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Long term heavy oil polluted soil microrflora from northern Bulgaria was investigated. Microbial community was stable with more than 10 6 CFU/gram dry soils. 80% of heterotrophic microorganisms are able to degrade or co-oxidate crude oil hydrocarbons as a sole carbon source. Each of physiological groups of isolated microorganisms contained over 50% strains (oil-oxidizing and fermenting bacteria, fungi, and streptomycetes) capable to grow on heavy or light crude oil as sole carbon source. Microflora was more abundant in soils with low concentrations of crude oil. Quantity of heterotrophic bacteria was comparable to oligotrophs. The least spore-forming bacteria were found in soil probes containing over 20 g/kg petroleum hydrocarbons. Bacillus, Pseudomonas and Sporosarcina genera representatives were predominated among the obtained bacterial isolates. The presence of family Enterobacteriaceae members was notable, and all investigated bacteria posess lipase. Pure cultures from genera Pseudomonas and Bacillus were chosen for further analyses as the most promising in bioremediation. Classical and molecular approaches were applied to determine the taxonomic status of investigated microbial isolates. PCR and sequence analyses of 16S ribosomal DNA were performed with genus-specific primers. As a result the strains B2-1, B2-2 and 468-1 were determined as members of Pseudomonas libanensis, Klebsiella planticola and Pseudomonas fluorescens, respectively.
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Cell & Plant Sci 2013 4(1): 12-17
A© ademy Journals 2013
Journal of Cell & Plant Sciences
Original Article
Microflora of Bulgarian Oil Contaminated Environments
Iliana A. IVANOVA1*, Svetla NIKOLOVA1, Hussein YEMENDZHIEV2, Alexandra KONIARSKA1, Zlatka
1 Sofia University “St. Kl. Ohridski”, Sofia, Bulgaria;
2 Institute of Microbiology, Bulgarian Academy of Sciences, Sofia, Bulgaria
Received: 03.09.2011 Accepted: 23.05.2012 Published: 25.12.2013
Long term heavy oil polluted soil microrflora from northern Bulgaria was investigated. Microbial community was stable with more than 106
CFU/gram dry soils. 80% of heterotrophic microorganisms are able to degrade or co-oxidate crude oil hydrocarbons as a sole carbon source.
Each of physiological groups of isolated microorganisms contained over 50% strains (oil-oxidizing and fermenting bacteria, fungi, and
streptomycetes) capable to grow on heavy or light crude oil as sole carbon source. Microflora was more abundant in soils with low
concentrations of crude oil. Quantity of heterotrophic bacteria was comparable to oligotrophs. The least spore-forming bacteria were found in
soil probes containing over 20 g/kg petroleum hydrocarbons. Bacillus, Pseudomonas and Sporosarcina genera representatives were
predominated among the obtained bacterial isolates. The presence of family Enterobacteriaceae members was notable, and all investigated
bacteria posess lipase. Pure cultures from genera Pseudomonas and Bacillus were chosen for further analyses as the most promising in
bioremediation. Classical and molecular approaches were applied to determine the taxonomic status of investigated microbial isolates. PCR
and sequence analyses of 16S ribosomal DNA were performed with genus-specific primers. As a result the strains B2-1, B2-2 and 468-1
were determined as members of Pseudomonas libanensis, Klebsiella planticola and Pseudomonas fluorescens, respectively.
Key words: 16S rDNA, Bioremediation, Oil-degrading bacteria, Taxonomy
*Corresponding Author: I.A.Ivanova., e-mail:, Phone: +35988593324, Fax+35928167255
Bioremediation is accepted as an effective, environmentally
harmless treatment for soils contaminated because of oil
spills. Despite of a relatively long history of research on oil-
spill bioremediation, it remains an essentially empirical
technology and many of the factors that control
bioremediation have not yet been adequately understood
(Head еt al.2003). A main role in the bioremediation process
plays group of microorganisms with oil-degrading activity,
which is significant part of total quantity of aerobic
heterotrophs, fermenting, anaerobic bacteria, fungi and
Streptomycetes (Il'inskii еt al., 1998; Atlas & Atlas, 1991;
Roy еt al., 2002; Ko еt al., 2005; Nwaugo еt al., 2006;
Sanchez еt al., 2006). Methods analyzing distribution of
microorganisms in polluted environment and their activity are
different and debatable (Walker & Colwell, 1976; Brown &
Braddock, 1990; Atlas & Atlas, 1991). Some of the most
popular microorganisms used in bioremediation technologies
are gram negative bacteria as those from genera Pseudomonas
and Klebsiella, applied in water and soil technologies. Some
Pseudomonas strains could transform fluorene till products as
3,4-dihydroxifluoren and 3,4-dihydrocumarine (Mrozik еt
al.2005). Pseudomonads are also capable to degrade
polycyclic aromatic hydrocarbons (PAHs) as anthracene and
phenanthrene which are typical components of heavy
Cell & Plant Sci 2013 4(1): 12-17 I. A. Ivanova et al.
13 A© ademy Journals 2013
Bulgarian crude oil (Nwaugo еt al., 2006; Ovchinnikova еt
al., 2009; Ashrafosadat еt al., 2009). Some Klebsiella strains
independently or in mixed bacterial cultures could degrade
PAHs, pesticides as atrazine and even crude oil
(Siripanattanacul еt al., 2009).
Molecular methods are widely used for rapid
determination of microbial communities in oil-polluted
environments. The microbial community shift from G (+)
with G (-) bacteria at oil-polluted beaches was demonstrated
by Macnaughton et al. (1999) with PCR-DGGE. Widmer et
al. (1998) described two primers (PsFor и PsRev) for 16S
rDNA, specific for bacteria, belonging to Pseudomonas spp.
(Widmer et al., 1998). One of the major advantages of the
primer usage is the opportunity to carry out a direct DNA
amplification from environmental samples (LaMontagne et
al., 2002).
The purpose of our research was to assess the biodiversity
in investigated microbial communities, to isolate and identify
different oil-degrading microorganisms with high ability to
use crude oil as a sole carbon source.
Microbial Isolation from Oil Polluted Environments
Soil samples from heavy and light oil polluted habitats
from northern Bulgaria (Dolni Dabnik and Shabla oil deposit)
were analyzed. The samples were collected from oil polluted
soil next to a heavy oil-well with over 20 g/kg dry soil crude
oil; microbial biofilm on the oil pipe; light oil polluted soil
under 10 g/kg dry soil crude oil; water samples from light oil-
well and unpolluted soil.
Growth Conditions and Media
Microorganisms from six different groups were tested
using classical microbiological nutrient media as follow: oil-
oxidizing and fermenting bacteria, Streptomycetes, fungi,
oligotrophs and methanogens (Atlas & Atlas, 1991;
Da Cunha, 2006; Tarasov, 2002). Three different methods
were used for determination the number of crude oil-
degrading microorganisms in all samples: classic and
miniaturized most probable number method and silica gel
plates with single carbon source crude heavy or light oil
(Walker & Colwell, 1976; Brown & Braddock, 1990; Atlas,
2004). Taxonomic determination was made using classical
microbiological methods: fungi (Domsch et al, 1995), oil-
oxidizing and fermenting bacteria (Palleroni et al., 2004). All
isolated strains were tested for ability to grow with 2% crude
oil as carbon source in the mineral nutrient medium.
Numerical taxonomic analyses by computer programs were
applied to the taxonomic data obtained (Zuberer, 1994;
Austin, 1977).
DNA Extraction and PCR Conditions
Total DNA from eleven strains G (-) and G (+) bacteria
was extracted [Manniatis]. PCR was performed with primers
for the bacterial 16S rDNA genes: a pair of primers for Bacilli
- BLS342F and 1392R (eubacterial); a pair of eubacterial
primers - fD1 and 1392R; and a pair of primers for
Pseudomonas - S-G-Psmn-0289-a-S-20 Ps-for and S-G-
Psmn-1258-a-A-18 Ps-rev (Weisburg et al., 1991; Widmer et
al., 1998; Blackwood et al., 2005).
PCR was performed on Eppendorf Mastercycler using
PuRe Taq Ready-To-GoTM PCR Beads (Amersham
Biosciences, NJ). The PCR reactions were carried out in a 25
μl final volume containing 10 pmol each primer and 50 ng
genomic DNA (1 μl).
PCR conditions include 95 0С for 5 min followed by 35
cycles: 95 0С for 30 s, 55 0С for 30 s, 60 0С for 30 s and final
extension at 72 0С for 10 min. PCR products were stored at 4
0C. The obtained PCR products were purified by GFXTM PCR
DNA and Gel Band Purification Kit (Amersham
Biosciences, Piscataway, NJ, USA).
RFLP Analysis
Restriction fragment length polymorphism (RFLP) of
amplicons obtained from Pseudomonas sp. was performed
with restriction endonucleases AluI и НinfI (Blackwood,
2005). 5 µl PCR-product was digested with 3-5U single
restrictase аnd 10 µl PCR product was double digested at
The sequencing of amplified fragments was performed on
ABI Prism 3100 Genetic Analyzer using BigDye®
Terminator Kit 3.1. The row data from Genetic Analyzer
were editing by Sequence scanner 1.0 (Applied Biosystems,
Foster City, CA, USA).
Reproducibility of Results
All experiments were repeated at least 3 times in a 3
consecutive years and the mean values are represented.
Establishing of the Most Abundant Groups in
Microbial Community
The quantity of microorganisms of six physiological
groups was tested in every soil and water sample: oil-
oxidizing, aerobic heterotrophs, fermenting bacteria,
oligothrophs, spore forming bacteria and methanogens.
Similar groups were tested in other investigations (Atlas &
Cell & Plant Sci 2013 4(1): 12-17 I. A. Ivanova et al.
14 A© ademy Journals 2013
Atlas, 1991; Da Cunha, 2006; Tarasov, 2002) and were
concerned as basic for bioremediation.
On elective media the quantity of different physiological
groups of microorganisms was determined (Atlas & Atlas,
1991; Atlas, 2004). Oligotrophic bacteria are ubiquitous with
105-106 CFU/ml. Quantity of oil-degrading bacteria was
comparable to the quantity of oligotrophic bacteria in most
samples and almost the same as this of aerobic heterotrophs,
no matter of origin of the sample or crude oil type pollution.
It could be suggested, that the role of oligothrophs is so
important and significant in polluted environments, as that of
aerobic heterotrophic oil-oxidizing bacteria (Fig.1).
Ferm bact.
sporef. Bact.
Lg CFU/ml
Figure 1 Quantity of microorganisms of six different physiological groups in
samples: a. oil polluted soil next to oil-well with over 20 g/kg dry soil
petroleum hydrocarbons; b. microbial biofilm on the oil pipe; c. unpolluted
soil; d. lightly oil polluted soil under 10 g/kg dry soil petroleum
hydrocarbons; e. oil- polluted water from deposit with less than 10 g/kg dry
soil petroleum hydrocarbons
This finding agreed with results of Yakimov et al, (2007)
about ecophysiologically unusual group of marine
hydrocarbon - oxidizing bacteria - the obligate
hydrocarbonoclastic bacteria that has been recognized and
shown to play a significant role in the biological removal of
petroleum hydrocarbons from polluted marine waters. It is
possible that similar situation exist in soil. As the introduction
of oil or oil constituents into seawater leads to successive
blooms of a relatively limited number of indigenous marine
bacterial genera, it is found similar change in the soil after oil
pollution and similar quantities of oil- oxidizing oligotrophs
and heterotrophs.
Fermenting bacteria were less than oligotrophs with 1-2
orders of magnitude. The quantities of spore-forming bacteria
and methanogens were between 10 and 100 CFU/ml.
Methanogens were not detected in heavy oil-polluted soil and
microbial biofilm on the oil pipe. Tested physiological groups
of oligotrophs, fermenting and oil-oxidizing bacteria have
significant quantity in all samples under investigation and are
important for microbial community activity, presented in
every heavy or light oil-polluted sample.
In our study different methods of quantity determination
of oil-oxidizing microorganisms were compared. Every soil
sample under investigation was tested using three methods:
classic (Atlas, 2004), miniaturized sheen-screen method
(Brown & Braddock, 1990) and silica-gel plates with single
carbon source crude light or heavy oil (Walker &
Colwell,1976). Results were similar and showed strong
decrease in microbial population in heavy oil polluted soil
and biofilm on the oil pipes (1 to 0, 01% in comparison with
control-unpolluted soil). In all soil samples, the quantity of
oil-oxidizing microorganisms was comparable as determined
with the classic and with the sheen-screen method (SSM).
The difference between liquid and solid media was about 10-
time less oil oxidizing bacteria in solid medium (Fig.2).
1 2 3 4 5
Figure 2 Quantity of oil degrading microorganisms, determined by different
methods: 1. oil polluted soil with over 20 g/kg petroleum; 2. microbial
biofilm on the oil pipe; 3. unpolluted soil; 4. soil <10 g/kg hydrocarbons; 5.
oil- polluted water
Isolation of Pure Cultures of Microorganisms
Several consecutive elective and selective procedures
were performed for isolation of pure cultures of oil-oxidizing
microorganisms from elective cultures received by three
methods. After determination the purity of isolated cultures,
they were tested again for oil-degrading activity. The most
prominent were taxonomically differentiated (Bergey’s
Manual). Oil-oxidizing and fermenting bacteria, as well as
some Streptomycetes and fungi were isolated from polluted
Estimation of Taxonomic Status of Purified Cultures
Taxonomic status of around twenty oil-oxidizing bacteria
was determined using morphological and biochemical
characteristics (Bergey’s Manual). Most of isolates were
motile rods, only 468-p was immobile coccus. Basic
physiological and biochemical characteristics of pure cultures
Cell & Plant Sci 2013 4(1): 12-17 I. A. Ivanova et al.
15 A© ademy Journals 2013
of oil-degrading bacteria are presented in table 1. Half of
them were colored and almost all could grow on 6.5% NaCl,
pH=7.5, 30˚C, potato- and Aikman agar. All bacteria were
catalase positive and possess nitrate reductase activity. They
could use wide spectrum of hydrocarbon sources and all use
cellobiose, melibiose and manose. Most of them are aerobes
or facultative anaerobes. Very interesting quality of isolated
strains was growth in nitrogen-free and crude oil-free
medium. Nitrogen fixation is important quality of microbial
community during bioremediation process. This could
diminish expenses for nutrients’ additions during
G (-) bacteria predominated among oil-oxidizing isolates.
About 30% of colored bacteria were determined as
Flavobacterium; the rest were belonging to genera Bacillus
and Sporosarcina. Among G (-) bacteria predominated genus
Pseudomonas, the other more abundant were Alcaligenes and
Aeromonas. Numerical taxonomy was applied to determine
the proximity of investigated bacteria to described type
species. Among oil-degrading genus Bacillus three isolates
were similar to Bacillus licheniformis 469, 359, 282-3, one
close to Paenibacillus macerans 282-2 and one to Bacillus
schlegelii- 282-1. Determined oil-oxidizing bacteria are
mentioned in other reports for microbial community of oil-
polluted environments (Atlas & Atlas, 1991; Da Cunha et al,
2006; Tarasov et al, 2002; Girvan et al, 2004; Kong et al,
2005; Ammar et al, 2005; Stoimenova et al, 2009).
Investigated characteristics of gram-negative fermenting
bacteria were as described in Bergey’s Manual: indol-
formation, Voges-Proskauer reaction, formation of H2S and
NH3, growth on acetate and citrate, hydrolysis of gelatin;
growth on 6, 5 and 13.5% NaCl, formation of amylase, lipase,
lecitinase, β–galactosidase, nitrate-reduction, and hemolytic
activity. Other tested features were aerobic/anaerobic acid and
gas formation from glucose; oxidation/fermentation of
lactose, sucrose, manitol, dulcitol, inositol, D-sorbitol, 2-
arabinose, rafinose, ramnose, maltose, D-xilose, trehalose,
esculine and galactose (Bergey’s Mannual).
In the group of fermenting bacteria, the genus
Enterobacter was dominant over than 50% of isolates
(strain 259 - Enterobacter cloacae, strain 282M and 159 -
Enterobacter aerogenes). The other isolates were determined
as follow: strain 298 belongs to genus Citrobacter, strain 95-1
to Serratia liquefaciens and strain 84 to Rahnella (Table 1).
Most of oil-degrading bacteria were determined as
aerobic or microaerophils, but the prevalent fermenting
bacteria were facultative anaerobes with formation of large
gas quantity during fermentation of sugars. This quality is
useful for microbial enhanced oil recovery (Tarasov et al,
2002). Obviously, fermenters are important together with oil-
degrading bacteria in providing of carbon dioxide for
methanogenic bacteria in microbial community.
About 28 different fungal strains were isolated from
polluted soil. Taxonomic differentiation of fungi was based
on characteristics, described in Compendium of Soil Fungi
(Domsch et al, 1995). The key characteristics were their
asexual and sexual reproduction, formation and appearance of
conidia and spores. The most often met oil-degrading fungi
were determined as Fusarium sp., Aspergillus niger,
Trichoderma sp. and Penicillium sp.
Oil-degrading microbial quantity is in correlation with
quantity of lipase producers in soil. Ko and colleagues (2005)
had determined 12 to 75% of bacterial, actinomycetes and
fungal isolates from oil-polluted soils as lipase- producers. In
their investigation, lipase producers were more common
among soil actinomycetes and fungi (Ko et al, 2005). In our
investigation, all of oil-degrading heterotrophic bacteria were
lipase-producers. Obviously, bacteria as well as the other
groups of microorganisms appear to be important in
decomposition of oils and organic matter in soils with long-
term petroleum pollution.
Results from classical biochemical and morphological
determinations and processing of results with a computer
program ESPS, proved taxonomic affiliation of strains B2-1,
B2-2, 4 and G-1 to genus Pseudomonas as could be seen by
dendrogramme (Figure 3). It shows that strains B2-1, B2-2
and G-1 had form separate cluster with a resemblance to the
genus Pseudomonas with 70%. Strain 4 is placed into a
cluster, formed by genera Ps. marina, Ps. doudoroffii and Ps.
aeruginosa with 80% similarity (Table 1).
Table 1 Taxonomic affiliation of isolated pure cultures
oil-degrading bacteria determined by morphological,
physiological and biochemical analyses
Pseudomonas sp.
Alcaligenes sp.
Aeromonas sp.
Rahnella sp.
Serratia marcescens
Enterobacter aerogenes
Bacillus schlegeli
Paenibacillus macerans
Bacillus licheniformis.
Enterobacter aerogenes
Enterobacter cloacae
Citrobacter sp.
Bacillus licheniformis
Flavobacterium sp.
Bacillus sp.
Sporosarcina sp.
Bacillus licheniformis
Pseudomonas sp.
Rhizobium radiobacter
Pseudomonas marginalis
Pseudomonas corrugata
Raoultella planticola
Cell & Plant Sci 2013 4(1): 12-17 I. A. Ivanova et al.
16 A© ademy Journals 2013
* * * * * * H I E R A R C H I C A L C L U S T E R A N A L Y S I S * * * * * *
Euclidean measure used.
Dendrogram using Ward Method
Rescaled Distance Cluster Combine
C A S E 0 20 40 60 80 100
Label Num +---------+---------+---------+---------+---------+
Ps hydrogenothermoph 29
Ps carboxydovorans 33
Ps compransoris 32
Ps palleronii 27
Ps testosteroni 21
Ps carboxydohydrogen 31
Ps acidovorans 20
Ps stutzeri 9
Ps mendocina 10
Ps pseudoalcaligenes 12
Ps nautica 36
Ps alcaligenes 11
Ps malei 13
Ps pseudomalei 14
Ps cepacia 16
Ps gladioli 17
Ps caryophylii 15
Ps flava 25
Ps pickettii 18
Ps solanacearum 19
Ps delafieldii 22
Ps facilis 23
Ps syringae 1
Ps viridiflava 2
b2-1 37
b2-2 38
g1 39
Ps saccharophila 24
Ps hydrogenovora 28
Ps putida 7
Ps carboxydoflava 30
Ps fluorescens 4
Ps chlororaphis 5
Ps aureofaciens 6
Ps aeruginosa 3
Ps marina 35
Ps doudoroffii 34
pr.4 40
Figure 3 Dendrogram of genus Pseudomonas and affiliation of isolates.
Strains 3-1, 3-2 were determined as different genera.
Strain 3-1 belongs to genus Aerococcus, 3-2 to family
Enterobacteriaceae, and 468-1 and 468-2 belong to genus
Total DNA from eleven strains G (-) and G (+) bacteria
was isolated. PCR with eubacterial pair of primers (fD1 and
1492R) were used (Weisburg et al, 1991). PCR analyses were
also carried out with specific primers for Bacilli and genus
Pseudomonas, respectively: BLS342F and 1392R
(Blackwood et al, 2005); S-G-Psmn-0289-a-S-20 Ps-for and
S-G-Psmn-1258-a-A-18 Ps-rev (Widmer et al.1998). PCR
products from Pseudomonas sp. were digested with HinfI and
AluI and Pseudomonas putida as a control was used (Figure
Figure 4 Restriction fragment length polymorphism of bacterial 16S rDNA
with Alu I _ _inf
Isolated strains were different and PCR products of
strains with most unique profiles were sequenced. Sequence
analysis of 16S r DNA differentiated strains B2-1, B2-2 and
468-1 as Pseudomonas libanensis, Raoultella planticola
(formerly Klebsiella planticola) and Pseudomonas
fluorescens with 99% probability. This is the first report
about Pseudomonas libanensis found in Bulgarial soils. The
sequences were registered in NCBI under following accession
numbers: GU227425, GU227426, and GU227427. The results
obtained in the present study correlated with previous works
reporting about members of P. libanensis, Raoultella
planticola (formerly K. planticola) and P. fluorescens. For
instance, Stoimenova et al. (2009) reported about surfactant
production of Pseudomonas fluorescens and possibility of its
applications. Klebsiella planticola strain DSZ, was described
as metabolically diverse and its ability to grow on a wide
range of s-triazine and aromatic compounds was reported
(Ammar et al., 2005; Sánchez et al., 2005; Li et al., 2008). P.
libanensis can be differentiated from P. fluorescens (all
biovars) by -aminobutyrate assimilation. These strains were
used in different experiments for environmental pollutants
biodegradation as producers of some effective biosurfactants
(Dabboussi et al., 1999; Saini et al., 2008). Our results show
formation of stable microbial community with high oil-
degrading potential after oil pollution.
Long term oil-polluted Bulgarian soils had stable
microbial community with more than 106 CFU/gram dry soil.
The quantity of bacteria was about 105-106 CFU/g soil, but
fungi and Streptomycetes were between 102-103 CFU/g dry
Heavily oil polluted soil with over 20 g/kg petroleum
hydrocarbons conteins less spore-forming bacteria. For the
first time investigated Bulgarian oil-degrading strains were
determined as Pseudomonas libanensis BF1, Raoultella
planticola (formerly Klebsiella planticola) BF2 and
Pseudomonas fluorescens BF3 with 99% probability. All of
them could be used for bioremediation of petroleum polluted
soil and water.
This study is supported by a Joint research project between
Bulgaria and Romania R-5B/05 and Scientific Fondation of Sofia
University project 173/2008.
Ammar E, Nasri M, Medhioub K, 2005. Isolation of Enterobacteria able to
Degrade Simple Aromatic Compounds from the Wastewater from Olive
Oil Extraction. World Journal Microbiology and Biotechnology21: 253-
Ashrafosadat HZ, Seyed AS, Ebrahim VF, Saman H, Emamzadeh A, 2009.
Cell & Plant Sci 2013 4(1): 12-17 I. A. Ivanova et al.
17 A© ademy Journals 2013
Extensive Biodegradation of Highly Chlorinated Biphenyl and Aroclor
1242 by Pseudomonas aeruginosa TMU56 Isolated from
ContaminatedSoils. International Biodeterioration and Biodegradation
63: 788794
Atlas RM, Atlas MC, 1991. Biodegradation of Oil and Bioremediation of Oil
Spills. Current Opinion in Biotechnology 2: 440-443 Atlas RM, 2004.
Handbook of Microbiological Media, Third Edition, CRC Press
Austin B, Allen DA, Mills A, Colwell RR, 1977. Numerical Taxonomy of
Heavy Metal-Tolerant Bacteria Isolated from an Estuary. Canadian
Journal Microbiology23: 14331447
Blackwood CB, Oaks A, Buyer JS, 2005. Phylum and Class-Specific PCR
Primers for General Microbial Community Analysis. Applied
Environmental Microbiology 71: 6193-6198
Brown EJ, Braddock JF, 1990. Sheen Screen, A Miniaturized Most-Probable-
Number Method for Enumeration of Oil Degrading Microorganisms.
Applied Environmental Microbiology 56: 3895-3896
Da Cunha CD, Rosado AS, Sebastián GV, Seldin L, von der Weid I, 2006.
Oil Biodegradation by Bacillus Strains Isolated from the Rock of an Oil
Reservoir Located in a Deep-Water Production Basin in Brazil. Journal
Applied Microbiology and Biotechnology 73: 949-959
Dabboussi F, Hamze M, Elomari M, Verhille S, Baida N, Izard D, Leclerc H,
1999. Pseudomonas libanensis sp. nov., a New Species Isolated from
Lebanese Spring Waters. International Journal Systematic Bacteriology
Domsch KH, Gams W, Anderson TH, 1995. Compendium of Soil Fungi.
Publisher(s): Lubrecht & Cramer Ltd.
Girvan MS, Bullimore J, Ball AS, Pretty J, Osborn AM, 2004. Response of
Active Bacterial and Fungal Communities in Soils under Winter Wheat
to Differing Fertiliser and Pesticide Regimes. Applied Environmental
Microbiology 70: 2692-2701
Head I, Singleton I, Milner M, 2003. Bioremediation; a critical review.
Il'inskii VV, Porshneva OV, Komarova TI, Coronelli T, 1998. The Effect of
Petroleum Hydrocarbons on the Hydrocarbon-Oxidizing
Bacteriocenosis in the Southeast Part of the Mozhaiskoe Water Storage
Basin. _icrobiology 67: 220-225
Ko WH, Wang IT, Ann PJ, 2005. A Simple Method for Detection of
Lipolytic Microorganisms in Soils, Soil Biology and Biochemistry 37:
Kong H, Blackwood C, Buyer JS, Gulya TJ Jr, Lydon J, 2005. The Genetic
Characterization of Pseudomonas syringae pv. tagetis Based on the
16S-23S rDNA Intergenic Spacer Regions. Biological Control 32: 356-
LaMontagne MG, Michel FCJ, Holden PA, Reddy CA, 2002. Evaluation of
Extraction and Purification Methods for Obtaining PCR-Amplifiable
DNA from Compost for Microbial Community Analysis Journal of
Microbiological Methods 49: 255-26
Li YG, Li WL, Huang JX, Xiong XC, Gao HS, Xing JM, Liu HZ, 2008.
Biodegradation of Carbazole in Oil/Water Biphasic System by a Newly
Isolated Bacterium Klebsiella sp. LSSE-H2. Biochemical Engineering
Journal 41: 166-170
MacNaughton SJ, Stephen JR, Venosa AD, Davis GA, Chang YJ, White DC,
1999. Microbial Population Changes during Bioremediation of an
Experimental Oil Spill. Applied and Environmental Microbiology 65:
Mrozik A, Labuzek S, Piotrowska SZ, 2005. Changes in Fatty Acid
Composition in Pseudomonas putida and Pseudomonas stutzeri during
Naphthalene Degradation. Microbiology Reviews 160: 149157
Nwaugo VO, Onyeagba RA, Nwahcukwu NC, 2006. Effect of Gas Tlaring of
Soil Microbial Spectrum in parts of Niger Delta Area of Southern
Nigeria. African Journal Biotechnology 19:1824-1826
Ovchinnikova AA, Vetrova AA, Filonov AE, Boronin AM, 2009.
Phenanthrene Biodegradation and the Interaction of Pseudomonas
putida BS3701 and Burkholderia sp. BS3702 in Plant Rhizosphere.
Microbiology 78:433-439
Palleroni NJ, 2004. Pseudomonas.In: Brenner DJ, Krieg NR, Staley JT (eds.)
Bergey’s Manual of Systematic Bacteriology. New York: Springer 2(2):
Roy S, Hens D, Bisis D, Kumar R, 2002. Survey of Petroleum-
DegradingBacteria in Coastal Waters of Sunderban Biosphere Reserve,
World Journal Microbiology and Biotechnology 18: 575-581
Saini HS, Barragan-Huerta BE, Lebron-Paler A , Pemberton JE, VazquezRR,
Burns AM , Marron MT, Seliga CJ, Gunatilaka AAL, Maier RM, 2008.
Efficient Purification of the Biosurfactant Viscosin from Pseudomonas
libanensis Strain M9-3 and Its Physicochemical and Biological
Properties Journal National Production 71: 10111015
Sánchez M, Garbi C, Martínez ÁR, Ortiz LT, Allende JL, Martín M, 2005.
Klebsiella planticola strain DSZ Mineralizes Simazine: Physiological
Adaptations Involved in the Process. Applied Microbiology and
Biotechnology 66:589-596
Sanchez O, Ferrera I, Vigues N, Garcia de Oteyza T, Grimalt JO, Mas J,
2006. Presence of Opportunistic Oil-Degrading Microorganisms
Operating at the Initial Steps of Oil Extraction and Handling.
International Microbiology 9: 119-24
Siripanattanacul S, Wirojanagud W, McEvoy J, Limpiyacom T, Khan E,
2009. Atrazine Degradation by Stable Mixed Cultures. Journal Applied
Microbiology 106: 986-992
Stoimenova E, Vasileva-Tonkova E, Sotirova A, Galabova D, Lalchev Z,
2009. Evaluation of Different Carbon Sources for Growth and
Biosurfactant Production by Pseudomonas fluorescens Isolated from
Wastewaters Zeitschrift fur Naturforshung 64C (1/2):96-102
Tarasov AL, Borzenkov IA, Milekhina EI, Belyaev SS, Ivanov MV, 2002.
Dynamics of Microbial Processes in the Stratal Waters of
thebRomashkinskoe Oil Field. Journal Mikrobiologiya, (in Russian) 71:
Walker YD, Colwell RR, 1976. Enumeration of Petroleum Degrading
Microorganisms. Applied Environmental Microbiology 31: 198-207
Weisburg WG, Barns SM, Pelletier DA, Lane DJ, 1991. 16S Ribosomal
DNA Amplification for Phylogenetic Study. Journal Bacteriology 173:
Widmer F, Seidler RJ, Gilleret PM, Watrud LS, Di Giovanni GD, 1998. A
Highly Selective Protocol for Detecting 16S rRNA Genes of the Genus
Pseudomonas (sensu stricto) in Environmental Samples, Applied and
Environmental Microbiology 64: 25452553
Yakimov M, Kenneth NT, Golyshin PN, 2007. Obligate Oil-Degrading
Marine Bacteria. Current Opinion in Biotechnology 18: 257-266
Zuberer DA, 1994. Recovery and Enumeration of Viable Bacteria. Methods
of Soil Analysis, Part 2. Microbiological and Biochemical Properties.
Madison, Wisconsin. Soil Science Society of America 119-144
Sudha P1, Smita SZ1, Shobha YB, Ameeta RK1 2011. Potent a-amylase
inhibitory activity of Indian Ayurvedic medicinal plants BMC
Complementary and Alternative Medicine 11:5
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ABSTRACT The lipases have ability to catalyze diverse reactions and are important in different biotechnological applications. The aim of this work was to isolate and characterize microorganisms that produce lipases, from different food industry effluents localized in Pelotas, RS/Brazil. Bacteria were identified using Gram stain and biochemical tests (Vitek 2(r)). Fungi were identified according to macro and micromorphology characteristics. The extracellular lipase production was evaluated using the Rhodamine B test and the enzymatic activity by titration. Twenty-one bacteria were isolated and identified as Klebsiella pneumoniae ssp. pneumoniae, Serratia marcescens, Enterobacter aerogenes, Raoultella ornithinolytica and Raoultella planticola. Were characterized isolated filamentous fungi by the following genera: Alternaria sp., Fusarium sp., Geotrichum sp., Gliocladium sp., Mucor sp., Paecilomyces sp. and Trichoderma sp. Extracellular lipase production was observed in 71.43% of the bacteria and 57.14% of the fungi. The bacterium that presented better promising enzymatic activity was E. aerogenes (1.54 U/ml) however between fungi there was not significant difference between the four isolates. This study indicated that microorganisms lipase producers are present in the industrial effluents, as well as these enzymes have potential of biodegradation of lipid compounds.
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To gain a better knowledge about reservoir petroleum biodegradation, it is essential to associate hydrocarbon degradation with specific organisms and metabolic processes. In this study, eighteen spore- forming,gram-positive bacteria were ,isolated from the rock of an ,oil reservoir located in a ,deep-water production basin in Brazil. These strains were identified as using,classical biochemical ,techniques and API 50CH kits and their identity was confirmed,by sequencing of part of their 16S rRNA gene. All strains were tested for their oil degradation ability in microplates using Arabe Leve and Marlin oils and only seven strains showed,positive results in both ,kinds of oils. They were ,further tested to their capability to grow ,in the presence of carbazol, quinoline and n-hexadecane as the only carbon source. These strains showed positive results to carbazol and n-hexadecane, but not to quinoline. The production of key enzymes involved with biodegradation process by Bacillus strains (catechol 1,2 dioxygenase and catechol 2,3 dioxygenase) was verified spectrophotometrically by detection of cis, cis-muconic acid and 2-hydroxymuconic semialdehyde. Preliminary results showed ,that the ortho ring cleavage pathway,is preferential. Biodegradation ,tests using Arabe Leve oil were carried out and only one strain (B. licheniformisT4.2) presented a good result, with 94% ofn-alkanes reduction. Furthermore, the seven Bacillus strains were screened for the presence of catabolic genes encoding alkane monooxygenase, catechol 1,2 dioxygenase and catechol 2,3 dioxygenase by using primers specific for these functional genes and PCR products were obtained with the DNA of different strains of B. licheniformis and B. subtilis. After DNA sequencing of the PCR products, they will be compared to previously well-known degradative genes. Keywords: Biodegradation, Bacillus, Oil reservoir
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The interaction between the strains degrading polycyclic aromatic hydrocarbons, Pseudomonas putida BS3701 and Burkholderia sp. BS3702, was studied in the course of phenanthrene degradation in plant rhizosphere. Strain BS3702 was shown to accumulate 1-hydroxy-2-naphthoic acid (which is toxic for plants); it was then utilized by strain BS3701, which thereby increased the resistance of plants to the pollutant and to the toxic intermediate. With this type of interaction (cooperation), the efficiency of phenanthrene degradation was noted to decrease.
Development of the natural hydrocarbon-oxidizing bacteriocenosis and alterations in its composition caused by the influence of petroleum and diesel fuel were studied in the Mozhaiskoe water storage basin during the summer period. The number of hydrocarbon-oxidizing bacteria was shown to vary between 25 and 250 cells/ml in the coastal area of the basin. The hydrocarbon-oxidizing community included bacteria of the genera Acinetobacter, Pseudomonas, Rhodococcus, and Micrococcus; the proportion of individual representatives of these genera varied at different periods in different parts of the reservoir. Model experiments revealed that the introduction of petroleum hydrocarbons into the reservoir water induced a rapid increase (by two or three orders of magnitude) in the total number of hydrocarbon-oxidizing microorganisms and alteration of their taxonomic composition, even with a change of dominating genera. Bacteria of the genus Rhodococcus were found to be the most resistant to the pollutants used.
Effects of gas-flaring on soil bacterial spectrum in parts of Niger-Delta area of Southern Nigeria was investigated using culture techniques and some ecological factors. While temperature decreased away from the flare points (60°C to 28°C), pH values, changed from acidic (4.0 - 4.2) to near neutral (6.4 - 6.6) away from the flare point. Moisture content also increased away from the flare. Bacterial load of Total Heterotrophic bacterial count (THBC), Petroleum degrading bacterial count (PDBC) and Total coliform count (TCC) also increased away from the flare points. The most affected by the Gas flaring was the coliforms. Bacterial species were also affected as only three Pseudomonas, and Bacillus species were found 10m away from the flare. The number increased to seven with the addition of E. coli, Enterobater, Flavobacterium and Micrococcus species at 100m away and finally 10, at 200m away with Citrobacter, Staphylococcus and Lactobacillus species. The same trend was observed in all the flaring sites examined. The results indicated adverse ecological and bacterial spectrum modifica-tions by the Gas flaring.
Several exciting findings about the biodegradation of hydrocarbons have come to light over the past year. The most significant developments include the discovery of anaerobic hydrocarbon metabolism, the isolation of 4- or 5-ring polycyclic aromatic hydrocarbon utilizers, the finding that aromatic and aliphatic hydrocarbons may be metabolized by totally different microbial populations, and the demonstration that induction of polycyclic aromatic hydrocarbon catabolic enzymes may depend upon the presence of simpler aromatic compounds. Bioremediation has been applied for the clean-up of major marine oil spills of refined petroleum products.
A novel Klebsiella sp. strain LSSE-H2 (CGMCC No. 1624) was isolated from dye-contaminated soil based on its ability to metabolize carbazole as a sole source of carbon and nitrogen. This strain efficiently degraded carbazole from either aqueous and biphasic aqueous–organic media, displaying a high denitrogenation activity and a high level of solvent tolerance. LSSE-H2 could completely degrade 12mmol/L carbazole after 56h of cultivation. The co-culture of LSSE-H2 and Pseudomonas delafieldii R-8 strains can degrade approximately 92% of carbazole (10mmol/L) and 94% of dibenzothiophene (3mmol/L) from model diesel in 12h.
Pseudomonas syringae pv. tagetis, a plant pathogen being considered as a biological control agent of Canada thistle (Cirsium arvense), produces tagetitoxin, an inhibitor of RNA polymerase which results in chlorosis of developing shoot tissues. Although the bacterium is known to affect several plant species in the Asteraceae and has been reported in several countries, little is known of its genetic diversity. The genetic relatedness of 24 strains of P. syringae pv. tagetis with respect to each other and to other P. syringae and Pseudomonas savastanoi pathovars was examined using 16S–23S rDNA intergenic spacer (ITS) sequence analysis. The size of the 16S–23S rDNA ITS regions ranged from 508 to 548bp in length for all 17 P. syringae and P. savastanoi pathovars examined. The size of the 16S–23S rDNA ITS regions for all the P. syringae pv. helianthi and all the P. syringae pv. tagetis strains examined were 526bp in length. Furthermore, the 16S–23S rDNA ITS regions of both P. syringae pv. tagetis and P. syringae pv. helianthi had DNA signatures at specific nucleotides that distinguished them from the 15 other P. syringae and P. savastanoi pathovars examined. These results provide strong evidence that P. syringae pv. helianthi is a nontoxigenic form of P. syringae pv. tagetis. The results also demonstrated that there is little genetic diversity among the known strains of P. syringae pv. tagetis. The genetic differences that do exist were not correlated with differences in host plant, geographical origin, or the ability to produce toxin.
Media selective for the isolation of bacteria, actinomycetes and fungi were amended with 0.1% sunflower oil emulsified with 0.01% Tween 80. Lipase-producing microorganisms produced clear zones on these media. When lipase-producing bacteria were cultured on a polycarbonate membrane laid on the selective medium for bacteria, clear zones were produced on the medium when the membrane along with bacteria was removed. The agar disc cut from the clear zone also produced a clear zone when placed on the fresh medium, indicating that clear zone formation is the result of the activity of extracellular lipases. The largest population of lipase-producing microorganisms in an agricultural soil was actinomycetes followed by bacteria and fungi. Ranging from 12 to 75% of bacteria, actinomycetes and fungi isolates from soils collected from three different locations were capable of producing lipases. In general, relatively small percentages of soil bacteria were lipase producers, and lipase producers were more common among soil actinomycetes and fungi. These three groups of microorganisms appear to be all important in decomposition of oils in organic matters in soils.
A survey of petroleum-degrading bacteria was carried out in the Indian part of deltaic Sunderbans to evaluate the distribution of the naturally occurring petroleum-degrading aerobic bacteria. Bacteriological analysis of surface water samples collected from five different locations in the Hooghly–Matla river mouth showed that, depending on the location, 0.08–2.0% of the heterotrophic bacteria culturable in marine agar medium could degrade crude petroleum hydrocarbons as the sole source of carbon. In the entire study area, the number of heterotrophic bacteria ranged from 1 103 to 3.8 105 c.f.u/ml, amongst which 2.7 101 to 6 103 c.f.u/ml were petroleum degraders. There was a maximum number of petroleum-degrading bacteria in the waters of Haldia Port and its surrounding areas, where the water is highly polluted by hydrocarbon discharges from a nearby oil refinery and from the ships docking at the port. Among the isolates, identified on the basis of their Gram reaction, morphological and biochemical tests including the use of API20E strips, Pseudomonas, Mycobacterium, Klebsiella, Acinetobacter, Micrococcus, and Nocardia were the most common petroleum degraders. Other heterotrophic bacteria included several species of Escherichia, Klebsiella, non-oil-degrading Pseudomonas, Vibrio, Streptococcus, Staphylococcus and Bacillus. Following preliminary selection, five strains, showing best growth in medium with oil fraction as sole carbon source, were chosen for estimation of the efficiency of crude oil biodegradation. The selected strains belonged to Pseudomonas (two strains), Mycobacterium (two strains), and Nocardia (one strain). These strains degraded 47–78% of Arab-Mix crude oil over a period of 20 days. The best oil-degrading isolate, a strain of Pseudomonas aeruginosa, (BBW1), was found to degrade and multiply more rapidly in crude oil than the rest. BBW1 showed profuse growth in Bushnell Haas medium containing crude oil (as sole source of carbon) at high concentrations ranging from 0.2 to 20% (v/v), with optimum at 10%. As much as 75% of the oil was degraded within 72 h of incubation with the bacteria. Physicochemical analysis showed considerable decrease in initial boiling point and carbon residue of the degraded oil. The ability to degrade crude oil was found to be associated with a single 70-kb plasmid, pBN70. Resistance to the metals Mn2+ (50 mM), Mg2+ (200 mM), Zn2+ (6 mM), Ni2+ (10 mM) and antibiotics like ampicillin (10 g/ml), cephalexin (30 g/ml), nitrofurantoin (300 g/ml) and penicillin (10 U/ml) were plasmid-mediated.