Role of Microbes in Petroleum Hydrocarbon Degradation

Twenty-one years after ZoBell's (1946) classic review, the super tanker Torrey Canyon sank in the English Channel. With this incident, attention of the scientific community was dramatically focused on problems of oil pollution. After this event, several studies were initiated to study the fate of oil pollution. Several studies were also initiated on the fate of petroleum in varied ecosystems. Biodegradation of petroleum products in natural ecosystem is no doubt a complex issue. The degradation of the hydrocarbon mixture depends on the nature of the oil, the nature of the microbial community, and a variety of environmental factors, which influence microbial activities (Gibson 1968; ZoBell 1973; Atlas 1981; Michaud et al. 2004). The ability to degrade petroleum hydrocarbons is not restricted to a few microbial genera (Whyte et al. 1998), but a diverse group of bacteria and fungi. ZoBell (1946), in his review, noted that more than 100 species representing 30 microbial genera had been shown capable of utilizing or degrading hydrocarbons. In a review, Bartha and Atlas (1977) also listed 22 genera of bacteria, one algal genus and 14 genera of fungi, which had been demonstrated to contain various members, utilize petroleum hydrocarbons. All of these microorganisms had been isolated from aquatic environments. The most important (based on frequency of isolation) genera of hydrocarbon utilizers in aquatic environments were Pseudomonas sp., Achromobacter sp., Micrococcus sp., Nocardia sp., Candida sp., Vibrio sp., Acinetobacter sp., Brevibacterium sp., Rhodotorula sp., Sporobolomyces sp., Corynebacterium sp., and Flavobacterium sp. (Bartha and

Atlas 1977). Walker and Colwell (1975) compared the abilities of bacteria and fungi in the degradation of hydrocarbons. The following genera were included in their study: Candida sp., Sporobolomyces sp., Hansenula sp., Rhodotorula sp., Cladosporium sp., Penicillium sp., Aspergillius sp., Pseudomonas sp., Vibrio sp., Nocardia sp. and Rhobium sp. Walker and Colwell (1976) isolated Vibrio sp., Pseudomonas sp. and Acinatobacter sp. from oil-contaminated sediments and Pseudomonas sp. and Coryneform sp. from oil free sediments. A large number of Pseudomonas sp. have been isolated that are capable of utilizing petroleum hydrocarbons. The genetics and enzymology of hydrocarbon degradation by Pseudomonas sp. have been extensively studied (Chakrabarty 1972; Chakrabarty et al. 1973; Dunn and Gunsalus 1973). The genetic information for hydrocarbon degradation in these organisms has been generally found to occur on plasmids. Numerical taxonomy has also been used to examine petroleum-degrading bacteria (Priest and Austin 1993). Recently, Rahman et al. (2002a) reported 130 bacterial cultures as crude oil degraders in Bombay high, India. Dela Fuente et al. (1991) studied the role of Serratia marcescens in oxidation of aromatic aldehydes into the corresponding acids in high yield under mild conditions. Verma et al. (2006) isolated Bacillus sp. SV9, Acinetobacter sp. SV17 and Pseudomonas sp. SV17 from the contaminated soil in Ankleshwar, India which were tested for their ability to degrade the complex mixture of petroleum hydrocarbons. This study revealed that after 5 days, the Bacillus strain was able to degrade oily sludge components of chain length of Ci2-C30 and aromatics more effectively than the other two strains. Degradation of diesel oil by Pseudomonas aeruginosa strain WatG was verified in soil microcosms. This strain exhibited a high diesel oil (mainly n-alkanes) degradation ability in soil provided with rich nutrients and also it produced dir-hamnolipid to facilitate the bioaugmentation ability (Wongsa et al. 2004). Degradation of polycyclic aromatic hydrocarbons by a newly discovered enteric bacterium Leclercia adecarboxylata was reported from oily sludge contaminated soil (Sarma et al. 2004). Bacterial consortium isolated from oil contaminated sites degraded alkanes in oil sludge and diesel contaminated soil (Rahman et al. 2002a, b, 2007, 2010). The introduction of 16S rRNA sequence analysis was heralded as the new era for bacterial systematics which could be used to rectify the mistakes in bacterial identification. Although numerical and chemotaxonomic methods were primarily employed to resolve these problems, 16S rRNA has been invaluable for the identification of evolutionary relationships not just in bacteria, but also in all living organisms. This technology was the first module examined for studying prokaryotic phylogenies, as a little was known about bacterial phylogeny earlier. One of the major successes was the reclassification of all life on this planet into three domains Bacteria, Archae and Eucarya (Woese et al. 1990). As a result of further work by Woese et al. (1990), the bacterial world is now divided into some 18 major lines of descent, referred to as phyla or divisions (Ludwig and Schleifer 1999). There are undoubtedly strong supports for 16S rRNA sequence analysis. Ludwig and Schleifer (1999) advocated the acceptance of 16S rRNA based phylogenetic trees as the ''gold standard'' which was supported by other workers

(Kimura 1980; Law et al. 1988; Weisburg et al. 1991; Larkin et al. 2007; Tamura et al. 2007).

The preliminary identification of bacteria by biochemical tests indicated that the isolates belonged to the genera Bacillus sp., Pseudomonas sp., Kelebsiella sp. and Serratia sp. The phenotypic profile of strains is shown in Tables 13.2 and 13.3. These bacteria were identified from the petroleum transporting pipeline.

Amplification of gene encoding for small subunit ribosomal RNA of DDB (diesel degrading bacteria) and NDB (naphtha degrading bacteria) was done using eubacterial 16S rDNA primers. The 16S rDNA amplicons derived from DDB and NDB was cloned in pTZ57R/T vectors. The recombinant plasmid (pACE2, harboring 16S rDNA insert) was partially sequenced. The sequence obtained was matched with the previously published sequences available in NCBI using BLAST. Sequence alignment of DDB and comparison revealed more than 99% similarity with S. marcescens ACE2, Bacillus cereus ACE4, Bacillus subtilis AR12, B. cereus AR14, P. aeruginosa AI1, Klebsiella sp. ACP, Pseudomonas sp. AP2, Bacillus litoralis AN1, B. cereus AN4 and Bacillus sp. AN5 and NDB comparison revealed more than 99% similarity with S. marcescens AR1, Bacillus pumilus AR2, Bacillus carboniphilus AR3, Bacillus megaterium AR4, and B. cereus AR5 has been deposited. The nucleotide sequences of 16S rDNA sequenced has been deposited in GenBank database under accession numbers DQ092416, AY912105, EF535590, EF535591, EF535592, EF535593, EF535594, EF535595, EF535596 and EF535597 (Rajasekar et al. 2010). Five naphtha degrading bacteria including representative strains of the two classified species S. marcescens AR1, B. pumilus AR2, B. carboniphilus AR3, B. megaterium AR4, and B. cereus AR5 were isolated and identified in a naphtha transporting pipeline. The 16S rDNA sequences are deposited in the gene bank with accession numbers DQ207558, DQ207559, DQ207560, DQ207561 and DQ207562.

The phylogenetic trees were constructed to find out the evolutionary interrelationship among various bacterial species present in the environment. The percentage distribution of individual species in diesel pipeline is presented in Fig. 13.2. 30% B. cereus, 10% of S. marcescens, 10% each species of Bacillus substilis, Klebiella sp., B. litoralis, Bacillus sp., Pseudomonas sp. and P. aeruginosa. Strains isolated from the corrosion product of naphtha transporting pipeline at South West India are presented in Fig. 13.3.

Sequence alignment and comparison revealed more than 99% similarity with S. marcescens AR1, B. pumilus AR2, B. carboniphilus AR3, B. megaterium AR4, B. cereus AR5, The nucleotides sequences data have been deposited in GenBank under the sequence numbers DQ207558, DQ207559, DQ207560, DQ207561 and DQ207562. The four isolates (AR2, AR3, AR4 and AR5) belong to the Bacilla-ceae family and one isolate AR1 to Enterbacteriacae. Bacillus sp. is the dominating genus in the pipeline, although all the five species distributed equally. Among the fifteen sequences in petroleum transporting pipeline, ten sequences were from Gram-positive bacteria. The dominant bacterial species in diesel and naphtha pipeline were identified as B. cereus and S. marcescens. All the bacterial strains exhibit catalase and oxidase positive. The naphtha and diesel degraders

Table 13.2 Biochemical characterization of isolates from diesel pipeline

Characteristics

ACE2

ACE4

ARI2

AR4

All

ACP

AP2

AN1

AN4

AN5

Cell morphology

Gram stain

Negative

Positive

Positive

Positive

Positive

Negative

Positive

Positive

Positive

Positive

Shape

Rod

Rod

Rod

Rod

Rod

Small rod

Rod

Rod

Rod

Rod

Motility

+

+

+

+

+

+

+

+

+

+

Sporulation

-

+

+

+

+

+

+

+

+

Growth at

20°C

+

+

+

+

+

+

+

+

+

+

30°C

+

+

+

+

+

+

+

+

+

+

40°C

+

+

+

+

+

+

+

+

+

+

Hydrocarbon utilization

Diesel

+

+

+

+

+

+

+

+

+

+

Biochemical reaction

Indole production test

-

+

-

-

-

-

+

+

+

Methyl red test

-

-

+

+

+

+

+

Voges Proskauer test

+

-

-

-

-

+

Citrate utilization test

+

-

+

+

+

+

-

+

+

Oxidase test

+

+

+

+

+

+

+

+

+

+

Catalase test

+

+

+

+

+

+

+

+

+

+

Production of acid from

Glucose

+

+

+

+

+

+

+

+

+

+

Galactose

-

-

+

+

-

+

-

-

Fructose

+

+

+

+

+

+

+

+

+

+

Sucrose

-

+

-

-

-

-

+

-

-

Mannitol

-

-

-

-

-

+

-

-

+

Lactose

-

-

+

+

+

-

-

+

+

+

Cellobiose

-

-

+

+

+

-

-

+

+

+

Adonitol

-

-

-

+

(continued)

Characteristics ACE2 ACE4 ARI2 AR4 All ACP AP2 AN1 AN4 AN5

Arabinose

+

+

+

+

Raffinose

-

-

-

-

-

+

-

-

+

-

Inositol

-

-

-

-

-

+

-

+

-

-

Hydrolysis of

Starch

-

+

+

+

+

+

+

+

+

+

Cellulose

-

-

-

-

-

+

+

-

+

-

Casein

-

+

-

-

-

+

+

+

-

+

Gelatin

-

+

+

+

+

-

-

+

+

+

Urea

-

+

-

-

-

-

+

-

-

-

Tween 80

+

+

-

-

-

+

+

+

+

+

ACE2—Serratia marcescens, ACE4—Bacillus cereus, AR12—Bacillus subtilis, AR14—Bacillus cereus, AP2—Pseudomonas stutzen, ACP—Klebsiella oxytoca, All—Pseudomonas aeruginosa, AN1—Bacillus litoralis, AN4—Bacillus cereus and AN5—Bacillus sp.

ACE2—Serratia marcescens, ACE4—Bacillus cereus, AR12—Bacillus subtilis, AR14—Bacillus cereus, AP2—Pseudomonas stutzen, ACP—Klebsiella oxytoca, All—Pseudomonas aeruginosa, AN1—Bacillus litoralis, AN4—Bacillus cereus and AN5—Bacillus sp.

Table 13.3 Partial biochemical characterization of isolates from naphtha pipeline

Characteristics

Cell morphology

Gram stain

Shape

Motility

Sporulation

Growth at

Hydrocarbon utilization Diesel

Biochemical reaction

Indole production test

Methyl red test

Voges Proskauer test

Citrate utilization test

Oxidase test

Catalase test

Production of acid from

Glucose

Fructose

Sucrose

Mannitol

Lactose

Cellobiose

Adonitol

Arabinose

Raffinose

Inositol

Hydrolysis of

Starch

Cellulose

Casein

Gelatin

Urea

Tween 80

Negative Rod

Positive

Positive

Positive

Positive

AR1—Serratia marcescens, AR2—Bacillus pumilus, AR3—Bacillus carboniphilus, AR4— Bacillus megaterium, AR5—Bacillus cereus

were able to growth at 20, 30 and 40°C. Most of the Gram-positive species identified are clustered while Gram-negative species, such as Pseudomonas sp. and Klebsiella sp. are grouped away from the former species (Fig. 13.4).

10% 10% Fig. 13.2 Distribution of diesel degrading bacteria isolated from diesel pipeline

□ Bacillus cereus

□ Serratia marcescens

□ Bacillus subtilis

□ Pseudomonas sp. s Bacillus litoralis

□ Pseudomonas aeruginosa

10% 10% Fig. 13.2 Distribution of diesel degrading bacteria isolated from diesel pipeline

20%

Fig. 13.3 Distribution of naphtha degrading bacteria isolated from naphtha pipeline

□ Bacillus cereus

□ Serratia marcescens El Bacillus pumilus

□ Bacillus carboniphilus

□ Bacillus megaterium

Fig. 13.3 Distribution of naphtha degrading bacteria isolated from naphtha pipeline

The similarity and species identified with the phylogenetic analysis are given in Table 13.3. In the Bacillus genera tree, most of the isolates belonging to Bacillus genus exhibited a high nucleotide sequence similarity with B. cereus species

7%

Fig. 13.4 Distribution of diesel/naphtha degrading bacteria isolated from diesel and naphtha pipeline

EH Bacillus cereus

□ Serratia marcescens E3 Bacillus subtilis

SI Pseudomonas sp.

□ Bacillus litoralis

S Pseudomonas aeruginosa

□ Bacillus cereus

□ Serratia marcescens

□ Bacillus pumilus

0 Bacillus carboniphilus

□ Bacillus megaterium

Fig. 13.4 Distribution of diesel/naphtha degrading bacteria isolated from diesel and naphtha pipeline

(99.5%). Similarly, in the tree corresponding to the Bacillus genus, most isolates were related to B. subtilis, B. pumilus, B. carboniphilus, B. litoralis and B. megaterium with high similarity values (98%). In the Classes Enterobacteriales, two isolates of the genus Serratia exhibited high sequence similarity with S. marcescens (99%) and one isolate of Klebsiella genus (Klebsiella oxytoca). Two isolates of genera Pseudomonas had high similarities with P. aeruginosa and Pseudomonas stutzeri (98%) in the Pseudomonadales tree. Phylogenetic relationship was analyzed between the 16S rDNA sequences of the bacterial phyla Firmicutes (Fig. 13.5) and Proteobacteria in the three phylogenetic trees (Figs. 13.6, 13.7). The phylogenetic analysis of the isolates showed the dominance of Bacillus sp., while B. cereus could be seen in dominant species among other species of genus Bacillus. Serratia sp., Pseudomonas sp. and Klebsiella sp. were present among phylum Gamma proteobacteria (Table 13.4).

The ability of enteric bacterium, S. marcescens to degrade hydrocarbon appears to be an unexpected finding since this feature has always been associated with typical soil bacteria. Besides, enteric bacterium is mainly regarded as an inhabitant of animal guts (Holt et al. 1994). Present understanding of role of hydrocarbon degradation on corrosion by enteric bacterium is still in its infancy. This study provides a new insight into the influence of degradation of diesel fuel on corrosion by enteric bacteria. Such studies can help in adopting efficient and predictable bioremediation strategies. However, only a few reports

Bsciflua ar>tt>rmci& (AM747220)

B*cHtu& tserovat ie-ns^/ow'-fi (EU429671) tfitiringtonaia serovar (ELM 2967 2;

Sscittua rhuririCfie(FJSS£030} Baciifua ap. (EF052509) BacUJua c«rava (EU115117) ACE«

GacMua smith» (AY373319) flec/Mjs sp. (FJ937S43) Bacillus cosgw/sn« (FJ978S33)

Sac'-Vy« f\)jrr»itj& (F".J434B57) ©■sctf/tvs t>umik)& (EU855216) AR2

(C3Q1S2134)

Baaftua magat&num (FJ52763B) Bacillus megalesiurr? (GQ152133) Saei'/ua AubfWs (FJ5276S9) Bso/lue sp (064670) BaciHac&ae bacterium (FJ1S97B3) ¿acfer/m) (FJ1S9761) B3C-///US sp (ABQ62678) Bsc^a Bp. (GQ249996)

-BeciV/ua i/iuwi¿»arts/a (E Ffi 169GS) -Bacittua sp. (AQ242B65)

BaciHua a/tack f&tor?// (AJ25031B) Baoiiiua piehiton\/i (A.F5194E3) Beciffua aubtitia (GO280380) Saciiiua thurr'nt)t&nais (FJ462697) B*c >ffuA /ae /a (A Y13Q303) Bacillua cereus (FJ6B6379) AR5

Baoiiua carbonipfitlua (ABO21192) 1—AR3

fn\>cokJ&a (FJ393303) BocffktQ C!>w«a (FJ393296) AN4

- Bscitiua ceraua (GQ1494Q1) IT«WUJ(SQ3448D6)

AR14

Ba (-BbciHus mafacitenata (AY6D3656)

-Gaciiiua p/chtrtoti/' (AF519464)

Baciitua acktocota (AFS472DS)

- axarqui+naia (AY6036S7)

86 fiiibiI'IVS(AJE337D9)

'AR12

Baciftua amt/foSiiyuef ec e (FJ370040)

-BacHiua iKbanifoffrtia (EU304938)

-Bacillus caraua (GQ14991 4)

yOcaanabacif/ua profundus (DCl3G©S35) .OcMnobsctf/us •soy.s-(AB473SG1) 11 Bacittu* itiurfngian*»'«(FJ393313)

IAN1

-Gfoatr/cfium sporogsnea (AM237439)

Fig. 13.5 Neighbor-joining tree based on 16S rRNA gene sequences, showing phylogenetic relationships between sequences of the bacterial phylum Firmicutes (Bacillus related species). Clostridium sporogenes was used as a bacterial outgroup. Numbers at nodes indicate bootstrap values >50% from 1000 replicates. GenBank accession numbers are given in parentheses. The scale bar indicate sequence divergence. ACE4—Bacillus cereus, AR2—Bacilluspumilus, AR4—Bacillus megaterium, AN5—Bacillus sp., AR5—Bacillus cereus, AR3—Bacillus carboniphilus, AN4— Bacillus megaterium, AR14—Bacillus cereus, AR12—Bacillus subtilis, AN1—Bacillus litoralis

Xenobiotics

Fig. 13.6 Neighbor-joining tree based on 16S rRNA gene sequences, showing phylogenetic relationships between sequences of the Order Enterobacteriales (phylum Proteobacteria). Pseudomonas aeruginosa was used as the out group sequence. Numbers at nodes indicate bootstrap values >50% from 1000 replicates. GenBank accession numbers are given in parentheses. The scale bar indicate sequence divergence. AR1—Serratia marcescens, ACE2— Serratia marcescens, ACP—Klebsiella oxytoca

Fig. 13.6 Neighbor-joining tree based on 16S rRNA gene sequences, showing phylogenetic relationships between sequences of the Order Enterobacteriales (phylum Proteobacteria). Pseudomonas aeruginosa was used as the out group sequence. Numbers at nodes indicate bootstrap values >50% from 1000 replicates. GenBank accession numbers are given in parentheses. The scale bar indicate sequence divergence. AR1—Serratia marcescens, ACE2— Serratia marcescens, ACP—Klebsiella oxytoca address utilization of aromatic compounds by Enterobacteria, particularly those of the genera Klebsiella, Escherichia and Hafnia (Grant 1967; Ijah 1998; Diaz et al. 2001). Although there are several reports of bioremediation of high-molecular-weight polyaromatic hydrocarbons (PAHs), research findings pertaining to biodegradation of these substances by enteric bacteria has been relatively rare (Diaz et al. 2001; Kanaly and Harayama 2000). Sarma et al. (2004) discovered a new enteric bacterium L. adecarboxylata PS 4040 in oil sludge-contaminated soil and reported the degradation of PAH at the Digboi Oil Refinery in Northeastern India.

911 Pseudomonas aeruginosa(EF535592) ' Pseudomonas aeruginosa^AY521230)

Pseudomonas aeruginosa (EU6617Q7) Pseudomonas aeruginosa (FJ4B3254) Pseudomonas aeruginosa^J2272BD) Pseudomonas aerag/nosa(AY4B6353) Pseudomonas okovorans (GQ365203) Pseudomonas sp.(EF523551) Pseudomonas f/uorescens bv. C (AF228367) Pseudomonas Jinaci(i™ssw(AF039489) Pseudomonas aicaliphila (EU0B2B32) Pseudomonas ofii/oi's(EF523547) Pseudomonas Blcatigenes(FJEi2A'\]9) Pseudomor&s borboritfkM] 14531) Pseudomonas f/avescens(EU221398) Pseudomonas o!M/s(EU239122) Pseudomonas antarctica(AM93351 B) Pseudomonas stutzeri (GQ1609DS) AP2

Pseudomonas sfufzeri (GQ28D051)

Pseudomonas stuizeri (GQ2S445B)

Pseudomonas atuizaw(FJ982924)

Pseudomonas puh'iii (EU849665) 88

I-Pseudomonas acetoxians (DQ981784)

'Pseudomonas sp,(FN377741)

-Pseudomonas sp. (GQ280063) -Escherichia coli (FJ405332)

Fig. 13.7 Neighbor-joining tree based on 16S rRNA gene sequences, showing phylogenetic relationships between sequences of the Order Pseudomonadales (phylum Proteobacteria). Escherichia coli was used as the out group sequence. Numbers at nodes indicate bootstrap values >50% from 1000 replicates. GenBank accession numbers are given in parentheses. The scale bar indicate sequence divergence. AI1—Pseudomonas aeruginosa, AP2—Pseudomonas stutzeri

Recently, Wongsa et al. (2004) identified S. marcescens which has the ability to degrade the broad spectrum of hydrocarbons present in crude oil-contaminated soil. Muthukumar et al. (2003a, b) reported that Brucella sp. and Gallionella sp. could degrade diesel in a transporting pipeline in Northwest India, while Rajasekar et al. (2005) detected bacterial genera Pseudomonas sp., Bacillus sp., Gallionella sp., Siderocapsa sp., Thiobacillus sp., Thiospira sp., Sulfolobus sp. and Vibrio sp. in a naphtha pipeline, and reported on

Table 13.4 Cultivable bacterial isolates associated with biodegradation in petroleum product pipeline

S. no.

Strain

GenBank accession number

Best match

Diesel degrading bacteria

1.

ACE2

DQ092416

Serratia marcescens

2.

ACE4

AY912105

Bacillus cereus

3.

AR12

EF535590

Bacillus subtilis

4.

AR14

EF535591

Bacillus cereus

5.

AI1

EF535592

Pseudomonas aeruginosa

6.

ACP

EF535593

Klebsiella sp.

7.

AP2

EF535594

Pseudomonas sp.

8.

AN1

EF535595

Bacillus litoralis

9.

AN4

EF535596

Bacillus cereus

10.

AN5

EF535597

Bacillus sp.

Naphtha degrading bacteria

11.

AR1

DQ207558

Serratia marcescens

12.

AR2

DQ207559

Bacillus pumilus

13.

AR3

DQ207560

Bacillus carboniphilus

14.

AR4

DQ207561

Bacillus megaterium

15.

AR5

DQ207562

Bacillus cereus

interactions between heterotrophs and chemolithotrophs in naphtha-transporting pipelines Southwest India. Hence, an investigation on the identification of bacteria in oil transporting pipelines is worthwhile for petroleum degradation control.

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