BIOTROPIA NO

BIOTROPIA NO. 17,2001 : 9 - 17

CHARACTERIZATION OF THREE BENZOATE DEGRADING
ANOXYGENIC PHOTOSYNTHETIC BACTERIA ISOLATED FROM THE

ENVIRONMENT

DWI SURYANTO1, ANTONIUS SUWANTO2'3*, and ANJA MERYANDINI3

; Dept. of Biology, Faculty of Science and Mathematics, North Sumatra University,
Medan, Indonesia 2 South East Asian Regional Center

for Tropical Biology (SEAMEO-BIOTROP),
Bogor, Indonesia

3 Dept. of Biology, Faculty of Science and Mathematics, Bogor Agricultural University,
Bogor, Indonesia

ABSTRACT

Three anoxygenic photosynthetic bacteria, DS-1, DS-4 and Cas-13, have been examinated for their
morphological and physiological properties. All strains were rod-shape cells with a swollen terminal end,
Gram negative, motile, non-halophilic, non-alkalophilic and non-acidophilic, and capable of utilizing
benzoate aerobically and photo-anaerobically. Sequence analysis of part of 16S rRNA genes showed that DS-
1 and Cas-13 were closely related to Rhodopseudomonas palustris Strain 7 with a similarity of 97%, whereas
DS-4 may not be closely related to the former two strains with a similarity of 78% based on the constructed
phylogenic tree. Spectral analysis indicated that the three bacteria had bacteriochlorophyl a and normal
spirilloxanthin series.

Growth in medium enriched with vitamin and supplemented with benzoate as their sole C-sources was
better than in medium without vitamin. Benzoate degradation in medium with vitamin was accelerated. The
ability to grow on benzoate without added vitamins indicated that the bacteria were able to synthesize their
own vitamins.

Key words: anoxygenic photosynthetic bacteria/ benzoate degradation/ 16S rRNA gene.

INTRODUCTION

Some toxic compounds are slowly degraded in polluted aerobic zones and,
therefore, may leach into anaerobic subsurface environments (Kohring et al. 1989).
Anaerobic degradation of such compounds may play an important role in
eliminating toxic substances. Anaerobic bioremediation has been proposed as an
inexpensive method for in situ removal of organic contaminants in the environment
(Kuo and Genthner 1996). Recent concern about the environmental fate of
industrially produced organic compounds has prompted a resurgence of interest in the
anaerobic degradation of aromatic compounds (Harwood and Gibson 1988).

Anoxygenic photosynthetic bacteria (APB) are nutritionally versatile in their
ability to utilize diverse sources of carbon ranging from simple aliphatic organic
acids to complex polysaccharides (Hiraishi et al. 1995). The occurrence of these
bacteria as common inhabitants in aquatic and some terrestrial habitats in nature

' Corresponding author: e-mail address : asuwanto@indo.net.id

BIOTROPIA NO. 17, 2001

(Hiraishi et al. 1995) might be explained partly by the fact that these bacteria
survive on various modes of energy-generating systems (Hiraishi et al. 1995). The
ability of phototrophic purple non-sulfur bacteria to grow aerobically and
anaerobically in the presence of diverse aromatic compounds makes these good
candidates for potential biodegradation of harmful compounds (Hanvood and Gibson
1988; Wright and Madigan 1991; Shoreit and Shaheb 1994). Commercialization of
these bacteria for purification treatment plants was initiated about 15 years ago
(Kobayashi and Kobayashi 1995).

Not many phototrophic purple non-sulfur bacteria species are known to degrade
aromatic compounds. Rhodopseudomonas palustris (Harwood and Gibson 1988;
Gibson and Gibson 1992; Shoreit and Shaheb 1994), Rhodomicrobium vannielii
(Wright and Madigan 1991), Rhodobacter capsulatus (Blasco and Castillo 1992;
Shoreit and Shaheb 1994), Rs. blastica, and Rhodospirilium rubrum (Shoreit and
Shaheb 1994) are able to degrade a variety of monocyclic aromatic compounds with or
without other C-sources.

Benzoate and its derivatives are among the common aromatic compounds that can be
completely mineralized (Harwood and Gibson 1988; Wright and Madigan 1991;
Shoreit and Shaheb 1994). However, Blasco and Castillo (1992) noted that
Rhodobacter capsulatus E1F1 is able to degrade mononitrophenol and dinitrophenol
with acetate as its carbon source. Rs. palustris utilized several phenolic compounds,
hydroxylated and methoxylated aromatic acids, aromatic aldehydes, and hydroaromatic
acids (Harwood and Gibson 1988). Other diverse aromatic compounds have also been
reported to be utilized by this group (Harwood and Gibson 1988; Wright and Madigan
1991; Shoreit and Shaheb 1994).

Degradation of aromatic compound by phototrophic purple non-sulfur bacteria in
general was emphasized in this investigation based on their ability to utilize
diverse aromatic compounds. To date, this group of bacteria has not been subjected to
intensive examination (Harwood and Gibson 1988). This study focused on the ability
of three new isolates of APB to utilize benzoate. Identification of the bacterial isolates
based on their 16S-rRNA genes as well as their morphological and physiological
properties was also carried out.

MATERIALS AND METHODS

Bacterial cultures and cultivation

Three isolates of APB, designated as DS-1, DS-4, and Cas-13 were studied. The
two former strains were isolated from Java, and the last was isolated from
Moluccas. All isolates were maintained on modified Sistrom medium with benzoate as
the sole carbon source.

To determine the ability of the bacteria to utilize and degrade aromatic
compounds, the isolates were grown in modified Sistrom by omitting all carbon
sources, including nitrilo-triacetic acid, supplemented with 5 mM benzoate as the C-

Characterization of three benzoate degrading anoxygenic photosynthetic bacteria -Dwi Suryanto et al.

source with or without vitamins in 100 ml completely filled screw-capped tubes.
Isolates also were grown in modified Sistrom supplemented with vitamins and 5
mM succinate, or 5 mM acetate as their carbon sources to serve as a means of
comparison.

For cultures grown on benzoate, the inocula were obtained from 3-day old
cultures of bacteria grown in modified Sistrom with benzoate with an initial cell
number of 5xl06 cells/ml. For cultures to test other C-sources, inocula were taken
from 2-day old cultures grown in Sistrom media with succinate as C-source. Unless
mentioned otherwise, all media were adjusted to pH 7.2.

Growth condition, measurement of growth, and quantitation of benzoate

Cultures were illuminated with a 40 W tungsten bulb at a distance of 30 cm.
Growth rates were determined by measuring turbidity at 660 nm every 24 hours. For
cultures grown in 5 mM succinate and 5 mM acetate, growth rates were measured
every 12 hours. For cultures grown in benzoate, residual benzoate levels were
measured at 120 hours of incubation. Benzoate concentration was measured at 276 nm
using a Hitachi Model U-2010 UV/Vis spectrophotometer (Hitachi Instrument, Inc.
Japan) according to Shoreit and Shabeb (1994). Cell density of the isolates grown in
other C-sources was measured after 72 hours of inoculation.

Spectral analysis

Cells were harvested from 7-day old anaerob-phototrophic cultures grown on
modified Sistrom supplemented with vitamins and 5 mM succinate as sole carbon
source, and suspended in ICM buffer (10 mM phosphate buffer pH 7.0 and 1 mM Na-
EDTA pH 7.0). Sonication was carried out using Soniprep 150 (MSB, UK) at an
amplitude of 2 u. for 2 minutes, three times with a time interval of 1 minute. Cell
extracts were centrifuged at 3000 rpm for 30 minutes at 4°C. Protein concentration was
determined according to the Pierce BCA* Protein Assay Kit (Rockford, III, USA).
Spectral analysis was performed using Hitachi U-2010 in protein concentration of ± 100
(ig/ml.

Amplification and sequencing of part of 16S rRNA genes

To sequence part of the 16S-rRNA genes, the 16S-rRNA genes were amplified by
PCR using specific primers of 63f and 1387r from genomic DNA (200 ng) on Ready-
To-GO PCR Beads (Pharmacia-Biotech, Uppsala, Sweden). Phenol-chloroform-
isoamylalcohol (25:24:1) treatment, ethanol precipitation, and agarose gel
electrophoresis were used to purify the genomic DNA. Total volume of the PCR reaction
(25 ul) consisted of 1.5 U Tag DNA Polymerase, lOmM Tris-HCl (pH 9 at room
temperature), 50 mM KC1, 1.5 mM MgCl2, 200 uM of each dNTPs and stabilizer
including BSA. The reaction was incubated in a Gene Amp PCR System 2400
Thermocycler (Perkin-Elmer Cetus, Norwalk, Conn.).

BIOTROPIA NO. 17, 2001

Part of 16S-rRNA gene was sequenced to infer the closest related organism
from the Ribosomal Database Project (RDP) maintained at the University of Illinois,
Urbana-Champaign. The sequencing reactions were done by using the Big Dye
Ready Reaction Dye Deoxy Terminator kit, purified by ethanol-sodium acetate
precipitation. The reactions were run on an ABI PRISM 377 DNA Sequencer (PE
Applied Biosystems, Foster City, CA.).

Construction of phylogenetic trees

For the construction of phylogenetic trees, cluster analysis of 16S-rRNA gene
was done by a computer program from the European Bioinformatics Institute
(http://www.ebi.ac.uk). The Treecon computer program (Yves Van de Peer of the
Department of Biochemistry, University of Antwerp) was used to determine their
phylogenetic relatedness based on their nucleotide sequences and MFLP profiles
obtained from pulsed-field gel electrophoresis.

RESULTS AND DISCUSSION

The three new isolates described in this study were similar in their morpho-
logical properties. All were Gram negative, non-halophilic, non-alkali or acido-
philic, aerobic and anaerob-phototrophs that have motile rod-shaped cells with
terminal swellings. Under anaerobic photothrophic growth conditions, all new
isolates produced brick-red and pink cultures in succinate and benzoate, respectively.

Spectral analysis of cell free extracts (Figure 1) of photosynthetic pigments
showed absorption maxima for DS-1 at 374, 493, 587, 803 and 852 nm, DS-4 at
371, 501, 585, 803 and 848 nm, and Cas-13 at 373, 502, 585, 802 and 871 nm
indicating the presence of bacteriochlorophyl a and normal spirilloxanthin series
including lycopene and rhodopin (Imhoff 1995). In this respect, the three isolates
were nearly identical to each other and might be considered as one group. However,
these isolates clearly differed from Rhodobacter sphaeroides 2.4.1., which has
different absorption maxima (376, 451, 453, 477, 507, 587, 746, 799, and 850 nm),
with yellow-brown colonies grown anaerobically in succinate.

The results from the analysis of partial sequencing of 16S-rRNA (c.a. 500 bp) of
DS-4, DS-1, and Cas-13 demonstrated that DS-4 and Cas-13 were closely related
(Figure 2). Partial sequencing of the first 500 bp of 16S rRNA gene could lead to a
main line of descent (Stackebrandt and Rainey 1995).

Comparison with the RDP database of the University of Illinois indicated that
both DS-1 and Cas-13 demonstrated 97% similarity with Rs. palustris Strain 7, and of
83% and 84% with Rb. sphaeroides IL106, respectively (Table 1). Similarity of those
of DS-4 to Rs. palustris Strain 7 was 78%. These results suggested that DS-1 and Cas-
13 are likely to be one species, Rs. palustris. DS-4, however, is significantly
different from DS-1 and Cas-13 by 22%, and from Rb. sphaeroides IL106 by 30%.
Complete sequence analysis is needed to give more definitive information about
the taxonomic position of these three isolates.

Characterization of three benzoate degrading anoxygenic photosynthetic bacteria -Dwi Suryanto et al.

Figure 1. Absorption spectrum of cell extracts DS-1, DS-4, Cas-13, and Rb. Sphaeroides 2.4.1.

Figure 2. Phylogenetic tree 16S rRNA gene sequences. The number at the tree lines represented bootstrap

values.

BIOTROPIA NO. 17, 2001

Table 1. Percent similarity of the DS-1, DS-4, and Cas-13 with other relative members of anoxygenic
photosynthetic bacteria.

Unlike Rb. sphaeroides 2.4.1, all of the three isolates were able to metabolize
benzoate (Figure 3). Among the members of APB, Rs. palustris is the most common
species capable of utilizing benzoate (Harwood and Gibson 1988; Gibson and Gibson
1992; Shoreit and Shaheb 1994).

The ability to grow in the presence of aromatic compounds such as benzoate
without vitamins (Figure 3) may suggest that the organisms were able to synthesize
their own vitamins. However, supplemented vitamins could increase their potential in
metabolizing benzoate. The rate of benzoate utilization for DS-1, DS-4, and Cas-13
were 0.024 mM/hour, 0.02 mM/hour, and 0.03 mM/hour in media with vitamin
supplements compared to 0.017 mM/hour, 0.012 mM/hour, and 0.018 mM/hour in
media without vitamins. A relatively similar pattern of extended lag phase was
observed in growth of the cultures. Time was needed in preparation of producing a
number of enzymes.

Carbon availability might affect cell growth. Cell density was observed to be
higher in media with benzoate (C7) (Figure 3), followed by succinate (C4) and
acetate (C2) (Figure 4). Relatively low cell density of all isolates was shown in 5 mM
benzoate with no vitamins. Vitamins were certainly necessary for their metabolic
activity.

Characterization of three benzoate degrading anoxygenic photosynthetic bacteria -Dwi Suryanto et al.

Figure 3. Growht of DS-01, DS-4 and Cas-13 in 5 mM benzoate with vitamins (above) and without

vitamins (below). Rb. Sphaeroides 2.4.1. was used as negative control.

BIOTROPIA NO. 17, 2001

Figure4. Growth of DS-1, DS-4, Cas-4, Cas-13, and R6. Sphaeroides 2.4.1. in 5 mM succinate

(above) and 5 mM acetate (below) with vitamins.

Characterization of three benzoate degrading anoxygenic photosynthetic bacteria -Dwi Suryanto et al.

ACNOWLEDGEMENT

This research was funded by the Center for Microbial Diversity, Faculty of
Science and Mathematics, Bogor Agricultural University, Bogor Indonesia.

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