Culture based approaches, dependent and independent, for microbial community fractions in petroleum oil reservoirs

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Culture based approaches, dependent and independent, for microbial community fractions in petroleum oil reservoirs

Sunita Varjani^1,2*, Vijay Kumar Srivastava³, Raveendran Sindhu^2,4, Indu Shekhar Thakur^2,5 & Edgard Gnansounou²

1Gujarat Pollution Control Board, Sector-10A, Gandhinagar-382 010, Gujarat, India

2Bioenergy and Energy Planning Research Group (BPE), IIC, ENAC, Station 18, Ecole Polytechnique Fédérale de Lausanne (EPFL), CH-1015 Lausanne, Switzerland

3GSFC University, Fertilizernagar, Vadodara-391 750, Gujarat, India

4Microbial Processes and Technology Division, CSIR-National Institute for Interdisciplinary Science and Technology (NIIST), Thiruvananthapuram-695 019, Kerala, India

5School of Environmental Sciences, Jawaharlal Nehru University, New Delhi-110 067, Delhi, India Received 15 November 2017; revised 08 January 2018

Petroleum reservoir is an ecosystem having extreme environmental conditions of temperature, pressure and salinity. They possess highly anoxic conditions. Major microbial communities present in this environment include fermentative bacteria, sulphate reducing bacteria (SRB), syntrophic bacteria and methanogens. Phylogenetic diversity of microorganisms as well as their ecological role play an important role in the petroleum reservoir ecosystem. Past and present efforts to characterize microbial communities in oil field ecosystem by culture or cultivation-dependent and -independent approaches are discussed with highlights of microbial ecology of petroleum oil reservoir ecosystem(s). Novel strategies used to study culture independent diversity of microorganisms using metagenomic techniques have also been narrated.

Keywords: Community genomics, Metagenomics, Microbial ecology, Sulfate reducers, Thermophiles

Branch of microbiology dealing with the study of microbes, which can either metabolize or alter crude or refined petroleum products is known as petroleum microbiology

. Sulfate-reducing bacteria, methanogens, fermentative bacteria as well as iron-, nitrate- and manganese- reducing bacteria are found in petroleum oil reservoir ecosystems

. Microbial groups depending on types of reservoirs, can be psychro-, meso- and thermophilic as well as halo- and barophilic

.

Importance of microbial community studies in petroleum oil field ecosystem has been studied earlier

. Our understanding of phylogenetic diversity, metabolic capabilities, ecological roles of microbial communities’ present in petroleum oil field reservoir ecosystem is in its infancy

. Petroleum oil reservoir microbial community studies are divided into major two parts: (a) cultivation dependent

; and (b) cultivation independent

. Only a small fraction of microorganisms is currently cultured from environmental samples including petroleum oil reservoirs

. If a microorganism is cultured then its role in a community and contribution to ecosystem

function is not necessarily revealed

. However, culture-independent 16S rDNA gene-based assays are useful to provide overall view of community composition in specific ecosystem, which is not dependent on metabolic abilities of community members

. Pros and cons of culture dependent and independent techniques are narrated earlier

. Cultivation independent studies also provide preliminary information about relative abundance of different groups within ecosystem and could be used to monitor temporal and spatial changes in particular ecosystem

. Various cultivation-independent 16S rDNA gene-sequencing assays have been reported in the petroleum reservoir ecosystems

. Worldwide, cultivation-dependent and cultivation-independent assays have been used to explore about microbial communities present in the oil fields viz. China

, India

, Tataria and Western Siberia

, Africa

, United Kingdom

, Argentina

, Canada

, United States of America

, Saudi Arabia

, Brazil

and Japan

.

This review article discusses about various types of microorganisms present in the petroleum oil reservoir ecosystem. It also provides updated information about the cultivation or culture-dependent and -independent

—————

*Correspondence:

E-mail: drsvs18@gmail.com

approaches used to study microbial communities of different oil fields.

Microbial ecology of petroleum oil reservoir ecosystem

Various groups of microorganisms belonging to diverse phylogenetic affiliations have been studied from petroleum oil reservoir ecosystem

. These microorganisms possess different physiological and metabolic activities

. The ability of microorganisms to survive in underground deep ecosystem is coupled with proved abilities of anaerobic microorganisms to utilize various components of crude oil

Presence of indigenous microbial communities (diverse bacterial and archaeal communities) in deep petroleum oil reservoirs has been proved by various research groups

. Sulphate reducing bacteria belonging to the genera Desulfotomaculum, Desulfacinum, Desulfovibrio, Desulfobacterium, Desulfovermiculus, Desulfotignum, Desulfonauticus, Marinobacterium, Oceanobacter, Halomonas, Thermodesulfobacterium, Desulfuromonas and Desulfobolus have been reported from petroleum oil reservoirs

. Methanogens from the genera Methanobacterium, Methanosarcina, Methanoplanus, Methanosaeta, Methanolobus and Methanothermobacter have been studied in different oil ecosystems

. Presence of fermentative bacteria from petroleum oil reservoirs belonging to the genera Geotoga, Thermotoga, Spirochaeta, Marinobacterium and Halomonas has been reported

. Apart from these major three groups, microorganisms of genera Deferribacter, Geobacillus, Shewanella and Thermovibrio having ability for iron, nitrate and manganese reduction have also been detected in petroleum oil fields

.

Studies on microorganisms in petroleum oil reservoir ecosystem

Petroleum hydrocarbon metabolizing microorganisms are widely distributed in petroleum reservoirs.

Approaches to catalogue microbial community diversity and function can be divided into cultivation- dependent and cultivation-independent studies

. This section explores various cultivation-dependent and cultivation-independent studies performed globally in petroleum oil reservoir ecosystem.

Cultivation-dependent and cultivation-independent techniques to study microorganisms present in petroleum oil reservoirs are enlisted in Table 1.

Cultivation-dependent studies in petroleum oil reservoirs

Cultivation-dependent studies are also known as traditional/culture-dependent methods to study microbial diversity of the ecosystem

. These

techniques have yielded valuable information about microbial interactions with petroleum hydrocarbons in oil fields

. Culture dependent methods are based on differential cellular, morphological, metabolic and physiologic traits of microorganisms

. These studies include isolation and cultivation of microorganisms from collected sample(s) using solid media, most probable number (MPN) type liquid media and Biolog plates to study substrate utilization profile of isolate for its identification

.

Enrichment, Isolation and characterization of bacterial and archaeal species having ability to perform their metabolic processes in petroleum oil fields have been reported

. Culture-dependent characterization of microbial communities depends on several factors that limit its microbial activities in oil reservoir ecosystems to completely describe ecosystem’s community

. Microorganisms that are easy to obtain in pure cultures are not necessarily abundant and/or metabolically active in situ, therefore isolation of a single microorganism which mediate a specific metabolic process generally do not represent the entire community present and performing in situ processes in petroleum oil reservoir

. The media routinely used for isolation of microorganisms present in the petroleum oil reservoirs are often carbon and nitrogen-rich compared to prevailing environmental

Table 1 — Cultivation-dependent and cultivation-independent techniques to study microorganisms in petroleum oil reservoirs Type of Technique Name of Technique and References Cultivation-dependent

studies

Cultivation of microorganisms using solid media20,42,61,62,63,66,67

Most probable number (MPN) using liquid media18,17,62,63,64,66,67

Biolog plates^18,16,71 Cultivation-

independent studies

Fluorescence in situ hybridization (FISH)⁷¹

DNA microarrays¹

Denaturing gradient gel electrophoresis (DGGE)7,15,22,23,72,73

16s rDNA sequencing^2,21,23,73 DNA re-association⁷³

Reverse sample genome probing^22,72 Ribosomal intergenic spacer analysis^23,27 Restriction fragment length

polymorphisms (RFLP)^7,15,22 Terminal-RFLP (T-RFLP)^7,22,36 Random amplified polymorphic DNA analysis (RPAD)27,31,36,72

Amplified ribosomal DNA restriction analysis (ARDRA)^27,72

Real time PCR (RT-PCR)/quantitative PCR (qPCR)^36,72

condition in the oil field ecosystem

. Culturing from samples collected at specific time and production stage of oil reservoir does not seize changes in microbial community occurring throughout entire oil exploration activities

. It has been reported that as compared to high temperature petroleum oil reservoirs less number of isolates are obtained from low- temperature oil reservoirs

. This limits to study distribution patterns of microbial communities in petroleum oil reservoir ecosystem. Isolation of Spirochaeta has been reported from oil field ecosystem already

. However, this study could not establish intra-phylum diversity, abundance and distribution pattern for phyla members in other oil reservoir ecosystems

. It is difficult for microbiologists to isolate a large fraction of microorganisms present in natural ecosystems including oil field reservoirs

.

Broadly distributed and diverse collection of bacteria, yeasts and fungi capable of hydrocarbon utilization from oil reservoirs and hydrocarbon- impacted environments globally has been reported by using plating using agar plates

. For enumeration and isolation of hydrocarbon metabolizing microorganisms from petroleum samples, it is necessary to keep proper control along with test samples

. Non-hydrocarbon metabolizing microorganisms were also reported along with hydrocarbon metabolizers on agar plate

. To overcome the problem with trace carbon in agar plates, some researchers have used silica gel as a solidifying agent. Due to tedious procedure of plate preparation it could not been established as widely used method to study petroleum hydrocarbon metabolizers. If isolates are not required, MPN test can be used to study metabolism of petroleum hydrocarbons based on emulsion formation that also avoids problem of trace carbon contamination

. Redox indicators can also be used to screen hydrocarbon degraders

. Various studies have been performed to describe interactions of microorganisms between themselves and petroleum hydrocarbon by detailed laboratory experimental studies with isolates from petroleum hydrocarbons polluted environments

. Researchers have also constructed consortia containing well-defined strains to identify processes occurring in petroleum hydrocarbon polluted environment

. These studies are primary for understanding general mechanisms of petroleum hydrocarbon degradation; however they do not reveal environmental importance

. To achieve a greater understanding of petroleum hydrocarbon degradation

mechanisms molecular biology and biochemistry of petroleum pollutants degradation processes are required to be understood in detail so that gene expression can be correlated to these activities

. In recent study, researchers have reported the application of halotolerant hydrocarbon degrading bacterial consortium that was obtained from on-shore sites of Gujarat, India; as bioremediation agent to manage terrestrial oil spills. The consortium was made up of six bacterial sp. of genera Ochrobactrum, Stenotro- phomonas and Pseudomonas. Authors have concluded that co-metabolic processes are required for effective crude oil degradation in petroleum hydrocarbon polluted sites

. Apart from isolation and identification of microorganisms present in petroleum hydrocarbon affected environments, microbial communities in such environments are described solely on functional characteristics

. If selective media are used, MPN assays can be applied to describe microbial communities into physiological types

. MPN assays have been widely used for studying anaerobic environments

. In situ bioremediation of waste oil- contaminated sites have been reported earlier, in the experiment researchers have used MPN technique to enumerate microbial communities present in petroleum oil polluted sample(s) and reported presence of oil- degrading denitrifiers, methylotrophs, facultative anaerobes, sulfate reducers and anaerobic vacuum gas- oil degraders

. However, some researchers have performed studies to reduce souring of sweet crude oil by sulphate reducing bacteria applying MPN technique in which they have used samples from western Canadian oil field waters. They have reported that nitrate addition in petroleum reservoir samples stimulates heterotrophic nitrate-reducing bacterial growth that outcompete sulfate-reducing bacteria

.

Total community analyses by culture dependent

method can be studied using Biolog substrate

utilization patterns

. Biolog substrate utilization

profiles to study metabolic and functional diversity

of microbial communities in continuous-flow

cultures containing hexadecane polluted intertidal

sediments. Hexadecane degradation rate, protein

production rate and oxygen consumption rate were

determined to study microbial activities. The cell

concentration of total heterotrophs, nitrate reducers,

sulfate reducers was determined by colony forming

units (CFU), and MPN. The metabolic diversity of

hexadecane utilizers was determined by the substrate

utilization profile using Biolog microtiter plates

.

Metagenomic studies: Cultivation-independent studies in petroleum reservoirs

Cultivation or culture-independent or metagenomic approaches to study microbial communities present in the crude oil impacted environments as well as petroleum oil reservoir ecosystem have become popular in the last two decades

. Molecular techniques to study microbial communities dominate the literature available in the petroleum oil reservoir microbiology

. Several culture- independent sequencing surveys have been conducted in high and low temperatures as well as in marine and terrestrial oil reservoirs

. Steps involved in cultivation independent studies of microbial communities present in the petroleum oil reservoir samples are schematically represented in Fig. 1.

Cultivation-independent approaches for community analysis began with direct examination of metabolically active microorganisms using fluorescence in situ hybridization (FISH), bulk analysis of total protein banding and phospholipid fatty acid analysis

. Nowadays, PCR-based approaches are applied in research to study specific microorganisms or groups of microorganisms and specific genes to evaluate overall community profiles in a petroleum oil field ecosystem

. Methods to evaluate community profiles include denaturing gradient gel electrophoresis (DGGE), reverse sample genome probing, ribosomal intergenic spacer analysis, restriction fragment length polymorphisms (RFLP), Terminal-RFLP (T-RFLP), single-strand conformation polymorphism, internal transcribed spacer-restriction fragment length poly- morphism, random amplified polymorphic DNA, amplified ribosomal DNA restriction analysis and Real time PCR (RT-PCR)/quantitative PCR (qPCR)

. Recently, developments to use DNA microarrays have attracted the attention of environmental microbiologists for more rapid throughput to allow tracking of thousands of genes at one time

. Cultivation-independent assays are also affected by contamination and re-inoculation during water flooding

.

It is important to study microbial diversity of petroleum oil reservoir ecosystems for studying indigenous microorganisms

. Analysis of 16S rDNA recovered from petroleum reservoir ecosystem has revealed unrecognized microbial diversity in different habitats including soils, sediments, hot springs and terrestrial subsurface environments

. DGGE, 16s rDNA sequencing and DNA re- association method in combination with measurement of methane and methanol oxidation to study diversity of methanogens have been used earlier

. Metagenomic/ culture independent approaches have been proved to be a boon for studying diversity of various petroleum oil field ecosystems to characterize microbial communities viz. (a) deep sea sediments/

subsurface water

(b) high temperature petroleum reservoirs

(c) low temperature oil fields

(d) oil field water

(e) oil well

(f) oil pipeline

and (g) petroleum hydrocarbons polluted soil

.

The 16S rDNA gene clone libraries to characterize microbial communities present in production water from a high-temperature oil reservoir ecosystem have been constructed by researchers. Pseudomonas, Petrobacter sp., Anareolineales, β-, γ-, and δ-Proteo- bacteria were detected in the ecosystem studied.

Presence of methanogens of the genus Methanosaeta and Methanothermobacter as well as Crenarchaeotes- related microorganisms in the petroleum oil field ecosystem have been reported

. Microbial communities present in heavy crude oil from Saudi Arabia using PCR-based culture independent methods have been studied earlier

. They have reported 30 distinct bacterial sequences in 177 clones obtained.

Bacterial species of genera Bacillus, Clostridia and Gamma-proteobacteria were reported in crude oil samples, however members of Alphaproteobacteria, Beta-proteobacteria, Gammaproteobacteria, Clostridia, Spingobacteria and Flavobacteria were reported in oily sludge sample studied

. Recently, diversity and abundance of potential functional genes of microbial communities present in oil field ecosystem of China using PCR based clone libraries construction coupled with quantitative real-time PCR analysis have been performed. Scientist have reported that bacteria of the genus Thermodesulfovibrio possessing the highest abundance and constituted 49% of all of the 81 clones detected. Apart from Thermodesulfovibrio other bacteria of genus Anaerolineaceae, Actinobacteria, Bacteroidetes, Synergistia and Firmicutes were also reported

.

Fig. 1 — Schematic representation of steps involved in cultivation independent/molecular characterization of the petroleum oil reservoir samples

Conclusion

Culture or cultivation-based approaches play important role in understanding the physiological potential of microorganisms. However, they may not provide comprehensive information about composition of ‘complex communities’ present in a petroleum reservoir ecosystem. Biodiversity of oil field ecosystem is often dominated by uncultured microorganisms.

Application of the molecular methods, such as nucleic acid probes, cloning & sequencing of 16S rRNA, gene amplification and DNA microarrays to study microbial ecology of petroleum reservoirs would help us to constitute important set of data regarding microbial communities in this ecosystem.

Acknowledgement

Authors Sunita Varjani, Raveendran Sindhu and Indu Shekhar Thakur are thankful to Ecole Polytechnique Federale de Lausanne, Switzerland for visiting fellowship.

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