Degradation of environmental toxins by a filamentous bacterium

This invention pertains to substantially purified cultures of a gram-negative, aerobic, filamentous bacterium with cells ranging in length from 20-200 .mu.m, that accumulates intracellular poly-.beta.-hydroxybutyrate in intracellular granules, and that degrades chlorinated aliphatic compounds such as trichloroethylene and dichloroethylene, as well as phenol and other substituted benzenes. The invention includes the representative strain A-1, which has been deposited at the American Type Culture Collection under the accession number 55581. Also included are methods for using the new bacterium for bioremediation of contaminated environmental sites.

FIELD OF THE INVENTION 
This invention relates to the biological degradation of halide-containing 
substances present in the environment such as trichloroethylene (TCE) and 
dichloroethylene (DCE) using a newly discovered filamentous bacterium. 
BACKGROUND OF THE INVENTION 
Environments contaminated with the chlorinated solvents trichloroethylene 
(TCE) and dichloroethylene (DCE) are major cleanup problems, and will 
involve hundreds of millions in Superfund expenditures in the coming 
years. TCE is a common ground-water contaminant in the United States as a 
result of solvent spills and dry-cleaning chemical disposal. 
Cis-1,2-dichloroethylene (DCE) is also a common ground-water contaminant 
that originates from anaerobic dehalogenation of TCE in the environment. 
These compounds are potential carcinogens and cannot be removed 
effectively from ground water using conventional water purification 
processes. For many sites, bioremediation is the only practical approach 
for cleanup, but the use of previously known solvent-metabolizing 
microorganisms has often been hindered by their extreme sensitivity to the 
toxic effects of intermediates produced as the result of these bacteria's 
degradation of TCE and related compounds, and by their sensitivity to high 
concentrations of TCE itself. 
Various types of bacteria have been shown to degrade TCE, including 
methanotrophs, toluene-degraders, phenol-degraders, propane oxidizers, and 
nitrifiers. (Fogel et al., 1986; Wackett et al., 1989; Ensley et al., 
1991). These bacteria degrade TCE by a process of "cometabolic 
degradation," in which substrates that support growth induce nonspecific 
monooxygenase or dioxygenase enzymes that fortuitously can also degrade 
TCE and other chlorinated aliphatic compounds (CACs). The dioxygenases and 
monooxygenases both require oxygen and reducing power in the form of NADH. 
When methanotrophs, the most extensively studied of this type of bacteria, 
metabolize trichloroethylene (TCE), dichloroethylene (DCE), or vinyl 
chloride by means of oxidative cometabolism, the resulting chemical 
intermediaries are known to be toxic to these microorganisms at relatively 
low levels (e.g., Alvarez-Cohen and McCarty, 1991a; Alvarez-Cohen and 
McCarty, 1991b; Alvarez-Cohen and McCarty, 1991c; Rasche et al., 1991; 
Oldenhuis et al., 1989). Moreover, the presence of methane competitively 
inhibits TCE degradation by methanotrophs (Strand et al., 1990). 
Consequently, the use of methanotrophs in bioremediation is severely 
limited. 
Bacteria other than methanotrophs are also known to degrade CACs. For 
example, Burkholderia (Pseudomonas) cepacia G4 (U.S. Pat. Nos. 4,925,802, 
and 5,071,755) expresses elevated levels of a CAC-degrading toluene 
ortho-monooxygenase enzyme in the presence of phenol, toluene, o-cresol, 
or m-cresol (originally called "Pseudomonas cepacia G4", this bacterium 
has been reclassified as "Burkholderia cepacia G4," Yabuuchi et al., 
Microbiol. Immunol. 36:1251-1275, 1981). This bacterium, isolated from an 
industrial waste site water sample, is strictly aerobic. The G4 isolate is 
reportedly a gram-negative, rod-shaped bacterium that grows predominantly 
in pairs and short chains, and can utilize carbohydrates as a carbon 
source. This organism reportedly consumes TCE at a rate of about 2.5 
nmol/minute per mg of protein with a K.sub.S of 3 .mu.M (Folsom et al., 
1990). "Intermediate toxicity," i.e., toxicity resulting from CAC 
degradation products, was observed in studies of Pseudomonas putida F1, as 
evidenced by a decrease in growth rate (Wackett and Householder, 1989). 
This study also reported that TCE damaged the P. putida's intracellular 
proteins. 
In other studies, phenol was utilized in situ to stimulate natural 
microorganisms to degrade CACs (Hopkins et al., 1993a; Hopkins et al., 
1993b). Phenol-degrading microorganisms were readily stimulated, and 
phenol removal from the injection site was complete after 170 hours. 
Cometabolic degradation of TCE and DCE was also observed, and 
phenol-degrading microorganisms were found to remove TCE more effectively 
than did methanotrophic organisms stimulated by methane addition in 
concurrent tests. Some laboratory studies with phenol/toluene oxidizers 
have been done (Folsom et al., 1990; Nelson et al., 1988; Shields et al., 
1989; Wackett and Householder, 1989), although such bacteria are less 
well-studied than the methanotrophs. However, it has been demonstrated 
that the toluene-degrader Pseudomanas putida F1 suffers toxic cellular 
effects from TCE degradation byproducts (Wackett and Householder, 1989). 
Another phenol-degrading bacteria reported to metabolize TCE is 
Alcaligenes eutrophus JMP134 (Harker and Kim, 1990). 
SUMMARY OF THE INVENTION 
Provided are substantially purified cultures of a new phenol-oxidizing 
bacteria that is capable of the cometabolic degradation of TCE and DCE and 
that is resistant to the toxic effects of metabolic intermediates produced 
by this degradation (Bielefeldt et al., 1995, which is hereby incorporated 
by reference). This new bacterium has a filamentous appearance and was 
first isolated from a surface-water sample grown under nitrogen-limiting 
conditions and with phenol provided as a carbon source. When used for 
bioremediation, this novel CAC-degrading microorganism has advantages over 
many of the previously known organisms that degrade TCE. The invention 
provides a straightforward method for obtaining the filamentous bacteria 
in enrichment cultures inoculated with environmental samples such as 
groundwater. Moreover, the growth advantage of this new microbe is shown 
to be so great that under nitrogen-limiting growth conditions it can 
overcome other microorganisms that have been inoculated into the cultures. 
The invention further provides a representative strain of the filamentous 
bacterium that was purified from a mixed culture by using standard 
microbiological techniques. This strain, called A-1, has been deposited 
with the American Type Culture Collection in accordance with the Budapest 
Treaty on May 20, 1994, at the American Type Culture Collection under the 
accession No. ATCC 55581. 
The present invention provides a method of bioremediation that involves 
contacting an environmental sample contaminated with chlorinated aliphatic 
compounds with a previously unknown filamentous bacterium capable of 
metabolizing chlorinated aliphatic compounds. This newly discovered 
filamentous bacterium can metabolize CACs in the presence or in the 
absence of phenol, although phenol induces elevated levels of the 
CAC-degrading enzyme. The filamentous bacteria can be used to 
decontaminate soil, water, and air samples. Isolates of the subject 
bacterium can be identified by their unique constellation of morphological 
and physiological properties. The new bacterium has non-sheathed 
rod-shaped cells filamentous in appearance and ranging in length from 
20-200 .mu.m, is aerobic, gram-negative, and when grown under 
nitrogen-limiting conditions accumulates poly-.beta.-hydroxybutyrate (PBB) 
in intracellular granules. Moreover, the bacterium is capable of growing 
in the presence of 0.2% phenol or 1% saturated toluene vapor, and does not 
grow on carbohydrates. 
The unique phylogenetic status of this novel bacterium has been confirmed 
by sequencing its 16S rRNA gene and comparing the sequences thus obtained 
with 16S rRNA sequences from other bacteria. Application of this 
well-established form of phylogenetic analysis indicated that this 
bacteria does not correspond to any known species for which 16S rRNA 
sequences are available. In light of the bacterium's unique morphology, 
these findings suggest that the filamentous bacterium may represent a new 
genus or species. 
The subject microorganism is more suited for bioremediation than 
methanotrophic bacteria due to its ability to tolerate metabolic 
intermediates produced from CACs, its tolerance for relatively high 
concentrations of substrate, and its having a higher rate of endogenous 
transformation of CACs. Moreover, it is shown here to tolerate higher TCE 
concentrations than other phenol-degrading microorganisms have been shown 
to tolerate. Thus, the subject microorganism can provide bioremediation in 
both aerobic treatment systems, suspended or fixed growth systems, or in 
gas biofilters.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT 
The subject invention provides methods of bioremediation comprising 
contacting an environmental sample contaminated with chlorinated aliphatic 
compounds with a unique bacterium capable of metabolizing chlorinated 
aliphatic compounds in the environmental sample. The improvement offered 
by this invention is the provision of substantially purified cultures of a 
previously unknown bacterium that is useful for bioremediation. This 
bacterium has non-sheathed rod-shaped cells filamentous in appearance and 
ranging in length from 20-200 .mu.m. The bacterium is aerobic, 
gram-negative, accumulates intracellular poly-.beta.-hydroxybutyrate in 
intracellular granules when grown under nitrogen-limiting conditions, is 
capable of growing in the presence of 0.2% phenol or 1% saturated toluene 
vapor, and does not grow on carbohydrates. Methods are provided for 
isolating substantially pure cultures of this new bacterium from 
environmental samples as well as methods for confirming that new isolates 
are substantially identical to the representative strain A-1 whose 
isolation is described here, and which was deposited in accord with the 
Budapest Treaty on May 20, 1995, at the American Type Culture Collection 
at 12301 Parklawn Drive, Rockville, Md. 20852, under the Accession No. 
55581. The new bacterium can be used to decontaminate soil, water, or air 
samples. 
In one embodiment of the methods provided by the invention, rather than 
contacting the contaminated sample directly with the bacterium, samples of 
soil or water are contacted with a stream of air into which the volatile 
chlorinated aliphatic compounds are thereby transferred. The 
contaminant-bearing air then is contacted with the subject bacterium. In 
other embodiments, contaminated soil or water are brought into direct 
contact with the bacterium. Alternatively, contaminated sites can be 
inoculated with the bacterium along with a single dose of phenol. 
The subject bacterium differs from previously known bacteria not only in 
its morphology and physiology, but with respect to sequences present in 
certain regions of its 16S ribosomal RNA (rRNA). In recent years, 16S rRNA 
sequence analysis has provided taxonomists with an invaluable tool for 
determining phylogenetic relationships among bacteria. Overall, the 16S 
rRNA molecule is highly conserved, but some regions of the molecule are 
subject to a relatively low degree of evolutionary constraint, hence can 
sustain numerous base sequence changes without loss of 16S rRNA function 
(see, e.g., Woese, 1987; Olsen et al., 1986; Gutell et al., 1994; Olsen et 
al., 1994, all of which are hereby incorporated by reference). These 
so-called "hypervariable regions" have been sequenced for a wide array of 
microorganisms, and the number and types of differences within these 
regions evaluated to determine evolutionary relationships among bacterial 
genera and species. A more modern approach to phylogenetic analysis of 
rRNA genes involves a computer-based comparison of the entire 16S rDNA 
sequence from different organisms, with the variable rates of evolution at 
each base position being taken into account by the computer program. 
Because of the sequence differences among bacteria, the 16S rRNA sequences 
have been exploited as a means for rapidly confirming whether field 
isolates of similar-appearing bacteria belong to the same or to different 
species (Moncla et al., 1990, which is hereby incorporated by reference). 
These investigators used radioactively-tagged 24-base oligonucleotide 
probes complementary to the hypervariable regions of the 16S rRNA of B. 
gingivalis to analyze numerous isolates of oral bacteria associated with 
periodontal disease. Several different probes were hybridized under 
stringent conditions with filter-immobilized DNA from a large number of 
bacteria isolated from the mouths of different patients. The hybridization 
conditions used by Moncla et al. were 0.6M NaCl, 90 mM Tris, pH 8.0, 10 mM 
EDTA, 30% deionized formamide, 0.5% sodium dodecyl sulfate (SDS), 
5.times.Denhardt solution (Sigma, pH 8.0), 100 .mu.g hydrolyzed carrier 
RNA per ml of hybridization buffer, with 4 hours or more incubation at 
42.degree. C. with gentle shaking, in the presence of about 5 ng of 
.sup.32 P-labeled oligonucleotide (about 1000-1200 cpm) per ml of 
hybridization buffer. These conditions ensured that the labeled probe 
would attach to the filter-bound DNA only if well-matched duplexes (i.e., 
hybrids) were formed. The filters were washed twice at 55.degree. C. (or 
at 50.degree. C.) with 0.09M NaCl, 9 mM Tris, 1 mM EDTA, 0.1% SDS, pH 8.0 
to remove unbound probes, and the hybridized probes were then detected by 
autoradiography. Other stringent hybridization conditions are known, i.e., 
conditions that permit only well-matched complementary nucleotide strands 
to form duplexes during the incubation period (see Sambrook et al., 1989, 
which is hereby incorporated by reference). A number of probes tested by 
Moncla et al. proved to be 100% specific when compared with standard 
microbiological methods for classifying these same bacteria. Thus, Moncla 
et al. demonstrated that hybridization under stringent conditions can 
provide a reliable means for determining whether bacterial isolates belong 
to the same or to different species. Similarly, probes selected from the 
16S rDNA sequences of strain A-1 can be used to confirm the identity of 
field isolates of the subject filamentous bacterium. 
The 16S rDNA of the representative strain A-1 has been sequenced using 
published methods (Dyksterhouse et al., 1995) and consists of the 
nucleotide sequences depicted in SEQ ID NO:3. SEQ ID NO:7 presents a 
subset of the sequences in SEQ ID NO:3. Strain A-1 was obtained by 
initially streaking a sample of the mixed culture of the filamentous 
bacteria, then repeatedly streaking and picking individual colonies. The 
A-1 strain resulting from these isolation procedures has been deposited at 
the American Type Culture Collection under the accession No. ATCC 55581. 
The scope of the invention includes microorganisms whose 16S ribosomal DNA 
is substantially identical to the sequences depicted in SEQ ID NO:3 and 
that are aerobic, gram-negative, have non-sheathed, filamentous, 
rod-shaped cells, under nitrogen-limiting conditions accumulate 
intracellular poly-.beta.-hydroxybutyrate in intracellular granules, that 
are capable of growing in the presence of 0.2% phenol or 1% saturated 
toluene vapor, and that do not grow on carbohydrates. For the purposes of 
this invention, nucleotide sequences "substantially identical" to SEQ ID 
NO:3 means that the DNA in question will hybridize with DNA from strain 
A-1 under stringent conditions. 
The novel microorganism of the subject invention became dominant when 
non-sterile culture medium was inoculated with surface water and 
maintained under aerobic nitrogen-limiting conditions in the presence of 
phenol, a substance capable of inducing phenol-degrading enzymes that also 
have the capacity to degrade CACs. "Nitrogen-limiting conditions" means 
that the culture media contains about 200 g NH.sub.4 Cl for every 1000 g 
phenol used as a carbon source. This is a lower ratio of carbon:nitrogen 
than is commonly used for cell growth. 
In several subsequent experiments, the subject microorganism became 
predominant in other cultures maintained under nitrogen-limiting 
conditions in the presence of phenol when the cultures were inoculated 
from a pure culture of Burkholderia cepacia G4. These cultures were not 
inoculated with the filamentous bacterium. 
A pure culture of the filamentous microorganism was obtained by repeatedly 
streaking the filamentous bacteria from a mixed culture using standard 
microbiological techniques, but the microorganism was observed to grow 
somewhat better in nitrogen-limited cultures in which sterility was not 
maintained. It is possible that the subject microorganism benefits in some 
way from the presence of small amounts of unidentified microorganisms in 
non-sterile cultures. Mixed cultures in which one microorganism enhances 
the growth of another are commonly termed "consortia," and the mixed 
cultures predominated by the filamentous bacteria are referred to as 
"consortia." However, in pure culture the subject microorganism grows 
reasonably well and can still degrade TCE. 
For the purposes of this invention, a "substantially-purified culture" of 
the novel bacterium includes a pure culture obtained by repeated 
streaking, as well as a non-sterile culture in which the filamentous 
bacterium predominates. The term "substantially-purified" indicates a 
culture which, when inspected microscopically at 1000.times. 
magnification, appears visually to contain at least 70% by mass of the 
filamentous bacterium. More typically, mixed cultures of the filamentous 
bacterium appear visually to contain about 90% by mass of the filamentous 
bacterium at this level of magnification. 
The physical appearance of this new microorganism is strikingly different 
from any morphological forms reported for the organism's nearest 
phylogenetic relatives. No CAC degradation work with a microorganism 
having this appearance has apparently been reported. Cultures enriched for 
this organism can be maintained for long periods under non-sterile 
conditions by providing intermittent doses of phenol and an inorganic 
nutrient solution in which nitrogen is limited. Established cultures can 
be maintained even under nitrogen-sufficient culture conditions. 
The lack of TCE intermediate toxicity effects on the enriched culture of 
the subject microorganism is a major advantage for its use over 
methanotrophs in bioremediation. The observed TCE transformation capacity 
of this new bacteria is substantially greater than values reported for 
most other cometabolic bacteria that are affected by intermediate toxicity 
(Bielefeldt et al., 1995). This advantage could be critical in terms of 
designing a treatment system, as phenol-metabolizing cultures could 
potentially maintain degradation for a longer period than methanotrophs, 
with whose use TCE degradation rates decline rather rapidly due to 
intermediate toxicity. Furthermore, when phenol is present, this new 
microorganism has been shown to tolerate and degrade concentrations of TCE 
as high as 130 mg/l, and concentrations of DCE up to 83 mg/l (Bielefeldt 
et al., 1995). Tolerance to high concentrations of contaminants would be 
critical for bacteria used to treat fairly concentrated waste streams. 
Moreover, the bacteria of the subject invention degrades TCE at 
concentrations higher than those previously reported to be degraded 
Burkholderia cepacia G4 (Bielefeldt et al., 1995). 
Besides its unique filamentous appearance, the subject microorganism has a 
constellation of physiological properties that set it apart from 
previously-known microorganisms. The new organism is aerobic, 
gram-negative, has non-sheathed rod-shaped filamentous cells, accumulates 
intracellular poly-.beta.-hydroxybutyrate in intracellular granules under 
nitrogen-limiting conditions, can grow in the presence of 0.2% phenol or 
1% saturated toluene vapor, and does not grow on carbohydrates. Based on 
these characteristics, the new bacteria is clearly different and distinct 
from its known phylogenetic relatives Comamonas acidovorans and Comamonas 
testosteroni. 
Bacterial isolates of the subject invention can be identified by their 
unique properties, i.e., their filamentous appearance combined with the 
constellation of traits listed above, but their identities can be further 
confirmed by analyzing the nucleotide sequence of their 16S rDNA. Using 
the A-1 strain as a representative source of the filamentous bacteria's 
DNA, the entire 16S rRNA gene of this new organism has been sequenced and 
compared with 16S rDNA sequences from other bacteria (Example 3, FIGS. 1 
and 2, and Tables 1 and 2). When the A-1 16S rDNA sequences were compared 
with three others for which all or most of the 16S rDNA sequence is known, 
results indicated that the filamentous bacteria's 16S rDNA gene differed 
from the nearest related bacteria by 4.62% (FIG. 2 and Table 1). A smaller 
region of A-1 16S rDNA sequence was compared with two additional bacteria 
for which only a limited region of 16S rDNA sequence was available (FIG. 3 
and Table 2). In this latter comparison, which also included the four 
bacteria from FIG. 2, the A-1 sequences differed from the most closely 
related bacterium at 4.18% of the nucleotide positions (Table 2). Thus, a 
16S rDNA analysis of this type can be applied to field isolates of the 
subject bacteria either as an alternative to a full determination of 
physiological properties, or as a means of further confirming an isolate's 
identity. 
The subject bacteria can be used in bioremediation applications in several 
ways. Treatment of air-stripping vapors is among the preferred 
applications. For this procedure, water or soil carrying TCE is first 
aerated, and the volatile TCE is transferred to the air stream with which 
the contaminated water or soil is aerated. For example, water is pumped 
out of the ground and aerated at the surface. The TCE-bearing air stream 
is passed subsequently through a biofilter or bioreactor in which the 
subject microorganisms are present. The biofilter or bioreactor is 
inoculated with a pure culture of the subject microorganism, which 
thereafter will remain the predominant microorganism under 
nitrogen-limiting conditions without any need to maintain the filter under 
sterile conditions. Alternatively, the bioreactor can be inoculated with a 
mixed culture in which the novel filamentous bacterium is the predominant 
organism. 
TABLE 1 
__________________________________________________________________________ 
Stripa research 
Brachymonas 
Strain 
C. testosteroni 
mine derived 
denitrificans 
A-1 
(ATCC 11996) 
environmental clone 
AS-P1 
__________________________________________________________________________ 
Strain A-1 1.000 
0.9538 0.9383 0.9449 
C. testosteroni 
1.000 0.9520 0.9463 
Stripa environmental clone 
1.000 0.9394 
Brachymonas denitrificans 1.000 
__________________________________________________________________________ 
TABLE 2 
__________________________________________________________________________ 
Stripa research 
Brachymonas 
Strain 
C. testosteroni 
C. terrigena 
C. acidovorans 
mine derived 
denitrificans 
A-1 
(ATCC 11996) 
(ATCC 8461) 
(ATCC 15668) 
environmental clone 
AS-P1 
__________________________________________________________________________ 
Strain A-1 1.000 
0.9425 0.9368 0.8908 0.9582 0.9454 
C. testosteroni 
1.000 0.9511 0.8908 0.9620 0.9454 
C. terrigena 1.000 0.9253 0.9468 0.9397 
C. acidovorans 1.000 0.9049 0.8994 
Stripa environmental clone 1.000 0.9544 
Brachymonas denitrificans 1.000 
__________________________________________________________________________ 
As an alternative to air-stripping, contaminated water can be treated 
directly with the subject bacteria. As an additional alternative, pure or 
enriched cultures of the subject bacteria can be injected into 
contaminated ground sites, in which case the growth of injected 
microorganisms may be enhanced by co-injection of substances such as 
phenol or toluene that induce the enzymes responsible for TCE degradation. 
The continuous presence of phenol is not necessary, as the phenol-induced 
enhancement of TCE-degradation persists for many hours after the phenol 
has been entirely degraded (see Example 5). The subject bacteria also have 
been found to be particularly effective in degrading benzene, toluene, 
ethylbenzene, and xylenes, substances common in gasoline spills, hence 
could be used for clean-up of such spills. 
The invention is further described in the following examples. 
EXAMPLES 
Example 1 
Isolation of Filamentous Bacteria Capable of Metabolizing Chlorinated 
Aliphatic Compounds 
Enrichment cultures of phenol-degrading bacteria were grown in mixed, 
flow-through reactors, and biomass from these reactors harvested for use 
in batch-fed, serum bottle tests to study the degradation of phenol and 
chlorinated aliphatic compounds. The cultures were grown at 20.degree. C. 
in continuously stirred 4L flasks stoppered with sterilized glass wool. 
However, the enrichment cultures were not intended to be sterile cultures, 
but rather were designed to encourage or select for the outgrowth of 
naturally-occurring bacteria that tended to thrive and become predominant 
under the provided growth conditions. 
(1) NL3 
A 4L flask was inoculated with water from a stream running through an old 
capped landfill in a parking lot located in Seattle, Wash. The pH was 
maintained at about 7.0 by adding sodium bicarbonate. The reactor feed 
solution contained 1000 mg/l phenol, 700 mg/l KH.sub.2 PO.sub.4, 1000 mg/l 
K.sub.2 HPO.sub.4, 200 mg/l NH.sub.4 Cl, 50 mg/l CaCl.sub.2, 30 mg/l 
MgSO.sub.4, 10 mg/l NaCl 0.055 mg/l CuCl.sub.2 H.sub.2 O, 0.148 mg/l 
ZnCl.sub.2, 0.022 mg/l NiCl.sub.2 6H.sub.2 0, 0.880 mg/l FeSO.sub.4 
7H.sub.2 O, 0.135 mg/l Al.sub.2 (SO.sub.4).sub.3 18.sub.2 O, 0.282 mg/l 
MnCl.sub.2 4H.sub.2 O, 0.056 mg/l CoCl.sub.2 6H.sub.2 O, 0.032 mg/l 
Na.sub.2 MoO.sub.4 2H.sub.2 O, 0.049 mg/l H.sub.3 BO.sub.3. The initial 
nutrient solution in the flask was the same as the feed solution except 
for the phenol. 
In the culture flask (or "bioreactor"), the chemical oxygen demand (COD) to 
N ratio in the feed (from phenol and NH.sub.4 Cl, respectively) was 
100:2.2 gCOD/g N. "Chemical oxygen demand" is a measure of the 
concentration of metabolizable carbon present in the culture. The 
concentration of phenol in the flask was maintained at about 0.5 mg/l, and 
phenol was the sole carbon source provided. A timer was used to 
intermittently feed the reactor at approximately 0.6 ml/min at an average 
phenol loading rate of 0.36 g/g volatile suspended solids per day (VSS-d) 
and to withdraw reactor liquid every 6 hours at a rate of about 7 ml/min 
for 12 minutes. The reactor liquid volume varied from 2-2.7 L and the 
concentration of VSS varied, but ranged from about 400-600 mg/L. Several 
weeks after the pH of this culture was temporarily dropped to 4.0 and the 
phenol concentration raised to 1 mg/L, a large filamentous bacterium was 
observed to dominate this culture. It is not known whether this temporary 
change in culture conditions affected the subsequent outgrowth of the 
subject bacteria. 
(2) Nitrogen-Limited (NL1) 
After the NL3 culture had been established, a separate non-sterile culture, 
designed NL1, was inoculated from a slant of Burkholderia cepacia strain 
G4, and fed with the above-described feed solution for 50 minutes every 
two hours at an overall loading of about 0.18 g phenol/L culture volume 
per day. Nitrogen-limiting conditions were maintained as described above. 
Effluent from the reactor was pumped out every six hours at a rate of 
about 7 ml/min for 12 minutes, and fresh medium pumped in at a similar 
rate. Instead of producing a culture predominated by B. cepacia G4 as 
expected, within three weeks the growth reactor became predominated by a 
filamentous bacteria that was morphologically very different from G4 and 
that was identical in appearance to the filamentous bacteria of NL3. The 
filamentous bacteria's outgrowth and predominance in NL1 apparently was a 
response to its selective advantage under the provided growth conditions. 
(3) NL2 
Inoculated from a B. cepacia G4 slant, this culture was fed with the feed 
solution described above for 2 min of every 5 min at an overall loading of 
about 0.18 g phenol/L-d; the feed COD:N ratio was about 45:1 gCOD/g N. 
This culture, like NL1, was intended to give rise to a mixed culture 
predominated by B. cepacia G4, but instead, became predominated within 
three weeks by a filamentous bacteria identical in appearance to that 
observed in NL3 and NL1. Under these growth conditions, the filamentous 
bacteria apparently has a reproducible growth advantage over B. cepacia G4 
and other microorganisms that may have been present in the non-sterile 
bioreactor. 
(4) NNL 
To test the capacity of the filamentous bacteria to tolerate increased 
nitrogen, after NL2 was maintained for three months under conditions 
described above, the COD:N ratio in the feed was changed to 100:5.6 by 
raising the NH.sub.4 Cl concentration to 300 mg/l and adding NaNO.sub.3 at 
300 mg/l. This culture was thereafter designated "NNL," or "non-nitrogen 
limited." Despite the increase in nitrogen concentration, the predominant 
filamentous bacteria initially present in this culture did not become 
displaced by other microorganisms. Thus, the filamentous bacteria does not 
absolutely require nitrogen-limiting conditions in order to remain the 
dominant organism in a culture. However, observations indicated that the 
organism grows somewhat better under nitrogen-limiting conditions. 
(5) Isolation of Strain A-1 
The large filamentous bacteria were further purified by streaking onto 1.5% 
agar medium containing 500 mg/l KNO.sub.3, 200 mg/l MgSO.sub.4, 15 mg/l 
CaSO.sub.4, 63 mg/l (NH.sub.4).sub.2 SO.sub.4, 425 mg/l Na.sub.2 
HPO.sub.4, 200 mg/l KH.sub.2 PO.sub.4, 1 mg/l FeSO.sub.4 H.sub.2 O, 0.5 
mg/l NaEDTA, 0.1 mg/l CuSO.sub.4, 0.1 mg/l ZnSO.sub.4. 7H.sub.2 O, 0.03 
mg/l NaMoO.sub.4.2H.sub.2 O, 0.02 mg/l MnSO.sub.4.2H.sub.2 O, 0.02 mg/l 
H.sub.3 BO.sub.3, 0.02 mg/l NiSO.sub.4.6H.sub.2 O, 0.01 mg/l 
CoSO.sub.4.7H.sub.2 O, 50 or 100 mg/l phenol. Phenol was the sole source 
of carbon and energy, and there was no significant growth if phenol was 
omitted from the medium. Colonies appeared within 2 days. Individual 
colonies were picked and streaked several times to obtain a colony that 
presumably originated from a single bacterial cell. The purified culture 
that arose from this colony was named strain A-1 (ATCC No. 55581). 
Example 2 
Characteristics of the Filamentous Phenol-Degrading Bacteria 
The predominant bacteria in NL3, NL1, NL2, NNL (described in Example 1), 
and strain A-1 were virtually indistinguishable from one another when 
examined under the microscope, although bacteria from the NNL culture had 
somewhat shorter filaments and fewer intracellular deposits. The 
filamentous bacteria exhibited long filaments ranging in length from 
20-200 .mu.m, contained large bluish intracellular deposits, and were 
capable of degrading TCE and utilizing phenol as their sole carbon source. 
Within the filaments, neither flagella nor septa were directly observed. 
The morphology of the subject bacteria was clearly very different from the 
much shorter inclusion-free rods of the phenol-degrader B. cepacia G4. 
The filamentous bacteria's capacity to degrade phenol was determined in 
sterile 160 ml serum bottles containing 10-70 ml of fresh nutrient medium 
brought up to a total of 80 ml from the enrichment cultures. The nutrient 
medium was the same as used for the growth reactor feed but without the 
phenol. The medium was pre-aerated with oxygen for 15-30 minutes before 
use. Phenol (10-40 mg/L) was spiked into bottles containing aliquots of 
the mixed cultures. After an appropriate interval to permit phenol 
degradation, the concentration was measured by removing 1 g of sample to a 
4 ml vial containing 4-chlorophenol as an internal standard, then adding 
40 =l each of K.sub.2 CO.sub.3 and acetic anhydride. After 30 min at room 
temperature to permit acetylation of non-degraded phenol, 1 ml of hexane 
was added and the sample vortexed for 2 min. After centrifugation to 
separate the layers, the hexane layer was collected and phenol measured by 
gas chromatography using standard procedures (Bielefeldt et al., 1995). 
Alternatively, phenol was measured colorimetrically by adjusting a culture 
sample to pH 7.9, then reacting with 4-aminoantipyrene and potassium 
ferricyanide to produce a yellow-to-red product. After 15 min, samples 
were analyzed spectrophotometrically at 500 nm. Concentrations of phenol 
were determined colorimetrically by comparison to standards containing 
known amounts of phenol. 
Samples of NL1, NL2, NL3, and NNL all were shown to be capable of degrading 
phenol. Extensive testing focused on NL1, NL2, and NNL. For those three 
cultures, degradation of phenol in batch tests was essentially linear, 
following zero-order kinetics down to less than 0.5 mg/L. For one 
experiment with samples from NL1, the phenol concentrations tested were 5, 
8, 16, and 26 mg/l. All the degradation curves were linear, and the rates 
observed for the highest and the lowest concentrations were very similar, 
consistent with zero-order kinetics. Overall, the rates of phenol 
degradation ranged from 2.2-16.4 g phenol/g-d. In general, rates were 
higher when the culture was grown under nitrogen-sufficient conditions 
rather than nitrogen-limiting conditions. In most batch test cultures, 
phenol degradation was complete in less than 1 h, and often in less than 
15 min. Moreover, when high concentrations of TCE were added, a decrease 
in the rate of phenol degradation was observed. 
These results were compared with published data describing phenol 
degradation by Burkholderia (Pseudomonas) cepacia G4 (Folsom et al., 
1990). This publication indicated that for G4, phenol degradation rates 
varied with phenol concentration, with increasing rates up to about 5 
mg/l, and then decreasing rates due to substrate toxicity. Moreover, 
Folsom et al. observed that TCE significantly inhibited phenol degradation 
by G4. 
Example 3 
Characterization of Strain A-1 
The representative strain A-1 is capable of growing on low concentrations 
of phenol and toluene, and does not grow when carbohydrates form the sole 
carbon source. While the filamentous bacterium occasionally appeared to be 
motile when grown in mixed cultures, strain A-1 is non-motile. A whole 
cell fatty acid composition of strain A-1 was performed using the MIDI gas 
chromatographic bacterial identification system. Based on the results, the 
closest match in the TSBA database was Comamonas (Pseudomonas) 
acidovorans. (The "TSBA database" is a commercially available database 
from MIDI in which identification of unknown bacterial strains is based on 
comparing the whole cell fatty acid composition of unknown strains to 
strains that are present in the MIDI database. Cultures to be identified 
must be grown under incubation conditions that are specified by MIDI). 
However, A-1 exhibited phenotypic properties quite different from 
previously described type strains of Comamonas, which are motile and have 
notably shorter rods. 
To perform phylogenetic analysis, the 16S rDNA of A-1 was amplified using 
primers specific for 16S rRNA genes, and the amplification products cloned 
and sequenced as described (Dyksterhouse et al., 1995). Sequencing was 
performed in both directions using the ABI automated sequencer and the 
Cycle-Sequencing kit. A computer program (fastDNAml) was used to compare 
the resulting sequences with other known 16S rDNA sequences and to create 
a phylogenetic tree using the maximum likelihood method. Results indicated 
that A-1 is a member of the .beta. subdivision of the Proteobacteria, but 
is not closely related to B. cepacia, or other Burkholderia species. 
The Ribosomal Database Project files indicated that A-1's closest relative 
based on 16S rDNA sequence is Comamonas testosteroni (the strain of C. 
testosteroni with which the A-1 sequence was compared was described in 
U.S. Pat. No. 5,120,652), a species closely related to Comamonas 
acidovorans, which MIDI analysis identified as the filamentous bacteria's 
closest relative. However, C. testosteroni and the filamentous bacterium 
differ significantly. C testosteroni can grow on carbohydrates while A-1 
cannot. Moreover, C. testosteroni exhibits gram-negative rods that are 
0.5-0.7 .mu.m wide and 1.5-3.0 .mu.m long (U.S. Pat. No. 5,120,652), in 
contrast to the long filaments (20-200 .mu.m) of the present isolate. The 
data in Table 1 suggests that A-1 may be a new species of Comamonas or may 
belong to a previously unknown genus. 
A computer-based comparison was performed of A-1 16S rDNA sequences to 16S 
rDNA from other bacteria as described in Dyksterhouse et al. (1995). 
Results for the most closely-related bacteria for which sequences were 
available are illustrated in Tables 1 and 2 and in FIGS. 2 and 3. These 
microorganisms, the closest known phylogenetic relatives to strain A-1, 
all belong to the .beta.-Proteobacteria. FIG. 2 shows the entire 16S rDNA 
sequence for C. testosteroni ATCC No. 11996; Brachymonas denitrificans 
AS-P1, strain A-1, and a partial sequence for the "Stripa-derived" 
environmental clone. These sequences correspond, respectively, to SEQ ID 
NOS:1-4. Except for A-1, the sequences in FIG. 2 were retrieved 
electronically from the Ribosomal Database Project. 
Table 1 summarizes the information of FIG. 2, providing a similarity matrix 
for the sequences. To facilitate the comparisons, the program introduced 
gaps into the sequences by maximizing the alignment of the highest 
possible number of identical sequences. The length of 16S rDNA sequence 
(without gaps) differed somewhat among these bacteria, as shown. The 
"Stripa-derived" sequence data in the database was determined originally 
from PCR-amplified rDNA using a DNA template that was extracted directly 
from an environmental sample. Hence, the organism that gave rise to the 
"Stripa-derived" DNA was never propagated or cultured; no physiological 
data are available for it, so its physiological properties could not be 
compared with those of the filamentous bacteria. 
Taken together with the unusual physiological profile of the filamentous 
bacterium, the data in Table 1 strongly suggest that it is a new species, 
and may be the first known member of a previously unknown genus. The 
16S-rDNA sequence of A-1 differs from the most closely-related match, C. 
testosteroni ATCC No. 11996, at 4.62% of the compared nucleotide residues. 
This extent of difference is comparable to that observed for other pairs 
of organisms that are known to be different species. TABLE 1 shows, for 
example, that C. testosteroni ATCC No. 11996 and B. denitrificans AS-P1 
differ at 5.47% of the residues in the compared region. This degree of 
difference is comparable to the 4.62% difference between A-1 and C. 
testosteroni ATCC No. 11996. The notion that strain A-1 is at least a new 
species is particularly compelling in view of the filamentous bacterium's 
unusual appearance (FIGS. 1A and 1B), which represents a morphology not 
previously reported for any member of the genus Comamonas. 
The sequence data of FIG. 3 suggest further that strain A-1 differs from 
previously known bacteria. FIG. 3 adds sequences from Comamonas terrigena 
ATCC No. 8461 and Comamonas acidovorans ATCC No. 15668 to the sequences 
shown in FIG. 2 and Table 1. These additional sequences were derived as 
described in Dyksterhouse et al (1995), using the SP3 primer. Only partial 
16S gene sequences were available for these two organisms. Accordingly, to 
compare it with A-1, the corresponding sequences from the organism listed 
in FIG. 2 were excerpted and aligned with C. testosteroni and C. 
acidovorens sequences as shown in FIG. 3. As for FIG. 2, a computer 
program was used to facilitate alignment of the sequences. The length of 
the ungapped fragments used for this comparison was 348 nucleotides for 
all of the included organisms except for the "Stripa-derived" bacterium, 
for which it was 263. The sequences of FIG. 3 correspond to SEQ ID 
NOS:5-10, and were derived, respectively, from C. testosteroni ATCC No. 
11996, "Stripa-derived" environmental clone, strain A-1, Brachymonas 
denitrificans AS-P1, C. terrigena ATCC No. 8461, and C. acidovorans ATCC 
No. 15668. 
Table 2 summarizes the sequences shown in FIG. 3, and provides a similarity 
matrix for the six organisms whose sequences are compared. Table 2 
indicates that for this region of the 16S rDNA sequence, the degree of 
difference between strain A-1 and the other microorganisms analyzed ranged 
from 4.18% to 10.92%. These differences exceed the differences seen 
between most pairs of organisms included in Table 2, again suggesting 
strongly that strain A-1 represents at least a new species of bacteria. 
Example 4 
Accumulation of Intracellular Poly-.beta.-Hydroxybutyrate (PHB) by Strain 
A-1 
Under the light microscope, the filamentous bacterium presents a 
characteristic granular appearance. Phase contrast microscopy revealed 
that the filaments of strain A-1 contain optically retractile inclusion 
bodies. Transmission electron micrographs also showed abundant large 
inclusions. Staining results suggested that these inclusions are comprised 
of PHB and possibly polyphosphate. When the culture was grown under 
nitrogen-limiting conditions, up to 80% of the culture dry weight was 
indeed determined to be PHB by gas chromatographic analysis. 
Example 5 
Degradation of TCE 
In these experiments, TCE degradation was measured using methods previously 
described (Folsom et al., 1990; Bielefeldt et al., 1995). In brief, TCE 
degradation was measured by extracting aliquots of the culture with 
pentane, and analyzing by gas chromatography. Concentrations of TCE in the 
samples were determined by comparison with a standard curve. 
Under endogenous conditions (i.e., with no inducer such as phenol 
concurrently present), all cultures of the filamentous bacteria as well as 
the A-1 isolate degraded TCE over a wide range of substrate 
concentrations. 
For tests with initial TCE concentrations ranging from about 5-25 mg/l, 
zero-order kinetics for initial degradation rates were observed. 
Zero-order kinetics were observed also for substrate concentrations as low 
or lower than 1 mg/L TCE. Rates of TCE consumption ranged from 0.11 to 
0.25 g TCE/g VSS-d. In one set of tests, the average TCE consumption rate 
was 0.18 g/g VSS-d with a standard deviation of 0.033 for 12 tests. There 
was no significant difference in TCE degradation rates after 0, 8, and 24 
h of pre-aeration using NL1, although some rate reduction was observed 
when NNL was tested. TCE degradation rates decreased at TCE concentrations 
above 30 mg/L, possibly due to TCE toxicity. Despite the filamentous 
bacteria's sensitivity to very high TCE concentrations, the consortia 
tolerated and degraded TCE at concentrations much higher than the 7.7 mg/L 
maximum tolerable concentration reported for methanotrophs (Strand et al., 
1990). 
The effect of phenol addition on TCE degradation rate was tested. Bottles 
that were spiked with 10-40 mg/l of phenol exhibited higher TCE 
degradation rates than those that did not receive phenol. Over the tested 
range of phenol concentrations, TCE degradation rates varied but were 
consistently higher than the average endogenous degradation rates at 
similar TCE concentrations. The higher degradation rates induced by adding 
phenol persisted for the duration of the TCE degradation (approximately 
eight hours), even though the added phenol was degraded completely within 
about an hour of its addition. Thus, there appeared to be no competitive 
inhibition of TCE degradation by phenol. Moreover, even at TCE 
concentrations above 40 mg/L, phenol addition caused a stimulation in the 
TCE degradation rates. 
The capacity of the filamentous bacteria to degrade TCE was further 
quantified to facilitate comparisons with other organisms. For this 
purpose, the filamentous bacteria's capacity to degrade TCE was expressed 
as grams of TCE degraded per gram of cells present, or "transformation 
quantity (Tq)." Values calculated for Tq ranged from &gt;0.31-0.51. These 
transformation quantities were higher than the reported values for 
endogenous batch TCE degradation tests with methanotrophs and phenol-grown 
mixed cultures (Bielefeldt et al., 1995, Table 4). 
To determine whether the filamentous bacteria could degrade TCE when grown 
in pure culture, tests were performed using strain A-1 grown in batch 
cultures with phenol as the sole source of carbon. Cells were washed and 
resuspended in chloride-free media in serum bottles with teflon-valved 
closures. TCE was added to produce an aqueous concentration of 
approximately 12 mg TCE/L. Total TCE in each bottle was approximately 3 
.mu.M. The rate of TCE degradation was assessed by using standard methods 
to measure chloride production over a period of several days (Nelson et 
al., 1987). Results indicated that strain A-1 degraded TCE at a slightly 
lower rate than those reported for pure Burkholderia cepacia G4. These 
results indicate that even though the filamentous bacteria grows somewhat 
better in mixed cultures, it is nevertheless capable of degrading TCE 
without requiring the presence of other bacteria. 
Other experiments indicated that the consortia could not degrade 
tetrachloroethylene, chloroform, and 111-trichloroethane, which were 
measured also by gas chromatography. However, for unknown reasons a 
concentration of 5 mg/L of 111-trichloroethane inhibited TCE degradation, 
although 111-trichloroethane at 1 mg/L did not affect TCE degradation. 
Neither tetrachloroethylene nor chloroform had any effect on TCE 
degradation rates at the concentrations tested. 
Example 6 
Intermediate Toxicity 
To determine whether the filamentous bacteria were susceptible to toxic 
effects from TCE metabolic intermediates seen with other TCE degraders, 
tests were conducted to compare degradation rates of TCE with and without 
prior TCE degradation in the same culture sample. The experiment also 
included tests to determine whether phenol would affect the filamentous 
bacterium's reaction to possible toxic effects of TCE breakdown products. 
Four culture bottles were used for this test. Two were initially spiked 
with about 25 mg/L TCE to generate intermediates, while two other bottles 
were put into the shaker without TCE. After 24 h, one TCE-fed culture was 
respiked with 25 mg/L TCE, and the other with 25 mg/L TCE plus 20 mg/L 
phenol. Also after 24 h, one of the remaining two cultures that had not 
been fed TCE was spiked with 25 mg/L TCE, and the other spiked with 25 
mg/L TCE plus 20 mg/L phenol. 
No effects of intermediate toxicity were seen, as the samples with prior 
exposure to TCE actually degraded the second dose of TCE at a slightly 
faster rate than their counterparts that were not pre-exposed to TCE. 
Faster degradation of the second dose of TCE was observed whether or not 
the second dose was accompanied by a dose of phenol. When the initial 
doses of TCE were increased up to 0.51 g TCE/g cells, still no toxic 
effects were observed. Moreover, no toxic effects were observed when 
similar tests were conducted with DCE instead of TCE, at loadings of DCE 
up to 0.3 g DCE/g cells (see Example 7). It was noted also that bottles 
respiked with phenol degraded TCE at a rate about three times higher than 
the cultures that did not receive the dose of phenol. 
Example 7 
Degradation of DCE 
The enrichment cultures were tested for their ability to degrade DCE under 
endogenous conditions using the methods previously described (Bielefeldt 
et al., 1995). DCE concentration was analyzed by purge and trap gas 
chromatography with a Hall detector in which samples were purged 10 
minutes with a 3-minute desorb time and 5-minute bake time. With initial 
DCE concentrations ranging from 14-83 mg/L, zero order kinetics were 
observed for DCE degradation under endogenous conditions. At the highest 
initial concentration tested, no significant decrease in initial 
degradation rate was evident in comparison with the lower concentrations 
tested concurrently. The initial degradation rates observed for DCE 
exceeded those observed for TCE degradation. When cultures were spiked 
with phenol, the observed rates of DCE degradation were 2-6-times higher 
than in the absence of phenol. 
To determine whether intermediates of DCE metabolism were toxic to the 
filamentous bacteria, DCE degradation rates with and without prior DCE 
degradation were compared using DCE respiking tests (Bielefeldt et al., 
1995). Several batch cultures from the NLL consortium were allowed to 
degrade an initial dose of DCE to completion, then respiked with a second 
dose eight hours after the first dose. The first dose was degraded at a 
rate of 0.85 g DCE/g VSS-d, and the second dose at a rate of 0.10 g DCE/g 
VSS-d. Thus, the second dose of DCE was degraded more slowly than the 
first. However, a control batch culture that received only the second dose 
of DCE also degraded it at a rate of 0.10 g DCE/g VSS-d. Thus, these 
results with the control cultures indicated that the prior degradation of 
DCE had had no effect on the later DCE degradation rate, indicating no 
intermediate toxicity. Instead, the decrease in degradation rate for the 
second dose of DCE most likely can be attributed to endogenous depletion 
of energy reserves. 
Further, if intermediate toxicity had significantly affected the cells, the 
addition of phenol would not have been expected to increase the rate 
because the cells would have been damaged and unable to utilize the 
electron donor. However, in experiments where phenol was added with the 
second dose of DCE, the culture's ability to degrade DCE was largely 
restored. 
In other experiments, it was observed that there was no decline in the 
degradation rate of either compound during the concurrent degradation of 
TCE and DCE by the enrichment cultures. 
Example 8 
Screening Test to Confirm the Identity of Field Isolates of Filamentous 
Phenol Degrading Bacteria 
The nucleotide sequences shown in SEQ ID NO:3 and SEQ ID NO:7 are used as 
the basis for designing oligonucleotide probes for a hybridization-based 
confirmation test. Probe design is based on information presently 
available in the prior art. The secondary structure of prokaryotic 16S 
rRNA has been elucidated and within this relatively invariant structure, 
several regions have been mapped within which the degree of sequence 
variability among species is relatively high (e.g., Autell and Fox, 1988, 
which is hereby incorporated by reference). These are known as 
"hypervariable regions." Other authors have disclosed methods of 
constructing phylogenetic trees by using a computer to align 16S rRNA 
sequences from different prokaryotes (Olsen et al., 1986). Such alignments 
make it possible to locate these hypervariable regions within any 
bacterial 16S rRNA sequence, given the demonstrated consistency of their 
locations. For example, hypervariable region probes have been used to 
successfully determine phylogenetic relationships by several different 
investigators (e.g., Moncla et al., 1990; Chuba et al., 1988; Gobel et 
al., 1987). Hence, one skilled in the art can readily identify the 
hypervariable regions of strain A-1 by aligning the sequences in SEQ ID 
NO:3 with previously determined bacterial 16S rRNA sequences. 
Oligonucleotide probes based on these sequences are used in diagnostic 
confirmation tests for field isolates of the filamentous bacterium. 
Probes corresponding to the hypervariable regions of strain A-1's 16S rRNA 
are synthesized by reference to SEQ ID NO:3 and the published literature 
(e.g., Noller, 1984; Moncla et al., 1990;; Gobel et al., 1987; Chuba et 
al., 1988; Autell and Fox, 1988; Olsen et al., 1986; Woese, 1987). 
Standard methodologies are applied to select the best probes and the 
optimum hybridization conditions for distinguishing the filamentous 
bacteria of the subject invention from other bacteria. Probes that are 
capable of differentiating strain A-1 from its nearest relatives are used 
for screening new isolates of filamentous phenol-degrading bacteria to 
determine whether they belong to the same phylogenetic position as the 
filamentous bacterium of the present invention. 
For determining the optimum hybridization conditions, filters are loaded 
with DNA extracted from strain A-1, as well as DNA from those bacteria 
that are known to be most closely related to the filamentous bacteria, 
namely Comamonas acidovorans, Comamonas testosteroni and Brachymonas 
dentrificans. Filters or membranes are also loaded with DNAs from 
distantly-related bacteria, such as Escherichia coli. A number of 
replicates of such membranes are prepared and used to test oligonucleotide 
probes based on the nucleotide regions shown in SEQ ID NO:3. The object of 
the tests is to identify probes that provide the requisite sensitivity and 
selectivity for distinguishing the filamentous bacteria from other known 
bacteria. Oligonucleotide probes are synthesized using a DNA synthesizer 
following the procedures provided by the manufacturer, and labeled with 
either a radioactive or nonradioactive reporter molecule. In choosing 
sequences for candidate oligonucleotide probes, probes are chosen based on 
the prior art and that contain the highest possible number of bases at 
which the sequence for A-1 differs from the organisms shown in Tables 1 
and 2. Using conventional techniques (e.g., see Sambrook et al., 1989), 
probes are radioactively labeled or labeled with a nonradioactive reporter 
molecule that will fluoresce or produce a colored product. Each labeled 
probe is hybridized under stringent conditions with one of the replicate 
filter strips. Probes that hybridize with strain A-1 DNA but not with DNA 
from the other bacteria are deemed to have the requisite specificity for 
differentiating the filamentous bacteria of the subject invention from 
other bacteria. 
The length of the probes is chosen using criteria based on established 
methodology in the prior art. Ten nucleotides is generally the lower limit 
for useful oligonucleotides because shorter probes sometimes cannot form 
stable hybrid duplexes regardless of hybridization conditions. Probes of 
about 20-30 nucleotides in length are preferred, as such probes are far 
more likely to form stable duplexes with their complementary sequences 
and, moreover, are very likely to be disruptable by mismatched bases. 
Sequences of this length are extremely unlikely to occur by chance 
anywhere within a bacterial genome. 
The probes are tested to determine whether each candidate probe provides 
the required specificity, i.e., the ability to form a stable duplex with 
strain A-1 DNA but not with DNA from C. testosteroni, C. acidovorans, or 
B. dentrificans or other non-filamentous bacteria. Stringent hybridization 
conditions are generally understood to mean conditions that permit only 
perfectly matched or nearly perfectly matched hybrids to form. The use of 
such conditions will facilitate identification of probes that hybridize 
specifically with 16S rDNA of the filamentous bacteria. However, the 
stability of duplexes formed with oligonucleotide probes can sometimes be 
disrupted by even a single mismatched base pair (Wallace et al., 1979; 
Wallace et al., 1981). The destabilizing effects on the duplex of 
mismatched bases becomes greater as probe length decreases so that for 
very short probes, for example, those shorter than 20 nucleotides, 
stringent hybridization conditions will rarely tolerate any mismatched 
bases. For longer probes, e.g., those &gt;50 bases long, a small number of 
mismatches may be tolerated even under stringent conditions. However, the 
procedures provided here nonetheless can identify probes having the 
requisite specificity even if such probes form stable duplexes containing 
a small number of mismatched bases. This is because the probe testing 
procedure identifies probes that react with strain A-1 DNA and not with 
the DNA from the other bacteria present on the filter. Probes meeting this 
criterion by definition have the requisite specificity to be used in the 
confirmation assay for testing new isolates of the filamentous bacterium. 
Hence, it is immaterial for the purposes of this assay whether the probes 
chosen for use form perfect or nearly perfect hybrids, as the ability to 
distinguish strain A-1 from the other bacteria on the filter is the only 
pertinent quality required for a test probe useful in the screening assay. 
A variety of useful hybridization conditions are available in the published 
literature (Sambrook et al., 1989). For example, it is known that the 
amount of formamide present in hybridization solutions has a predictable 
effect on the stringency of hybridization reactions. By varying the 
percentage of formamide present, one can vary the percentage of mismatched 
bases that will be tolerated in duplexes that form at a given incubation 
temperature. To determine the optimal hybridization conditions for the 
probes for the present screening assay, a series of hybridization 
reactions is conducted with the replicate filters described above. Each 
filter is hybridized with a different percentage of formamide present in 
the hybridization solution. The optimal hybridization condition for each 
probe is that in which the relative signal strengths between strain A-1 
DNA and the other DNA on the filter shows the greatest difference. Using 
the hybridization conditions that provide the greatest relative signal 
strength differences, or specificity, filters are prepared that contain, 
in addition to strain A-1 and the Comamonas and Brachymonas DNAs, DNAs 
from NL1 and NNL in which the filamentous bacterium predominates. Probes 
that hybridize strongly with DNA from strain A-1, NL1 and NNL but weakly 
or not at all with the other DNAs on the filter are the test probes used 
further in screening field isolates of phenol-degrading bacteria. The best 
probes for this assay are those that hybridize with DNA from the subject 
filamentous bacteria and not at all with DNA from the other bacteria whose 
DNA is present on the filter. Probes that hybridize slightly with DNA 
besides the filamentous bacteria are useful also, provided the signal 
strength with filamentous bacterial DNA is measurably greater than with 
the other DNAs. For example, densitometry can be used to quantify signal 
strength differences. 
In the screening test, the signal obtained by hybridizing the test probe 
with A-1 DNA will be compared with the signal strength obtained when the 
test probe is hybridized with DNA from the new field isolates. The field 
isolate is regarded as being within the scope of the present invention 
when its DNA hybridizes with the test probes to a similar extent as the 
probes hybridize with A-1 DNA. For the screening test, the filters contain 
control DNAs as well as DNA from the new isolates. Control DNAs include C. 
testosteroni, C. acidovorans, B. dentrificans, and E. coli DNAs, and other 
DNAs if desired. Thus, the reagents provided here, when combined with 
conventional methodology, are readily used to develop a rapid screening 
test to verify the identity of any field isolate of a phenol-degrading 
filamentous bacteria. 
Example 9 
Methods for Using the Filamentous Bacterium for Bioremediation 
Water or soil carrying TCE or DCE is aerated, and the TCE is transferred to 
the air stream to which the contaminated water or soil is subjected. The 
TCE-bearing air stream is passed subsequently through a biofilter or 
bioreactor in which the subject microorganisms are present. The biofilter 
or bioreactor is inoculated with a pure culture of strain A-1, or with an 
aliquot of a consortium in which the filamentous bacteria of the subject 
invention is the predominant microorganism. The biofilter or bioreactor is 
supplied with the enrichment medium described in Example 1. 
While the preferred embodiment of the invention has been illustrated and 
described, it will be appreciated that various changes can be made therein 
without departing from the spirit and scope of the invention. 
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21. Olsen, G. J. et al. "Microbial Ecology and Evolution: A Ribosomal RNA 
Approach." Ann. Rev. Microbiol, 40:337-365 (1986). 
22. Gutell, R. R. et al. "Lessons From an Evolving rRNA: 16S and 23S rRNA 
Structures From a Comparative Perspective." Microbiological Reviews, 
58:10-26 (1994). 
23. Olsen, G. J. et al. "The Winds of (evolutionary) Change: Breathing New 
Life Into Microbiology." J Bacteriol., 176:1-6 (1994). 
24. Moncla, B. -J. et al. "Use of Synthetic Oligonucleotide DNA Probes for 
the Identification of Bacteroides gingivalis." J Clin. Microbiol., 
28(2):324-327 (1990). 
25. Sambrook, J., Fritsch, E. F., and Mariates, T. Molecular Cloning, 
Second Ed., Cold Spring Harbor Press (1989). 
26. Dyksterhouse, S. E. et al. "Cycloclasticus pugetii gen. nov., sp. nov., 
an Aromatic Hydrocarbon-Degrading Bacterium from Marine Sediments." Intl. 
J Systematic Bacteriology, 45(1): 116-123 (1995). 
27. U.S. Pat. No. 5,120,652. 
28. Nelson, M. J. K., Montgomery, S. O., Mahaffey, W. R., and Pritchard, P. 
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Biodegradative Pathway." Appl. and Environ. Microbiol., 53:949-954 (1987). 
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__________________________________________________________________________ 
SEQUENCE LISTING 
(1) GENERAL INFORMATION: 
(iii) NUMBER OF SEQUENCES: 10 
(2) INFORMATION FOR SEQ ID NO:1: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 1536 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: nucleic acid 
(A) DESCRIPTION: "16S ribosomal DNA" 
(iii) HYPOTHETICAL: NO 
(iv) ANTI-SENSE: NO 
(vi) ORIGINAL SOURCE: 
(A) ORGANISM: Comamonas testosteroni ATCC No. 11996 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
CGAACTATAGAGTTTGATCCTGGCTCAGATTGAACGCTGGCGGCATGCTTTACACATGCA60 
AGTCGAACGGTAACAGGTCTTCGGATGCTGACGAGTGGCGAACGGGTGAGTAATACATCG120 
GAACGTGCCTAGTAGTGGGGGATAACTACTCGAAAGAGTAGCTAATACCGCATGAGATCT180 
ACGGATGAAAGCAGGGGACCTTCGGGCCTTGTGCTACTAGAGCGGCTGATGGCAGATTAG240 
GTAGTTGGTGGGGTAAAGGCTTACCAAGCCTGCGATCTGTAGCTGGTCTGAGAGGACGAC300 
CAGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATTT360 
TGGACAATGGGCGAAAGCCTGATCCAGCAATGCCGCGTGCAGGATGAAGGCCCTCGGGTT420 
GTAAACTGCTTTTGTACGGAACGAAAAGCCTGGGGCTAATATCCCCGGGTCATGACGGTA480 
CCGTAAGAATAAGCACCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGTGCAA540 
GCGTTAATCGGAATTACTGGGCGTAAAGCGTGCGCAGGCGGTTTTGTAAGACAGTGGTGA600 
AATCCCCGGGCTCAACCTGGGAACTGCCATTGTGACTGCAAGGCTAGAGTGCGGCAGAGG660 
GGGATGGAATTCCGCGTGTAGCAGTGAAATGCGTAGATATGCGGAGGAACACCGATGGCG720 
AAGGCAATCCCCTGGGCCTGCACTGACGCTCATGCACGAAAGCGTGGGGAGCAAACAGGA780 
TTAGATACCCTGGTAGTCCACGCCCTAAACGATGTCAACTGGTTGTTGGGTCTTAACTGA840 
CTCAGTAACGAAGCTAACGCGTGAAGTTGACCGCCTGGGGAGTACGGCCGCAAGGTTGAA900 
ACTCAAAGGAATTGACGGGGACCCGCACAAGCGGTGGATGATGTGGTTTAATTCGATGCA960 
ACGCGAAAAACCTTACCCACCTTTGACATGGCAGGAACTTACCAGAGATGGTTTGGTGCT1020 
CGAAAGAGAACCTGCACACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTT1080 
GGGTTAAGTCCCGCAACGAGCGCAACCCTTGCCATTAGTTGCTACATTCAGTTGAGCACT1140 
CTAATGGGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAGTCCTCATGGC1200 
CCTTATAGGTGGGGCTACACACGTCATACAATGGCTGGTACAAAGGGTTGCCAACCCGCG1260 
AGGGGGAGCTAATCCCATAAAGCCAGTCGTAGTCCGGATCGCAGTCTGCAACTCGACTGC1320 
GTGAAGTCGGAATCGCTAGTAATCGTGGATCAGAATGTCACGGTGAATACGTTCCCGGGT1380 
CTTGTACACACCGCCCGTCACACCATGGGAGCGGGTCTCGCCAGAAGTAGGTAGCCTAAC1440 
CGTAAGGAGGGCGCTTACCACGGCGGGGTTCGTGACTGGGGTGAAGTCGTAACAAGGTAG1500 
CCGTATCGGAAGGTGCGGCTGGATCACCTCCTTTCT1536 
(2) INFORMATION FOR SEQ ID NO:2: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 1452 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: nucleic acid 
(A) DESCRIPTION: "16S ribosomal DNA" 
(iii) HYPOTHETICAL: NO 
(iv) ANTI-SENSE: NO 
(vi) ORIGINAL SOURCE: 
(A) ORGANISM: Brachymonas denitrificans AS-P1 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: 
ATTGAACGCTGGCGGCATGCTTTACACATGCAAGTCGAACGGTAACAGGTCCTTCGGATG60 
CTGACGAGTGGCGAACGGGTGAGTAATGTATCGGAACGTGCCCAGTAGTGGGGGATAACT120 
ACTCGAAAGAGTGGCTAATACCGCATGAGAACTGAGGTTGAAAGCGGGGGACCTTTGGGC180 
CTCGCGCTACTGGAGCGGCCGATATCAGATTAGGTAGTTGGTGGGGTAAAGGCCTACCAA240 
GCCGACGATCTGTAGCTGGTCTGAGAGGACGACCAGCCACACTGGGACTGAGACACGGCC300 
CAGACTCCTACGGGAGGCAGCAGTGGGGAATTTTGGACAATGGACGCAAGTCTGATCCAG360 
CAATGCCGCGTGCAGGACGAAGGCCTTCGGGTTGTAAACTGCTTTTGTACAGAACGAAAA420 
GGCTCTGGTTAATACCTGGGGCTCATGACGGTACTGTAAGAATAAGCACCGGCTAACTAC480 
GTGCCAGCAGCCGCGGTAATACGTAGGGTGCGAGCGTTAATCGGAATTACTGGGCGTAAA540 
GCGTGCGCAGGCGGTTTTGTAAGACCGATGTGAAATCCCCGGGCTCAACCTGGGAACTGC600 
ATTGGTGACTGCAAGGCTGGAGTGCGGCAGAGGGGGATGGAATTCCGCGTGTAGCAGTGA660 
AATGCGTAGATATGCGGAGGAACACCGATGGCGAAGGCAATCCCCTGGGCCTGCACTGAC720 
GCTCATGCACGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCCTA780 
AACGATGTCAACTGGTTGTTGGGTATTTGCTTACTCAGTAACGAAGCTAACGCGTGAAGT840 
TGACCGCCTGGGGAGTACGGCCGCAAGGTTGAAACTCAAAGGAATTGACGGGGACCCGCA900 
CAAGCGGTGGATGATGTGGTTTAATTCGATGCAACGCGAAAAACCTTACCCACCTTTGAC960 
ATGGCAGGAATTCCGAAGAGATTTGGAAGTGCTCGTAAGAGAACCTGCACACAGGTGCTG1020 
CATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACC1080 
CTTGCCATTAGTTGCTACGAAAGGGCACTCTAATGGGACTGCCGGTGACAAACCGGAGGA1140 
AGGTGGGGATGACGTCAAGTCCTCATGGCCCTTATAGGTGGGGCTACACACGTCATACAA1200 
TGGCCGGTACAAAGGGTAGCCAACCCGCGAGGGGGAGCCAATCCCATAAAGCCGGTCGTA1260 
GTCCGGATCGCAGTCTGCAACTCGACTGCGTGAAGTCGGAATCGCTAGTAATCGTGGATC1320 
AGCATGTCACGGTGAATACGTTCCCGGGTCTTGTACACACCGCCCGTCACACCATGGGAG1380 
CGGGTTCTGCCAGAAGTGGTTAGCCTAACCGTAAGGAGGGCGATCACCACGGCAGGGTTC1440 
GTGACTGGGGTG1452 
(2) INFORMATION FOR SEQ ID NO:3: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 1455 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: nucleic acid 
(A) DESCRIPTION: "16S ribosomal DNA" 
(iii) HYPOTHETICAL: NO 
(iv) ANTI-SENSE: NO 
(vi) ORIGINAL SOURCE: 
(A) ORGANISM: Unknown. Possibly new species 
(B) STRAIN: A-1 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: 
ATTGAACGCTGGCGGCATGCTTTACACATGCAAGTCGAACGGCAGCATGGGCTTCGGCCT60 
GATGGCGAGTGGCGAACGGGTGAGTAATACATCGGAACGTGCCTGGTAGTGGGGGATAAC120 
TACTCGAAAGAGTAGCTAATACCGCATGAGATCTACGGATGAAAGCGGGGGATCGCAAGA180 
CCTCGCGCTACCAGAGCGGCTGGTGGCAGATTAGGTAGTTGGTGGGATAAAAGCTTACCA240 
AGCCGACGATCTGTAGCTGGTCTGAGAGGACGACCAGCCCACACTGGGACTGAGACWCGG300 
CCCAGACTCCTACGGGAGGCAGCAGTGGGGAATTTTGGACAATGGGCGCAAGCCTGATCC360 
AGCAATGCCGCGTNGCAGGATGAAGGCCTTCGGGTTGTAAACTGCTTTTGTACGGAACGA420 
AAAGGCTCTCTCTAATACAGAGAGCCGATGACGGTACCGTAAGAATAAGCACCGGCTAAC480 
TACGTGCCAGCAGCCGCGGTAATACGTAGGGTGCAAGCGTTAATCGGAATTACTGGGCGT540 
AAAGCGTGCGCAGGCGGTCTTGTAAGACAGAGGTGAAATCCCCGGGCTCAACCTGGGAAC600 
GGCCTTTGTGACTGCAAGGCTGGAGTGCGGCAGAGGGGGATGGAATTCCGCGTGTAGCAG660 
TGAAATGCGTAGATATGCGGAGGAACACCGATGGCGAAGGCAATCCCCTGGGCCTGCACT720 
GACGCTCATGCACGAAAGCGTGGGGAGCACACAGGATTAGATACCCTGGTAGTCCACGCC780 
CTAAACGATGTCANCTGGTTGTTGGGTCTTCACTGACTCAGTAACGAAGCTAACGCGTGA840 
AGTTGACCGCCTGGGGAGTACGGCCGCAAGGTTGAAACTCAAAGGAATTGACGGGGACCC900 
GCACAAGCGGTGGATGATGTGGTTTAATTCGATGCAACGCGAAAAACCTTACCCACCTTT960 
GACATGGCAGGAATCCTTTAGAGATAGAGGAGTGCTCGAAAGAGAACCTGCACACAGGTG1020 
CTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCA1080 
ACCCTTGCCATTAGTTGCTACGAAAGGGCACTCTAATGGGACTGCCGGTGACAAACCGGA1140 
GGAAGGTGGGGATGACGTCAAGTCCTCATGGCCCTTATAGGTGGGGCTACACACGTCATA1200 
CAATGGCTGGTACAAAGGGTTGCCAACCCGCGAGGGGGAGCCAATCCCATAAAGCCAGTC1260 
GTAGTCCGGATCGCAGTCTGCAACTCGACTGCGTGAAGTCGGAATCGCTAGTAATCGTGG1320 
ATCAGAATGTCACGGTGAATACGTTCCCGGGTCTTGTACACACCGCCCGTCACACCATGG1380 
GAGCGGGTCTCGCCAGAAGTAGGTAGCCTAACCGCAAGGAGGGCGCTTACCACGGCGGGG1440 
TTCGTGACTGGGGTG1455 
(2) INFORMATION FOR SEQ ID NO:4: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 876 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: nucleic acid 
(A) DESCRIPTION: "16S ribosomal DNA" 
(iii) HYPOTHETICAL: NO 
(iv) ANTI-SENSE: NO 
(vi) ORIGINAL SOURCE: 
(A) ORGANISM: Stripa research mine environmental clone. 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: 
GCGGTAATACGTAGGGTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGCGTGCGCAGGC60 
GGTGATGTAAGACAGGCGTGAAATCCCCGGGCTCAACCTGGGAATTGCGCTTGTGACTGC120 
ATCGCTGGAGTGCGGCAGAGGGGGATGGAATTCCGCGTGTAGCAGTGAAATGCGTAGATA180 
TGCGGAGGAACACCGATGGCGAAGGCAATCCCCTGGGCCTGCACTGACGCTCATGCACGA240 
AAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCCTAAACGATGTCAAC300 
TGGTTGTTTGGGTCTCTTTCTGACTCAGTAACGAGCTAACGCGTGAAGTTGACCGCCTGG360 
GGAGTACGGCCGCAAGGTTGAAACTCAAAGGAATTGACGGGGACCCGCACAAGCGGTGGA420 
TGATGTGGTTTAATTCGATGCAACGCGAAAAACCTTACCCACCTTTGACATGTACGGAAT480 
TTGCCAGAGATGGCTTAGTGCTCGAAAGAGAGCCGTAACACAGGTGCTGCATGGCTGTCG540 
TCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGTCATTAG600 
TTGCTACATTCAGTTGGGCACTCTAATGAGACTGCCGGTGACAAGCCGGAGGAAGGTGGG660 
GATGACGTCAAGTCCTCATGGCCCTTATAGGTGGGGCTACACACGTCATACAATGGCCGG720 
TACAAAGGGTCGCAAACCCGCGAGGGGGAGCCAATCCATCAAAGCCGGTCGTAGTCCGGA780 
TCGCAGTCTGCAACTCGACTGCGTGAAGTCGGAATCGCTAGTAATCGTGGATCAGCATGT840 
CACGGTGAATACGTTCCCGGGTCTTGTACACACCGC876 
(2) INFORMATION FOR SEQ ID NO:5: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 348 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: nucleic acid 
(A) DESCRIPTION: "16S ribosomal DNA" 
(iii) HYPOTHETICAL: NO 
(iv) ANTI-SENSE: NO 
(vi) ORIGINAL SOURCE: 
(A) ORGANISM: Comamonas testosteroni ATCC No. 11996 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: 
TACGGAACGAAAAGCCTGGGGCTAATATCCCCGGGTCATGACGGTACCGTAAGAATAAGC60 
ACCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGTGCAAGCGTTAATCGGAAT120 
TACTGGGCGTAAAGCGTGCGCAGGCGGTTTTGTAAGACAGTGGTGAAATCCCCGGGCTCA180 
ACCTGGGAACTGCCATTGTGACTGCAAGGCTAGAGTGCGGCAGAGGGGGATGGAATTCCG240 
CGTGTAGCAGTGAAATGCGTAGATATGCGGAGGAACACCGATGGCGAAGGCAATCCCCTG300 
GGCCTGCACTGACGCTCATGCACGAAAGCGTGGGGAGCAAACAGGATT348 
(2) INFORMATION FOR SEQ ID NO:6: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 263 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: nucleic acid 
(A) DESCRIPTION: "16S ribosomal DNA" 
(iii) HYPOTHETICAL: NO 
(iv) ANTI-SENSE: NO 
(vi) ORIGINAL SOURCE: 
(A) ORGANISM: Unknown. Stripa research mine environmental 
clone. 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: 
GCGGTAATACGTAGGGTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGCGTGCGCAGGC60 
GGTGATGTAAGACAGGCGTGAAATCCCCGGGCTCAACCTGGGAATTGCGCTTGTGACTGC120 
ATCGCTGGAGTGCGGCAGAGGGGGATGGAATTCCGCGTGTAGCAGTGAAATGCGTAGATA180 
TGCGGAGGAACACCGATGGCGAAGGCAATCCCCTGGGCCTGCACTGACGCTCATGCACGA240 
AAGCGTGGGGAGCAAACAGGATT263 
(2) INFORMATION FOR SEQ ID NO:7: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 348 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: nucleic acid 
(A) DESCRIPTION: "16S ribosomal DNA" 
(iii) HYPOTHETICAL: NO 
(iv) ANTI-SENSE: NO 
(vi) ORIGINAL SOURCE: 
(A) ORGANISM: Unknown. Possibly new species. 
(B) STRAIN: A-1 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: 
TACGGAACGAAAAGGCTCTCTCTAATACAGAGAGCCGATGACGGTACCGTAAGAATAAGC60 
ACCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGTGCAAGCGTTAATCGGAAT120 
TACTGGGCGTAAAGCGTGCGCAGGCGGTCTTGTAAGACAGAGGTGAAATCCCCGGGCTCA180 
ACCTGGGAACGGCCTTTGTGACTGCAAGGCTGGAGTGCGGCAGAGGGGGATGGAATTCCG240 
CGTGTAGCAGTGAAATGCGTAGATATGCGGAGGAACACCGATGGCGAAGGCAATCCCCTG300 
GGCCTGCACTGACGCTCATGCACGAAAGCGTGGGGAGCACACAGGATT348 
(2) INFORMATION FOR SEQ ID NO:8: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 348 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: nucleic acid 
(A) DESCRIPTION: "16S ribosomal DNA" 
(iii) HYPOTHETICAL: NO 
(iv) ANTI-SENSE: NO 
(vi) ORIGINAL SOURCE: 
(A) ORGANISM: Brachymonas denitrificans AS-P1 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: 
TACAGAACGAAAAGGCTCTGGTTAATACCTGGGGCTCATGACGGTACTGTAAGAATAAGC60 
ACCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGTGCGAGCGTTAATCGGAAT120 
TACTGGGCGTAAAGCGTGCGCAGGCGGTTTTGTAAGACCGATGTGAAATCCCCGGGCTCA180 
ACCTGGGAACTGCATTGGTGACTGCAAGGCTGGAGTGCGGCAGAGGGGGATGGAATTCCG240 
CGTGTAGCAGTGAAATGCGTAGATATGCGGAGGAACACCGATGGCGAAGGCAATCCCCTG300 
GGCCTGCACTGACGCTCATGCACGAAAGCGTGGGGAGCAAACAGGATT348 
(2) INFORMATION FOR SEQ ID NO:9: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 348 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: nucleic acid 
(A) DESCRIPTION: "16S ribosomal DNA" 
(iii) HYPOTHETICAL: NO 
(iv) ANTI-SENSE: NO 
(vi) ORIGINAL SOURCE: 
(A) ORGANISM: Comamonas terrigena ATCC No. 8461 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: 
TACGGAACGAAAAGCTTCGGGTTAATACCCTGGAGTCATGACGGNACCGTAAGAATAAGC60 
ACCGTNTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGTNCAAGCGTTANTCGGNAT120 
TACTGGGCGTAAAGCGTGCGCAGGCGGTCTTGTAAGACAGAGGTGANNTCCCCGGNCTCA180 
NCCTGGGAACTGCCTNTGTGACTACAAGGCTGGAGTGCGGNAGAGGGGGATCGANTTCCG240 
CGTGTAGCAGTGANATGCGTNGATATGCGGAGGAACACCGATGGCGAAGGCACTCCCCTG300 
GGCCTGCACTGACGCTCATACACGAANGCGTGGGGAGCAAACAGTATT348 
(2) INFORMATION FOR SEQ ID NO:10: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 348 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: nucleic acid 
(A) DESCRIPTION: "16S ribosomal DNA" 
(iii) HYPOTHETICAL: NO 
(iv) ANTI-SENSE: NO 
(vi) ORIGINAL SOURCE: 
(A) ORGANISM: Comamonas acidovorans ATCC No. 15668 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10: 
TACGGAACGAAAANGCTTCTCCTAATACGAGAGGCCCATGACGGCACCGTAAGAATAAGC60 
ACCGTATANCTACGTGCCAGCAGCCGCGGTAATACGTAGGGTGCGAGCGTTACTCGGTAT120 
TACTGGGCGTAAAGCGTGCGCAGGCGGTTATGTAAGACAGATGTGACCTCCCCGGTCTCA180 
NCCTGGGAACTGCATGTGTGACTGCATGGCTAGAGTACGGGAGAGGGGGATCGAATTCCG240 
CGTGTAGCAGTGATATGCGTAGATATGCGGAGGAACACCGATGGCGAAGGCACTCCCCTG300 
GCCCTGTTCTGACGCTCATACACGAAAGCGTGGGGAGCAAACAGTATT348 
__________________________________________________________________________