Hydrogenophaga carboriunda sp. nov., a Tertiary ButylAlcohol-Oxidizing, Psychrotolerant Aerobe Derived fromGranular-Activated Carbon (GAC)

Kimberly M. Reinauer • Jovan Popovic • Christopher D. Weber •

Kayleigh A. Millerick • Man Jae Kwon • Na Wei • Yang Zhang •

Kevin T. Finneran

Received: 28 May 2013 / Accepted: 13 October 2013 / Published online: 17 December 2013

� Springer Science+Business Media New York 2013

Abstract A Gram-negative, rod-shaped bacterium was

isolated from a mixed culture that degraded tert-butyl

alcohol (TBA) in a granular-activated carbon (GAC)

sample from a Biological-GAC reactor. Strain YZ2T was

assigned to the Betaproteobacteria within the family

Comamonadaceae based on 16S rRNA gene similarities.

The nearest phylogenetic relative (95.0 % similarity) with

a valid name was Hydrogenophaga taeniospiralis. The

DNA G?C content was 66.4 mol%. DNA:DNA hybrid-

ization indicated that the level of relatedness to members of

the genus Hydrogenophaga ranged from 1.1 to 10.8 %.

The dominant cellular fatty acids were: 18:1 w7c (75 %),

16:0 (4.9 %), 17:0 (3.85 %), 18:0 (2.93 %), 11 methyl 18:1

w7c (2.69 %), Summed Feature 2 (2.27 %), and 18:0

3OH (1.35 %). The primary substrate used was TBA,

which is a fuel oxygenate and groundwater contaminant.

YZ2T was non-motile, without apparent flagella. It is a

psychrotolerant, facultative aerobe that grew between pH

6.5 and 9.5, and 4 and 30 �C. The culture grew on and

mineralized TBA at 4 �C, which is the first report of psy-

chrotolerant TBA degradation. Hydrogen was used as an

alternative electron donor. The culture also grew well in

defined freshwater medium with ethanol, butanol, hydroxy

isobutyric acid, acetate, pyruvate, citrate, lactate, isopro-

panol, and benzoic acid as electron donors. Nitrate was

reduced with hydrogen as the sole electron donor. On the

basis of morphological, physiological, and chemotaxo-

nomic data, a new species, Hydrogenophaga carboriunda

is proposed, with YZ2T as the type strain.

Introduction

Tert-butyl alcohol (TBA) is an industrial chemical, gaso-

line additive, and a metabolite of the gasoline oxygenate

methyl tert-butyl ether (MTBE) [6, 29]. Currently, there

are a limited number of pure bacterial cultures that degrade

TBA. These cultures are Hydrogenophaga flava ENV735

[8, 28], Mycobacterium austroafricanum IFP2012 [7],

Aquincola tertiaricarbonis L10T and L108 [21], Burk-

holderia cepacia CIP I-2052 [24], and Methylibium pe-

troleiphilum PM1 [23]. Several aerobic mixed cultures

have also been reported [5].

Only one species within the Hydrogenophaga has been

reported to utilize TBA as the sole carbon and energy

source: H. flava ENV735. However, the Hydrogenophaga

are a diverse genus with organisms that degrade several

xenobiotic compounds [17, 19, 29]. Based on the 16S

rRNA gene phylogenetic placement, alternate chemotaxo-

nomic data, morphological characteristics, and unique

physiological data, a new species Hydrogenophaga car-

boriunda is proposed here with YZ2T as the type strain.

Electronic supplementary material The online version of thisarticle (doi:10.1007/s00284-013-0501-8) contains supplementarymaterial, which is available to authorized users.

K. M. Reinauer

Hart Crowser and Associates, Seattle, WA, USA

K. M. Reinauer � K. A. Millerick � N. Wei � Y. Zhang

Civil and Environmental Engineering, University of Illinois,

205 North Mathews Avenue, Urbana, IL 61801, USA

J. Popovic � C. D. Weber � K. A. Millerick � K. T. Finneran (&)

Environmental Engineering and Earth Sciences, Clemson

University, 312 Biosystems Research Complex, 105 Collings

Street, Clemson, SC 29634, USA

e-mail: ktf@clemson.edu

M. J. Kwon

Korea Institute of Science and Technology (KIST),

Gangneung 210-340, South Korea

123

Curr Microbiol (2014) 68:510–517

DOI 10.1007/s00284-013-0501-8

Materials and Methods

Isolation of Strain YZ2

Strain YZ2 was isolated from a sample of granular-acti-

vated carbon (GAC) that was used in an aboveground GAC

unit treating groundwater at a petroleum contaminated

former refinery facility in Fountain Valley, California. The

sample was collected in September 2005, and the culture

was isolated from a TBA-grown enrichment culture that

was previously reported [25].

Isolates were maintained on modified Wolfe’s fresh water

(FW) medium, containing (per liter unless otherwise stated)

2.5 g NaHCO3, 0.25 g NH4Cl, 0.6 g NaH2PO4�H2O, 0.1 g

KCl, 20 lg biotin, 20 lg folic acid, 100 lg pyridoxine HCl,

50 lg riboflavin, 50 lg thiamine, 50 lg nicotinic acid,

50 lg pantothenic acid, 1 lg vitamin B-12, 50 lg p-ami-

nobenzoic acid, 50 lg thioctic acid, 15 mg NTA, 30 mg

MgSO4, 5 mg MnSO4�H2O, 10 mg NaCl, 1 mg FeSO4�7H2O, 1 mg CaCl2�2H2O, 1 mg CoCl2�6H2O, 1.3 mg

ZnCl2, 100 lg CuSO4�5H2O, 100 lg AlK(SO4)2•12H2O,

100 lg H3BO3, 250 lg Na2MoO4, 240 lg NiCl�6H2O,

250 lg Na2WO4�2H2O, and 189 lg Na2SeO4. 2 mM TBA

(grade 99.3 %, Sigma-Aldrich Inc.) was added as the sole

carbon source. The culture was grown in either 125 ml

conical flasks (50 ml culture) with mini-Nert screw tops or

with 25 ml tubes (10 ml culture) with aerobic caps. Bottles

and tubes were placed on a rotary shaker (150 RPM) bed at

30 �C in the dark. TBA degradation was confirmed by

monitoring the concentration of TBA using gas chroma-

tography equipped with mass spectrometry (GC–MS; Var-

ian 4000, Varian Inc.). TBA mineralization was determined

by measuring 14CO2 production using uniformly labeled

[14C]-TBA (4 mCi/mmol, Moravek Biochemicals) by GC-

radiochromatography (GC-GPC; IN/US, System Inc.).

Alternatively, the culture was also grown in LB medium

during the characterization process or FW medium with

hydrogen as the sole electron donor.

Phylogenetic Analysis of the Complete 16S rRNA

Gene

The complete 16S rRNA gene (bases 27–1492 using the

E. coli numbering [9] of strain YZ2T) was used to establish

phylogenetic placement. DNA was extracted using the Fast

DNA extraction kit (MP Biomedicals, LLC) with a bead-

beating apparatus according to the manufacturer’s instruc-

tions. The 16S rRNA gene was amplified with primers

Eub27F [20] and Eub1492R [26] with an initial denaturation

step at 94 �C for 4 min, followed by 35 cycles of 94 �C (30 s),

50 �C (30 s), and 72 �C (1 min 30 s) with a final extension at

72 �C for 7 min, and holding at 4 �C. The sequence was

purified by QiaQuick PCR purification kit (QIAGEN) and

sequenced at the University of Illinois Core Sequencing

Center. The GenBank Accession Number for the YZ2T full

16S rRNA gene sequence is EU095331. G?C content,

DNA:DNA hybridization, and total cellular fatty acid content

were performed by the Deutsche Sammlung von Mikroor-

ganismen (DSMZ) characterization services using their

standard methods [13, 15, 22].

The initial percent of identity was determined using the

National Center for Biotechnology Information (NCBI)

Basic local alignment search tool (BLAST), which com-

pares the sequence to the Genbank database. A phyloge-

netic tree was constructed for the dominant clone in TBA-

degrading microcosms using MEGA4 software. Multiple

sequence alignment was conducted using the ClustalW

algorithm in MEGA4. The tree was obtained using the

Neighbor-Joining method and genetic distances were esti-

mated by using the maximum composite likelihood method

estimated by the Tamura–Nei model. The confidence of

phylogeny was tested by bootstrap re-sampling for 1,000

replicates. Reference microorganisms selected were closely

related Hydrogenophaga species.

DNA:DNA Hybridization

Cells were grown in modified Wolfe’s freshwater medium

with TBA as the sole carbon and energy source for

DNA:DNA hybridization analysis. Total genomic DNA

was extracted as described above. DNA was submitted to

the DSMZ for DNA:DNA hybridization using their stan-

dard protocols. The Hydrogenophaga included in the

DNA:DNA hybridization were Hydrogenophaga defluvii,

H. atypica [14], and H. flava.

Fatty Acid Methyl Ester Analyses

Cells were grown in modified Wolfe’s FW medium with

TBA as the sole carbon and energy source for total cellular

fatty acid analysis. Cells were submitted to the DSMZ for

fatty acid analysis using their published protocols [13, 15,

22].

Morphological Characterization

Gram staining was performed according to a standard

procedure [11]. Cell morphology and motility were

investigated with phase contrast microscope (Zeiss Axio-

skop, Zeiss Inc.) and scanning electron microscope (Philips

XL30 ESEM-FEG, FEI Company) at 5 kV with cells

harvested from a log phase culture. SEM photos were

produced by the University of Illinois core microscopy

facility.

K. M. Reinauer et al.: Hydrogenophaga carboriunda sp. nov. 511

123

Physiological and Metabolic Properties

Microbial growth with cell counting was quantified by

inoculating 0.5 ml of TBA-grown YZ2T into 9.5 ml FW

media, with 2 mM TBA as the sole carbon and energy

source. The associated control was the same transfer

without TBA amendment. GC–MS was used to monitor the

concentration of TBA. A 1–100 ll culture aliquot was

sampled every 6–12 h, and diluted in a final volume of

1 ml for cell counting. 100 ll of 25 % glutaraldehyde was

used to fix the cells, and 4 ll of 0.1 g l-1 acridine orange

was applied later for cell staining [10]. The samples were

vacuum filtered through a nucleopore filter (0.22 lm, SPI

Inc.) underlain by a membrane filter (0.22 lm, Millipore

Inc.). The nucleopore filter was then placed on a micro-

scope slide and covered with a coverslip and counted using

an epifluorescence microscope (Zeiss Axioskop, Zeiss

Inc.). Seven areas were counted for each sample and the

average number was recorded for all grid locations.

YZ2T growth was tested at various temperatures (4, 18,

30, 37, and 60 �C) and pH values (6.0–10.5 in 0.5 pH unit

increments) in FW media with TBA as the sole carbon and

energy source. Additional carbon (and electron donor)

sources tested included glycerol, erythritol, D-arabinose,

L-arabinose, D-ribose, D-xylose, L-xylose, D-adonitol,

methyl-b d-xylopyranoside, D-galactose, D-glucose, D-

fructose, D-mannose, L-sorbose, L-rhamnose, dulcitol,

inositol, D-mannitol, D-sorbitol, methyl-a d-mannopyran-

oside, methyl-a d-glucopyranoside, n-acetylglucosamine,

amygdalin, arbutin, esculin ferric citrate, salicin, D-cello-

biose, D-maltose, D-lactose (bovine origin), D-melibiose, D-

saccharose (sucrose), D-trehalose, inulin, D-melezitose, D-

raffinose, amidon (starch), glycogen, xylitol, gentiobiose,

D-turanose, D-lyxose, D-tagatose, D-fucose, L-fucose, D-

arabitol, L-arabitol, potassium gluconate, potassium 2-ke-

togluconate, and potassium 5-ketogluconate using the api�

50CH Carbohydrate Kit (bioMerieux, Inc.). To test growth,

0.15 mL of actively growing culture in freshwater medium

was distributed to each cupule within the kit. The culture

and each substrate were mixed, transferred from the cupule

to a 96-well plate, and incubated at 30 �C. MTBE, ben-

zene, toluene, butanol, HIBA, ethanol, methanol, formal-

dehyde, formate, acetate, pyruvate, acetone, citrate, lactate,

isopropanol, and benzoic acid were also tested as electron

donors. Anaerobic growth was tested at 30 �C in FW

media, with carbon dioxide as the carbon source, and

hydrogen as the energy source. An H2/N2/CO2 mixture

(60:30:10) was applied in the headspace, within blue rub-

ber stopper-sealed 25-ml tubes. 10 mM nitrate, nitrite, or

fumarate was added (respectively) as the electron accep-

tors. The same H2/N2/CO2 mixture was also applied with

Fe(III)–citrate FW media [16] to test the usage of Fe(III) as

the electron acceptor. 100 mg l-1 penicillin, streptomycin,

and tetracycline were applied to determine if they inhibited

the growth of YZ2T in TBA–FW media. Growth tests were

performed either in triplicate (for temperature values, pH

values, and antibiotics tests), in a 96-well plate (for the

api� 50CH Carbohydrate Kit), or in single tube (for addi-

tional alternative electron donors and acceptors) from 70 h

to 2 weeks. Growth (in any experiment) was monitored by

measuring the turbidity (optical density) by spectropho-

tometer at 600 nm (GENESYSTM2, Thermo Spectronic

Inc.). To measure growth using the substrates provided in

the api� 50CH Carbohydrate Kit, a spectrophotometer with

a microplate drawer (SpectraMax Plus 384, Molecular

Devices) was used to measure turbidity at 600 nm.

An oxidase test was performed using an oxidase kit

(Biochemika, Sigma-Aldrich Inc.). A catalase test was

performed by placing a single colony of YZ2T into a 3 %

hydrogen peroxide solution.

Results and Discussion

Strain YZ2T completely mineralizes TBA to CO2 and

conserves energy for growth by this metabolism, which has

been previously reported [25]. The culture temporarily lost

the ability to degrade TBA when grown in LB media, or

freshwater (minimal) medium plus H2. It is possible that

genes required for TBA degradation require continuous

TBA to be induced; although this was not directly tested.

TBA degradation was recovered after a lag of 7–10 days

by providing TBA as the sole substrate. Growth curves of

YZ2T indicated that 97 % of the TBA was degraded within

68 h (Supplementary Fig. 1), with a small amount of ace-

tone detected; however, acetone alone did not support

growth (Supplementary Table 1). Previously reported data

demonstrate that YZ2 mineralized 80 % of U-[14C]-TBA

to 14CO2 [25]. While a full degradation pathway was not

elucidated during this characterization, it is possible that all

TBA carbon would eventually be recovered between the

biomass and mineralization fractions.

Different substrates were tested as the sole carbon and

energy source for growth, and data indicate some overlapping

intermediates when compared to the previously reported

pathway for TBA degradation [27], primarily that strain YZ2

grew with hydroxyisobutyric acid (HIBA) as the sole sub-

strate (Supplementary Table 1). Strain YZ2 did not grow on

acetone, suggesting an alternative pathway for TBA miner-

alization relative to ENV735. The inability to use MTBE as a

carbon or energy source is relatively unique among organisms

that degrade TBA. TBA typically arises in aquifers as a result

of MTBE biodegradation, and very often it is the same aerobic

microorganism catalyzing the initial step (MTBE ? TBA)

and subsequent transformations. Only B. cepacia is an

512 K. M. Reinauer et al.: Hydrogenophaga carboriunda sp. nov.

123

obligate TBA degrader similar to YZ2T. Supplementary

Table 1 summarizes the similarities and differences among

the nearest TBA-degrading strains.

Cells stained Gram-negative and were curved rods; this

agrees with the majority of Hydrogenophaga, which stain

Gram-negative. The cells were approximately 0.8–2 lm long

and 0.3–0.5 lm in diameter (Fig. 1). Flagella were not

identified. YZ2T was oxidase and catalase positive. The G?C

content was 66.4 mol%. Growth was completely inhibited by

100 mg l-1 streptomycin and tetracycline. YZ2T had an 80-h

lag phase with 100 mg l-1 penicillin prior to the onset of

growth. YZ2T grew best at pH 6.0 and 6.5, but growth was

quantifiable between pH 6.0 and 9.5. Table 1 summarizes the

metabolic, morphological, and physiological attributes of

Fig. 1 Scanning electron microscopy (SEM) image of strain YZ2.

Cells were approximately 0.8–2 lm long and 0.3–0.5 lm in diameter.

No flagella were identified. Cells in the center of the micrograph are

seen mid cell division. The bar is 500 nm

Table 1 Differential phenotypic characteristics between H. carboriunda strain YZ2 and recognized species within the genus Hydrogenophaga

Species name H.

carboriunda

strain YZ2T

H. canei H.

defluvii

H.

atypica

H.

intermedia

H.

flava

H.

pseudoflava

H.

taeniospiralis

H.

palleronii

H.

bisanensis

Autotrophic

Growth on

H2

? – ? – – ? ? ? ? –

Nitrate

reduction

? ? ? ? ? ? ? ? – ?

Oxidase ? ? ? ? ? ? ? ? ? ?

Catalase ? ? ? ? ? – – – NA1 ?

Maximum/

minimum

growth

temperature

(�C)

30/4 35/15 37/28 38/28 20/20 NA 37/2 30/min not

available

NA 46/15

Mol% G?G 66.4 61.6 65.0 64.0 68.6 67.0 66.0 65.0 67.0 64.8

Motile No Yes Yes Yes Yes Yes Yes Yes Yes Yes

Flagella

visible

No 1 1 or 2 1 or 2 1 or 2 1 or 2 1 or 2 1 or 2 1 or 2 NA

Shape Rod Rod Rod Rod Rod Rod to

cocci

Rod Rod Rod to

bean

shape

Rod

1 Not available or not reported

Taxa include: H. caeni [2], H. defluvii [14], H. atypica [14], H. intermedia [4], H. flava [8, 28, 31], H. pseudoflava [31], H. taeniospiralis [18, 19,

31], H. palleronii [31], and H. bisanensis [32]

Fig. 2 Growth curves at varying temperatures for strain YZ2 with

tert-butyl alcohol (TBA) as the sole carbon and energy source.

Results are the means of triplicate analyses; bars indicate one

standard deviation

K. M. Reinauer et al.: Hydrogenophaga carboriunda sp. nov. 513

123

strain YZ2 versus the most closely related members of the

genus Hydrogenophaga.

Strain YZ2 grew well between 4 and 30 �C; the culture

did not grow at 37 �C or above (Table 1). The optimal

growth temperature of cells tested with TBA as the sole

carbon and energy source was 30 �C, with maximal bio-

mass by 180 h and a doubling time of 33 h. However, the

most unique physiological attribute for this culture is

psychrotolerant growth (4 �C) with TBA as the sole carbon

and energy source (Fig. 2); it also grew well at 18 �C.

Doubling times were much longer, 103 and 136 h at 4 and

18 �C, respectively, and biomass did not plateau until

closer to 400 h. However, only one other Hydrogenophaga

species (H. pseudoflava) is reported to grow as low as 4 �C,

and this is the first report of a pure culture degrading TBA

at 4 �C. These combined data make it unique within this

genus. While evidence using aquifer material suggests

psychrotolerant MTBE biodegradation, it was never con-

firmed with liquid cultures developed from the aquifer

material [1]. This opens the possibility for using YZ2T in

specific bioaugmentation applications for ex situ treatment

in cold groundwater environments.

The nearest phylogenetic relative for which a cultured

isolate is available was Hydrogenophaga taeniospiralis

(ATCC 49743) with 95 % identity based on 16S rRNA gene

sequence similarity. Other related Hydrogenophaga species

included Hydrogenophaga palleronii (ATCC 17728), Hy-

drogenophaga pseudoflava (ATCC 33668), Hydrogenoph-

aga sp. YED6-4 (ATCC BAA-304), H. flava (CCUG 1658),

Hydrogenophaga sp. TRS-05, and H. flava DSM 619 (ATCC

33667), all with approximately 94–95 % identity. Within

those sequences, H. flava DSM619 is a TBA degrader. Other

Hydrogenophaga included in the phylogenetic analysis

(Fig. 3, based on 16S rRNA gene sequence) were H. defluvii,

H. atypica [14], H. intermedia [4], H. flava, H. pseudoflava, H.

taeniospiralis, H. caeni [2], H. bisanensis [32], and H. pal-

leronii [31].

The data demonstrated that the dominant fatty acids were:

18:1 w7c (75 %), 16:0 (4.9 %), 17:0 (3.85 %), 18:0 (2.93 %),

11 methyl 18:1 w7c (2.69 %), Summed Feature 2 (2.27 %),

Fig. 3 Phylogenetic analysis

using the maximum likelihood

method. The evolutionary

history was inferred by using

the maximum likelihood

method based on the Kimura

2-parameter model. The

percentage of trees in which the

associated taxa clustered

together is shown next to the

branches. Initial tree(s) for the

heuristic search were obtained

automatically by applying

Neighbor-Join and BioNJ

algorithms to a matrix of

pairwise distances estimated

using the maximum composite

likelihood (MCL) approach, and

then selecting the topology with

superior log likelihood value.

The tree is drawn to scale, with

branch lengths measured in the

number of substitutions per site.

The analysis involved 27

nucleotide sequences. All

positions containing gaps and

missing data were eliminated.

Evolutionary analyses were

conducted in MEGA5. The bar

represents 0.01 substitutions per

nucleotide position

514 K. M. Reinauer et al.: Hydrogenophaga carboriunda sp. nov.

123

unknown peak at 18.814 (2.03 %), and 18:0 3OH (1.35 %).

Other fatty acids were present at 1 % or less for a total percent

of 99.15 %. Summed Feature 3 was present at 0.52 %. These

fatty acids have been identified previously within the genus

Hydrogenophaga [3, 32, 33]. Fatty acids 16:0, 18:1 w7c, and

Summed Feature 3 (with 16:1 w7c as the dominant fatty acid)

are characteristic for members of this genus [12, 30]. The

hydroxylated fatty acids 8:0 3-OH and 9:0 3-OH were not

identified, but this lack of this characteristic hydroxylated fatty

acids has been reported previously [14, 32, 33].

The G?C content of YZ2T was 66.4 mol%, which is

similar to all reported Hydrogenophaga strains. However,

YZ2T exhibited differences with respect to substrate utili-

zation, temperature maximum, and motility. DNA:DNA

hybridization between YZ2T and selected and available

strains demonstrated limited relatedness to H. defluvii

(1.1 %), H. atypica (8.0 %), and H. flava (10.8 %); this

indicates that YZ2T is distinct from each of those species.

The nearest relative based on 16S rRNA gene analysis (H.

taeniospiralis) was not available for DNA:DNA hybridiza-

tion, nor was H. intermedia due to culturing difficulties for

these type strains. Taken with alternate chemotaxonomic

data plus physiological and metabolic data, these hybrid-

ization data are consistent with strain YZ2T being proposed

as a new species within the genus Hydrogenophaga.

YZ2T was also compared to all other major MTBE/TBA-

degrading microorganisms. Supplementary Table 1 summa-

rizes the similarities and differences among these strains.

Gram stain results varied among the strains; YZ2T stained

negative as did all others with the exception of IFP2012. YZ2T

was not motile; PM1, L10/L108, and CIP I-2052 are motile

each with a single polar flagellum. YZ2T degraded TBA but

did not degrade MTBE, which is only characteristic of B.

cepacia CIP I-2052; all others degraded both MTBE and

TBA. YZ2T did not grow on formate, while PM1, IFP2012,

and CIP I-2052 all grew on formate. Methanol was not used by

YZ2T, demonstrating that YZ2T is not a methylotroph; PM1

and CIP I-2052 both can grow with methanol as the sole

carbon and energy source. Nitrate was reduced by YZ2T,

while L10/L108 cannot use it. Strain PM1 will reduce nitrate

to nitrite with H2 as the sole electron donor [23].

In summary, YZ2T is a tertiary butyl alcohol-oxidizing

microorganism, and TBA is used as the sole carbon and energy

source. This substrate utilization ability alone places the isolate

within a group that has a limited number of known microor-

ganisms. However, its alternate physiological characteristics

taken with the chemotaxonomic data also warrant its designa-

tion as a new species. It grows at 4 �C, which makes YZ2T the

second reported psychrotolerant Hydrogenophaga and the first

reported psychrotolerant TBA degrader. Based on its 95 %

similarity to several Hydrogenophaga as well as the chemo-

taxonomic and physiological attributes described above (e.g.,

H2 oxidation, fatty acid composition, mol% G?C, and substrate

use patterns), this isolate is proposed as a new species, H.

carboriunda, within the genus Hydrogenophaga.

Description of Hydrogenophaga carboriunda sp. nov

Hydrogenophaga carboriunda (carbo, L. n. carbon; L. fem.

adj. oriunda, arising from; N. L. adj. carboriunda, arising

from carbon, to signify that the microorganism was isolated

from GAC).

Cells are non-motile, Gram-negative, short rods of

approximately 2 lm in length by 0.5 lm in diameter. Col-

onies on solid medium are smooth, opaque to clear, rounded,

and flat with a diameter of approximately 1 mm. The cells

grow well in rich LB medium and minimal freshwater

medium with defined electron donors under aerobic condi-

tions. The culture is a facultative anaerobe that will utilize

nitrate as an anaerobic electron acceptor with H2 as the sole

electron donor; growth with nitrate plus H2 is chemoauto-

trophic. Growth with the primary carbon substrate, TBA, is

slow with a doubling time of approximately 5 h and a total

cell yield of 59108 cells ml-1. In addition to TBA, YZ2T

utilizes ethanol, butanol, hydroxy isobutyric acid, acetate,

pyruvate, citrate, lactate, isopropanol, benzoic acid, glyc-

erol, erythritol, D-arabinose, L-arabinose, D-ribose, D-xylose, L-

xylose, D-adonitol, D-galactose, D-glucose, D-fructose, D-man-

nose, L-sorbose, L-rhamnose, n-acetylglucosamine, arbutin,

esculin with ferric citrate, salicin, D-cellobiose, D-maltose, D-

lactose (bovine origin), D-melibiose, D-saccharose (sucrose),

D-trehalose, amidon (starch), xylitol, gentiobiose, D-lyxose,

D-tagatose, D-fucose, and L-fucose. MTBE, benzene, toluene,

methanol, formaldehyde, formate, acetone, methyl-b D-

xylopyranoside, dulcitol, inositol, D-mannitol, D-sorbitol,

methyl-a D-mannopyranoside, methyl-a D-glucopyranoside,

amygdalin, inulin, D-melezitose, D-raffinose, glycogen, D-

turanose, D-arabitol, L-arabitol, potassium gluconate, potas-

sium 2-ketogluconate, and potassium 5-ketogluconate did

not support growth. Fumarate, nitrite, and Fe(III) were not

used as electron acceptors. Cells grew well and degraded

TBA between 4 and 30 �C. Cells grew within a pH range of

6.0–9.5; however, the growth rate was fastest between 6.0

and 6.5. Cells were oxidase and catalase positive. The G?C

content was 66.4 mol%. The major cellular fatty acid were

18:1 w7c (75 %), 16:0 (4.9 %), 17:0 (3.85 %), and 18:0

(2.93 %). The type strain YZ2T for this culture was origi-

nally isolated from GAC used to treat TBA. Strain YZ2T is

the type strain and has been submitted to the American Type

Culture Collection (ATCC; Deposition Number BAA-1569)

and the Deutsche Sammlung von Mikroorganismen (DSMZ;

Deposition Number Pending).

Acknowledgments This work was funded by British Petroleum, an

Atlantic Richfield Company (BP/ARCO); we thank Xiaomin Yang of

K. M. Reinauer et al.: Hydrogenophaga carboriunda sp. nov. 515

123

BP/ARCO for providing the GAC samples. We thank Rachel Whi-

taker and Angela Kent of the University of Illinois (Microbiology and

Natural Resources, respectively) for suggestions regarding phyloge-

netic analyses. We also thank Cate Wallace of the Imaging Tech-

nology Group of Beckman Institute for Advanced Science and

Technology at the University of Illinois and Lou Ann Miller of the

University of Illinois for SEM microscopy and cell culture prepara-

tion for SEM.

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