Thomson (discoverer of the electron), in a speech delivered
in 1916 [quoted on p. 198 of The Life of Sir J.J. Thomson,
Lord Rayleigh, Cambridge University Press, 1942]: “I will
give just one example of the ‘utility’ of […] (basic) research,
one that has been brought into great prominence by the
War—I mean the use of X-rays in surgery. Now how was this
method discovered? It was not the result of a research in
applied science starting to find an improved method of locat-
ing bullet wounds. […] No, this method is due to an investi-
gation in pure science, made with the object of discovering
what is the nature of Electricity.” Also, as C.H. Llewellyn
Smith wrote: “the reasons we have practical computers now,
and did not have them 100 years ago, […] is because of dis-
coveries in fundamental physics which underwrite modern
electronics, developments in mathematical logic, and the
need of nuclear physicists in the 1930s to develop ways of
counting particles” [What’s the use of basic science? by C.H.
Llewellyn Smith, former Director-General of the CERN. At:
http://public.web.cern.ch]. Such examples are countless.
So, basic research leads to fundamental results that may
later be used in developing and applying technological im-
provements (Fig. 1). However, results that are or will become
Xavier Maymó-Gatell
Credit Initiatives, Andorra la
Vella, Principality of Andorra
INTERNATIONAL MICROBIOLOGY (2005) 8:137-140
www.im.microbios.org
e-mail: xmaymo@creditandorra.ad
138 INT. MICROBIOL. Vol. 8, 2005
fundamental are not usually predictable. Might Watson and
Crick heve predictal the cloning of dinosaur DNA? Did
Rutherford foresee nuclear power? Since this was obviously
not the case, then why do institutions controlling research
funds exert such powerful control over deciding what should
be investigated? A few lines of applied research will clearly
deserve right of passage, but by no means should the bulk of
basic research funding be obliterated by a short-term, finan-
cially or commercially derived vision of knowledge. The
constant four-dimensional (vertical, horizontal, in depth, and
across time) transfer of scientific information rules out the
prospect of a world researched only by applied scientists to
the detriment of basic-research scientists, as postulated by
some. Carl Sagan (1934-1996), in a meeting at Cornell
University celebrating his 60th birthday, two years before his
early death, conveyed a clear message: nothing applied may
be pursued successfully without its fundamental base being
well-developed. Basic research is the key to obtaining funda-
mental results that, at some point in time, will become essen-
tial to progress in applied research and to the well-being of
society. As a consequence, public institutions should pursuit
a merit-based approach to supporting basic research (funda-
mental results do not yield immediate revenues), which
means that long-term investments with an assumed long pay-
back must be made using tax revenues, with the payback
occurring later in the form of either a financial return on the
initial investment or an application that improves society’s
well-being. If this strategy were followed, both basic science
and applied research would thrive.
A case report
Within my previous field of study (microbial ecology),
results compiled from decades of basic research have been
fundamental to a wide variety of applications, including gold
biomining (Thiobacillus ferrooxidans), the production of
biodegradable plastics (polyhydroxyalkanoates-producing
bacteria), the improvement of rumen and soil-plant-microbe
symbiotic interactions, sewage clean-up, and bioremediation
of recalcitrant toxic compounds in contaminated sites.
On January 7, 2005, a paper was published in Science [7]
reporting the genome sequence of Dehalococcoides etheno-
genes strain 195 (Fig. 2), the only bacterium known that is
capable of reductively dechlorinating tetrachloroethene
(PCE) and trichloroethene (TCE)—two cancer-producing
groundwater pollutants—to the non-toxic form ethene.
Deciphering the D. ethenogenes genome sequence con-
tributes to a better understanding of the complex nutrient
requirements, respiratory processes, phylogenic relation-
MAYMÓ-GATELL
Int. Microbiol.
Fig. 1. Roadmap to the application of basic
science.
139
INT. MICROBIOL. Vol. 8, 2005
ships, and enzymatic reactions of this bacterium, and lays the
foundations for synthesizing specific probes and for genetic
studies. The Science article has surely improved our scientif-
ic knowledge on how life works at some of its smallest lev-
els, but it will also allow researchers to elucidate the regula-
tory network involved in the dechlorination of PCE, TCE,
both dichloroethene (DCE) isomers, and vinyl chloride (VC)
to innocuous compounds. Together with previous studies
[1–6] of the growth requirements and other characteristics of
D. ethenogenes strain 195, the basis for the effective biore-
mediation of PCE-, TCE- and DCE-polluted environments
has been established.
The successful use of D. ethenogenes in bioremediation is
the result of both basic and applied science, in an essential,
intense, and extended team effort that has been going on for
more than 15 years. In 1997, I isolated D. ethenogenes strain
195 [4], the genus-defining strain, while carrying out doctoral
thesis research under the direction of Stephen H. Zinder, at
the Department of Microbiology of Cornell University. Our
progress would have been slower and less fruitful if it were
not for continuous collaboration with the laboratory of James
M. Gossett, of the Engineering Department at the same uni-
versity, more involved in applied science. The professional
relationship among the two laboratories made it impossible
to divide the work into basic and applied efforts. Working as
a multidisciplinary team not only improved the quality of the
output, but also—as the published articles show—allowed us
to maximally benefit from results, derived either directly or
indirectly, of basic research carried out over the previous
decades. This earlier research, comprising studies on
methanogenic and acetogenic bacteria, anaerobic culture
enrichment techniques, and the microbiology and biochem-
istry of electron donors and acceptors, supplied crucial infor-
mation, without which neither the remediation of environ-
ments polluted with chloroethenes nor the sequencing of the
D. ethenogenes strain 195 genome would have been possible.
Final thoughts
The Casablanca song says, intelligently, “the fundamental
things apply, as time goes by”. In science, this should not be
interpreted as meaning that everything considered funda-
mental must have a practical application, or that everything
resulting from applied research is directly derived from basic
research. It does imply, however, that we must strongly sup-
port and encourage basic science in order to improve the
general social welfare and living conditions on Earth. The
fact is that, regretfully, we walk through our days immersed
in a society ruled by governments and financial markets that,
because of their chronic short-term interests, are only willing
to understand fundamental efforts in the light of their applied
and thus almost-immediate commercial potential. Needless
to say, this is not a good strategy for facing the future.
Public institutions must support basic research regardless
of its less than probable immediate returns (and also because
of this). However, this is not an opinion likely to win votes,
nor does it produce quick economic and financial gains.
Nevertheless, the results that society expects from applied
science depend on the priorities that society sets for itself. By
neglecting or annihilating basic science research, we shut
ourselves out of a better world if our priorities, as a whole,
DEHALOCOCCOIDES ETHENOGENES
Fig. 2. Dehalococcoides ethenogenes strain 195. Mechanism and genes in a schematic genome (See Science 7 Jan. 2005, p. 107.)
MAYMÓ-GATELL
2. Fennell DE, Gossett JM, Zinder SH (1997) Comparison of butyric acid,
ethanol, lactic acid, and propionic acid as hydrogen donors for the
reductive dechlorination of thetrachloroethene. Environ Sci Techn
31:918-926
3. Maymó-Gatell X, Anguish T, Zinder SH (1999) Reductive dechlorina-
tion of chlorinated ethenes and 1,2-dichloroethane by “Dehalococcoides
ethenogenes” 195. Appl Environ Microbiol 65:3108-3113
4. Maymó-Gatell X, Chien Y-T, Gossett JM, Zinder SH (1997) Isolation of
a bacterium that reductively dechlorinates tetrachloroethene to ethene.
Science 276:1568-1571
5. Maymó-Gatell X, Nijenhuis I, Zinder SH (2001) Reductive dechlorina-
tion of cis-1,2-dichloroethene and vinyl chloride by “Dehalococcoides
ethenogenes”. Environ Sci Techn 35:516-521
6. Maymó-Gatell X, Tandoi V, Gossett JM, Zinder SH (1995) Charac-
terization of an H2-utilizing enrichment culture that reductively dechlo-
rinates tetrachloroethene to vinyl chloride and ethene in the absence of
methanogenesis and acetogenesis. Appl Environ Microbiol 61:3928-
3933
7. Seshadri R, Adrian L, Fouts DE, et al. (2005) Genome sequence of the
PCE-dechlorinating bacterium Dehalococcoides ethenogenes. Science
307:105-108
were to change someday (and let us hope they will). Should
we continue to pursue material prosperity only out of individ-
ual interest? Or should we also aim at achieving further cul-
tural and education advances? As Robert Wilson (1914-
2000), first director of the Fermilab, stated in front of a
Congressional Committee, “my research may not contribute
to the defense of our country, but it will make it worth
defending” [What’s the use of basic science? by C. H.
Llewellyn Smith, former Director-General of CERN. At:
http://public.web.cern.ch]
References
1. DiStefano TD, Gossett JM, Zinder SH (1992) Hydrogen as an electron
donor for dechlorination of tetrachloroethene by an anaerobic mixed
culture. Appl Environ Microbiol 58:3622-3622
INT. MICROBIOL. Vol. 8, 2005
140
... Dehalococcoides ethenogenes strain 195 is the only bacterium known that is capable of reductively dechlorinating tetrachloroethene (PCE) and trichloroethene (TCE) to the nontoxic form ethane (Maymo-Gatell 2005). Reductive dehalogenase (RD) gene transcript levels in Dehalococcoides ethenogenes strain 195 were investigated using reverse transcriptase quantitative PCR during growth and reductive dechlorination of tetrachloroethene (PCE), trichloroethene (TCE), or 2,3-dichlorophenol (2,3-DCP) (Fung et al. 2007). ...
Chapter
Full-text available
Soils serve as a shelter for wide range of bacteria and virus monotypes, which include tailed, spherical, and filamentous particle. The existence of temperate phages, i.e., prophages, and their significance in soil hosts is not well understood. Whole genome sequence data and outcome of prophage detection methods in bacterial genomes indicate that prophage sequences pervade prokaryotic genomes. Lysogenic state is considered as mutual coexistence of host bacteria and prophage which improves host fitness leading to phage-mediated horizontal gene transfer and also favors the prophage genome to be a permanent associate of the host. Using the database of cryptic prophage elements and phage remnants available at http://bicmku.in:8082, we focus on the methods for systematic and definitive identification of prophages in a collection of soil bacteria and the importance of phage-mediated gene transfer in the evolution of prokaryotes. A total of 200 bacterial genomes with no prophage reports were taken up for the study. Employing a proteome comparison method using protein similarity approach (PSA) yielded 30 prophage-like elements. By the genome comparison method using dinucleotides relative abundance difference (DRAD), 52 prophage elements were identified. Comparative analysis of other available methods against the above approaches developed here will be discussed. Detailed analysis of locus will help in understanding the contribution of prophages to the microbial communities in soil.
Article
Full-text available
Hydrogen served as an electron donor in the reductive dechlorination of tetrachloroethene to vinyl chloride and ethene over periods of 14 to 40 days in anaerobic enrichment cultures; however, sustained dechlorination for more extended periods required the addition of filtered supernatant from a methanol-fed culture. This result suggests a nutritional dependency of hydrogen-utilizing dechlorinators on the metabolic products of other organisms in the more diverse, methanol-fed system. Vancomycin, an inhibitor of cell wall synthesis in eubacteria, was found to inhibit acetogenesis when added at 100 mg/liter to both methanol-fed and hydrogen-fed cultures. The effect of vancomycin on dechlorination was more complex. Methanol could not sustain dechlorination when vancomycin inhibited acetogenesis, while hydrogen could. These results are consistent with a model in which hydrogen is the electron donor directly used for dechlorination by organisms resistant to vancomycin and with the hypothesis that the role of acetogens in methanol-fed cultures is to metabolize a portion of the methanol to hydrogen. Methanol and other substrates shown to support dechlorination in pure and mixed cultures may merely serve as precursors for the formation of an intermediate hydrogen pool. This hypothesis suggests that, for bioremediation of high levels of tetrachloroethene, electron donors that cause the production of a large hydrogen pool should be selected or methods that directly use H2 should be devised.
Article
Full-text available
We have been studying an anaerobic enrichment culture which, by using methanol as an electron donor, dechlorinates tetrachloroethene (PCE) to vinyl chloride and ethene. Our previous results indicated that H2 was the direct electron donor for rductive dechlorination of PCE by the methanol-PCE culture. Most-probable-number counts performed on this culture indicated low numbers (< or equal to 10(4)/ml)) of methanogens and PCE dechlorinators using methanol and high numbers (> or equal to 10(6)/ml)) of sulfidogens, methanol-utilizing acetogens, fermentative heterotrophs, and PCE dechlorinators using H2. An anaerobic H2-PCE enrichment culture was derived from a 10(-6) dilution of the methanol-PCE culture. This H2-PCE culture used PCE at increasing rates over time when transferred to fresh medium and could be transferred indefinitely with H2 as the electron donor for the PCE dechlorination, indicating that H2-PCE can serve as an electron donor-acceptor pair for energy conservation and growth. Sustained PCE dechlorination by this culture was supported by supplementation with 0.05 mg of vitamin B12 per liter, 25% (vol/vol) anaerobic digestor sludge supernatant, and 2 mM acetate, which presumably served as a carbon source. Neither methanol nor acetate could serve as an electron donor for dechlorination by the H2-PCE culture, and it did not produce CH4 or acetate from H2-CO2 or methanol, indicating the absence of methanogenic and acetogenic bacteria. Microscopic observatios of the pruified H2-PCE culture showed only two major morphotypes: irregular cocci and small rods.
Article
Full-text available
Dehalococcoides ethenogenes is the only bacterium known to reductively dechlorinate the groundwater pollutants, tetrachloroethene (PCE) and trichloroethene, to ethene. Its 1,469,720-base pair chromosome contains large dynamic duplicated regions and integrated elements. Genes encoding 17 putative reductive dehalogenases, nearly all of which were adjacent to genes for transcription regulators, and five hydrogenase complexes were identified. These findings, plus a limited repertoire of other metabolic modes, indicate that D. ethenogenes is highly evolved to utilize halogenated organic compounds and H2. Diversification of reductive dehalogenase functions appears to have been mediated by recent genetic exchange and amplification. Genome analysis provides insights into the organism's complex nutrient requirements and suggests that an ancestor was a nitrogen-fixing autotroph.
Article
Tetrachloroethene is a prominent groundwater pollutant that can be reductively dechlorinated by mixed anaerobic microbial populations to the nontoxic product ethene. Strain 195, a coccoid bacterium that dechlorinates tetrachloroethene to ethene, was isolated and characterized. Growth of strain 195 with H2 and tetrachloroethene as the electron donor and acceptor pair required extracts from mixed microbial cultures. Growth of strain 195 was resistant to ampicillin and vancomycin; its cell wall did not react with a peptidoglycan-specific lectin and its ultrastructure resembled S-layers of Archaea. Analysis of the 16S ribosomal DNA sequence of strain 195 indicated that it is a eubacterium without close affiliation to any known groups.
Article
Previous studies indicated that dechlorinators can utilize H2 at lower concentrations than can methanogens. This suggests a strategy for selective enhancement of dechlorinationmanaging H2 delivery so as to impart a competitive advantage to dechlorinators. Four H2 donorsbutyric and propionic acids, which can only be fermented when the H2 partial pressure is lower than 10-3.5 or 10-4.4 atm, respectively, and ethanol and lactic acid, which are readily fermented at H2 partial pressures 2−3 orders of magnitude higherwere administered to anaerobic mixed cultures. Comparison of the resulting enrichment cultures during time-intensive, short-term tests showed significant differences in patterns of donor degradation, H2 production and use, and distribution of reduction equivalents between dechlorination and competing methanogenesis. Amendment with butyric and propionic acids resulted in less methanogenesis than did amendment with ethanol or lactic acid, which generated much higher H2 levels. Ethanol did not support complete dechlorination during short-term tests, but it was a viable donor over long-term testing because a portion was converted to a pool of slowly degraded propionic acid and because during long-term tests, cultures were routinely co-amended with pre-fermented yeast extract, a source of slowly fermented volatile fatty acids. Understanding the fate of electron donors and their fermentation products is an important component in understanding dechlorinating communities.
Article
Tetrachloroethene is a prominent groundwater pollutant that can be reductively dechlorinated by mixed anaerobic microbial populations to the nontoxic product ethene. Strain 195, a coccoid bacterium that dechlorinates tetrachloroethene to ethene, was isolated and characterized. Growth of strain 195 with H2 and tetrachloroethene as the electron donor and acceptor pair required extracts from mixed microbial cultures. Growth of strain 195 was resistant to ampicillin and vancomycin; its cell wall did not react with a peptidoglycan-specific lectin and its ultrastructure resembled S-layers of Archaea. Analysis of the 16S ribosomal DNA sequence of strain 195 indicated that it is a eubacterium without close affiliation to any known groups.
Article
"Dehalococcoides ethenogenes" 195 can reductively dechlorinate tetrachloroethene (PCE) completely to ethene (ETH). When PCE-grown strain 195 was transferred (2% [vol/vol] inoculum) into growth medium amended with trichloroethene (TCE), cis-dichloroethene (DCE), 1,1-DCE, or 1,2-dichloroethane (DCA) as an electron acceptor, these chlorinated compounds were consumed at increasing rates over time, which indicated that growth occurred. Moreover, the number of cells increased when TCE, 1,1-DCE, or DCA was present. PCE, TCE, 1,1-DCE, and cis-DCE were converted mainly to vinyl chloride (VC) and then to ETH, while DCA was converted to ca. 99% ETH and 1% VC. cis-DCE was used at lower rates than PCE, TCE, 1,1-DCE, or DCA was used. When PCE-grown cultures were transferred to media containing VC or trans-DCE, products accumulated slowly, and there was no increase in the rate, which indicated that these two compounds did not support growth. When the intermediates in PCE dechlorination by strain 195 were monitored, TCE was detected first, followed by cis-DCE. After a lag, VC, 1,1-DCE, and trans-DCE accumulated, which is consistent with the hypothesis that cis-DCE is the precursor of these compounds. Both cis-DCE and 1,1-DCE were eventually consumed, and both of these compounds could be considered intermediates in PCE dechlorination, whereas the small amount of trans-DCE that was produced persisted. Cultures grown on TCE, 1,1-DCE, or DCA could immediately dechlorinate PCE, which indicated that PCE reductive dehalogenase activity was constitutive when these electron acceptors were used.
Article
cis-Dichloroethene (DCE) and vinyl chloride (VC) often accumulate in contaminated aquifers in which tetrachloroethene (PCE) or trichloroethene (TCE) undergo reductive dechlorination. "Dehalococcoides ethenogenes" strain 195 is the first isolate capable of dechlorinating chloroethenes past cis-DCE. Strain 195 could utilize commercially synthesized cis-DCE as an electron acceptor, but doses greater than 0.2 mmol/L were inhibitory, especially to PCE utilization. To test whether the cis-DCE itself was toxic, or whether the toxicity was due to impurities in the commercial preparation (97% nominal purity), we produced cis-DCE biologically from PCE using a Desulfitobacterium sp. culture. The biogenic cis-DCE was readily utilized at high concentrations by strain 195 indicating that cis-DCE was not intrinsically inhibitory. Analysis of the commercially synthesized cis-DCE by GC/mass spectrometry indicated the presence of approximately 0.4% mol/mol chloroform. Chloroform was found to be inhibitory to chloroethene utilization by strain 195 and at least partially accounts for the inhibitory activity of the synthetic cis-DCE. VC, a human carcinogen that accumulates to a large extent in cultures of strain 195, was not utilized as a growth substrate, and cultures inoculated into medium with VC required a growth substrate, such as PCE, for substantial VC dechlorination. However, high concentrations of PCE or TCE inhibited VC dechlorination. Use of a hexadecane phase to keep the aqueous PCE concentration low in cultures allowed simultaneous utilization of PCE and VC. At contaminated sites in which "D. ethenogenes" or similar organisms are present, biogenic cis-DCE should be readily dechlorinated, chloroform as a co-contaminant may be inhibitory, and concentrations of PCE and TCE, except perhaps those near the source zone, should allow substantial VC dechlorination.