Literature Review

This paper suggests a standard protocol for sampling and extracting biological material from natural asphalts. I have used the most prominent primary research in DNA extraction from natural asphalt reservoirs and compiled the experimental procedures. The results are an in depth review on the successes and shortcomings of two main experimental procedures: the sampling of petroleum and the successful extraction of DNA. I suggest these protocols with the hope to provide the community with a guide that can make performing experiments in difficult conditions more accessible. Through this I seek to show the benefits of conducting research in extreme environments with the hope of learning more about the microorganisms that inhibit them.   

Introduction

The exploration of extreme environments and the resilient organisms that inhabit them has progressed over the past decade. Extremophiles have sparked curiosity in a range of disciplines. The search for extraterrestrial life and biotech applications by analyzing organisms’ unique adaptations are popular fields of study (Rothschild & Mancinelli, 2001). Naturally occurring oil seeps and asphalts host communities of extremophiles displaying their economic potential through bold adaptations (Kim & Crowley, 2007). Petroleum hydrocarbons, in the form of liquid and gas, migrate through cracks and fissures and spill along the surface of the earth (Kim & Crowley, 2001; Palabiyik & Ozdemir, 2019). 

Natural asphalts are commonly referred to as tar pits and come in different shapes and sizes (Fig. 1 & 2). With this diversity of structure comes microbial diversity (Schilze-Makuch, 2011). Active communities – many of them novel - include bacteria and archaea (Kim & Crowley, 2001; Schilze-Makuch, 2011). The methodologies used to sample and extract DNA from the recalcitrant natural asphalt are as novel as some of these strains. Therefore, liquid petroleum hydrocarbon sampling and extraction presents an opportunity to explore different techniques as no standard exists. Instead, previous studies adopt soil sampling and DNA extraction protocols (Kim & Crowley, 2001). Evidently, soil and petroleum have distinct textures and compositions and so the effectiveness of using soil protocols is in question. This paper analyzes different methods used to gain a better understanding of petroleum microbiology. 



Figure 1. (A) Overview of Pitch Lake, a naturally-occurring asphalt seep. (B, C and D) Varying representations of tar pits. (Schulze-Makuch et al., 2011) 



Figure 2. (A, B, and C)  Varying representations of Rozel Point Tar Seeps (Picture credit: Cayla Martin) 

Methodology 

Sampling Protocols 

The uncooperative nature of asphalt makes sampling difficult. Additionally, the behavior and composition of tar pits vary and therefore influence characteristics that affect sampling. For example, Kim and Crowley (2007) sample 10 cm subsurface, which is possible because the asphalt is soil-permeated. On the other hand, Schulze-Makuch et al. (2011) work with higher velocity asphalt, which makes subsurface sampling challenging. Unique characteristics of tar pits allow researchers to exercise creativity in sampling protocols as studies of important applications of microbial diversity in natural asphalts are still in their infancy (Magot, 2000). However, suggested sampling protocols can be useful to new researchers in this field, or even individuals interested in expanding on existing studies. Discussion items are reviewed and developed from studies published in accredited journals that proved successful in sampling and extracting DNA from natural asphalts. 

Asphalt Sampling Protocol Discussion Points: 

• As mentioned, the depth of sampling can depend on the viscosity of the tar pits. However, consistency between each sample is recommended. Suggested sampling depth increments are 0-5, 5-10, 10-20 cm (USDA-NRCS, 2004). 
• Roughly 500g should be collected for each sample. Obtaining high-quality DNA to be used in cloning and sequencing can be challenging and therefore samples may need to be pooled (Kim & Crowley, 2007). 
• Autoclaved spatulas and sterile 50-ml plastic tubes with screw caps ensure minimal contamination (Kim & Crowley, 2007; Schulze-Makuch et al., 2011) 
• Samples should be placed on ice and kept cool during fieldwork and transportation. The optimal temperature is maintained at 4℃ as much as possible (GRACEnet, 2005). 
• Frequency and timing are dependent on the unique attributes of the study. 

Extraction Protocols 

DNA extraction calls for creativity. As of now, no asphalt DNA extraction kits exist. Previous studies have used soil extraction kits according to the manufacturer’s protocols (Kim & Crowley, 2007; Schulze-Makuch et al., 2011; Balcom & Crowley, 2010). Experiments conducted at the Rancho La Brea Tar Pits (2007) is an example of extracting DNA from asphalt using soil kits.  Kim and Crowley (2007) were able to successfully extract DNA and create phylogenetic analyses, which will be expanded upon in the discussion of microbiology. However, it is important to note that adaptations needed to be made to the soil extraction protocols. As a result, it is reported that it was difficult to obtain high-quality DNA. This highlights the need to review and compare different methods and suggest helpful recommendations. 

For example, the first step, which is not listed in the soil kit’s protocols, is crucial in achieving successful DNA extraction. Namely, the manipulation of the texture of the liquid hydrocarbon to resemble soil. Kabir et al. (2003) discuss using liquid nitrogen to help grind the samples, which is largely utilized. However, if working in warmer conditions, the asphalt, being volatile, could melt before the extraction process is complete. The sticky texture thus interferes with the first listed step in the manufacturer’s procedure of homogenization and lysis (Mo Bio Isolation Handbook). Balcom and Crowley (2010) report that working quickly and using physical lysis is the most successful method in producing higher-quality DNA. Furthermore, some recommended modifications are suggested: 

• First use liquid nitrogen to ground the sample into a soil-like texture.  

• The sample is then mixed with a kit lysis buffer containing silica beads.   
• Complete extraction according to the manufactures protocols and treat the product with aseERASE. 
• Purify product following a standard phenol-chloroform extraction and ethanol precipitation.    
• Store at -20℃. 

Even with modifications, the soil extraction kit is not as effective at extracting DNA from asphalt as it is soil. As a result, samples have low concentrations of DNA (Kim & Crowley, 2007). Asphalt extraction presents the challenge of producing DNA that is of a high enough quality to move on to the next step of amplification and cloning. Suggested combative methods include purification and combination of subsamples. While such recommendations are useful in analyzing the microbial diversity of tar pits, future studies could investigate creating asphalt-specific extraction kits. In the meantime, the absence of asphalt-specific extraction kits present opportunities for scientists to exercise creativity with forging new methodology.  

The final step includes phylogenetic analysis of the isolated DNA (Stackebrandt et al., 1993). Once DNA has been extracted, it can be subjected to a range of applications including amplification and cloning. The specific methodology of analysis is dependent on the unique questions being asked in individual studies. Therefore, rather than discussing the well-reviewed topics of phylogenetic analysis methodology, I will explore the unique microbiology results. 

Microbiology 

The first paper to describe microbial communities in oil-producing wells was published in 1926 (Bastin et al., 1926). However, until recently, the general understanding was that naturally occurring asphalt reservoirs were lifeless (Magot et al., 2000). Our appreciation of extremophiles has blossomed over the past decade (Rothschild & Mancinelli, 2001). With this, our perceptions of metabolic pathways have broadened too, resulting in scientists exploring harsh environments. The microbial communities inhabiting natural asphalts are a platform to study unique adaptations to extreme environments and to change the discrimination that tar pits are lifeless. 

Magot et al. (2000) discuss natural asphalt environmental factors that are extreme, driving adaptation curves. High temperatures are characteristic of hydrocarbon reservoirs and increase with depth. While geothermal gradients can significantly differ between tar pits, evidence show a mean temperature increase of 3℃ per 100 m. No current studies have sampled and extracted from tar seeps at greater depths. Magot et al. (2000) argue that no microbial growth can be sustained at greater depths due to excessive temperatures. Namely, deep hydrocarbon reservoirs can surpass temperatures in situ of 130-150℃. Stability issues of biological molecules at such extreme conditions would make it surprising to discover microbial life at temperatures close to 150 ℃ (Charlier & Droogmans, 2005). However, since Tom Brock’s reports in1967, hyperthermophiles have optimum temperatures above 80℃. Therefore, future studies could include sampling at greater depths. This would further contribute to the tar pit microbial community database and add to the understanding of how resilient these extremophiles can be. 

Another adaptation is seen in how extremophiles have evolved unique metabolic machinery to use different sources of energy (Van Hamme et al., 2003). For example, microbial communities in tar pits obtain carbon and energy from petroleum. More general mechanisms have been explored and are represented in the data from the microbial analysis conducted at the Rancho La Brea tar pits (Kim & Crowley, 2007). The discovery of hundreds of new strains of bacteria and archaea and three new groups of enzymes were described for the first time. Some of the dominant groups revealed in sequencing analysis were halophilic archaea and two clusters of closely related species. Unclassified genera were similar to Nantonorubrum spp and Natrialba spp. The idea of closely related Archaea indicated that there must have been a selection for traits such as efficient hydrocarbon-degrading enzymes that enable these organisms to survive the extreme conditions (Baquiran, 2010). 

Bacteria were also evident in Kim and Crowley’s (2007) analysis with Gammaproteobacteria being the most dominant. Included in the clones were families in the order Chromaiales, Xanthomanadaceae, and Pseudomonadaceae. The identification of clones representing Rubroacteracea suggested that La Brea tar pits, and potentially other natural asphalts, select for organisms that can survive in mutagenic habitats (Baquiran, 2010). Furthermore, there were also dominant amounts of Pseudomonas sp. representing bacteria that possess diverse catabolic pathways (Kim & Crowley, 2007; Baquiran, 2010). In continuation, there were many unclassified clones including 7 new families of bacteria and one new order. The discovery of new communities is encouraging the expansion of research being conducted on microbiology at naturally occurring asphalt (Kim & Crowley, 2007). The extension of such research could bring the development of important applications. Creating protocols and directed kits could catalyze this and stimulate future research.   

Implications

Over the past decade, scientists have started to explore unlikely places for life to exist (De Vera et. al, 2012). With a deeper understanding of extremophiles has come a wave of diverse applications of the discovered knowledge (Van Hamme, 2003). This ranges from astrobiology applications and the exploration of life outside of earth to bioremediation and biotechnology (Schulze-Makuch 2011, Baquiran 2010). 

Areas that are polluted with industrial wastes or toxic compounds can potentially be treated with extremophiles that have the adaptive ability to use these compounds as sources of energy (Baquiran, 2010). Organisms with specialized enzymes can not only survive but thrive in these environments. Green chemistry and biotechnology can be used to help with the restoration and remediation of pollution that is otherwise difficult to clean up (Schmid et al., 2001). Enzymes available in nature to aid with this have been discovered in habitats such as tar pits, where communities of microbes have evolved. Some of these genes can be utilized in creating genetically modified organisms (GMOs), specialized for these applications (Baquiran, 2010). There have already been successfully constructed GMOs, combining different organisms’ enzymes and pathways (Dua et al., 2002, Chen et al., 1999, Paul et al., 2005, Timmis and Pieper, 1999). 

On the other hand, moving away from Earth, Astrobiologists have used organisms thriving in tar pits as a starting point to analyzing limits for life not only here on earth, but in the Universe (Rothschild & Mancinelli, 2001).  Microbial communities thriving in tar pits are modern-day analogs for life in similar extraterrestrial environments (Schulze-Makuch et al, 2011). For example, the hydrocarbon lakes on Titan, Saturn’s largest moon, are similar environments to the natural asphalts here on earth. Schulze-Makuch et al. (2011) argue that if life can be described as an intrinsic property of chemical reactivity, then it is largely possible for life to exist in conditions such as Titan. 

Conclusion 

Diverse and rich microbial communities exist in places where we least expect it (Rothschild & Mancinelli, 2001, Schulze-Makucj 2011, Kim and Crowley, 2007). The beneficial applications of the organisms that inhabit natural asphalts should encourage more protocols and procedures to develop around hydrocarbon biology. Creating kits and standards that are more easily accessible and successful could encourage additional research in this field. As a result, the hydrocarbon microbial database would expand, bringing more attention to the idea of looking to nature to help solve issues such as pollution. 

Literature Cited  

Balcom, I. N., & Crowley, D. E. (2010). Microbial diversity of asphalt-soil mixtures in the Rancho LA Brea asphalt seeps. International Journal of Phytoremediation, 12(6), 599-615. 

Bastin, E. S., Greer, F. E., Merritt, C. A., & Moulton, G. (1926). The presence of sulphate reducing bacteria in oil field waters. Science, 63(1618), 21-24. 

Baquiran, J. P. M. (2010). Application of metagenomics for identification of novel petroleum hydrocarbon degrading enzymes in natural asphalts from the Rancho La Brea Tar Pits. University of California, Riverside 

Brock, T. D., & Freeze, H. (1969). Thermus aquaticus gen. n. and sp. n., a nonsporulating extreme thermophile. Journal of bacteriology, 98(1), 289-297. 

Brock, T. D. (2012). Thermophilic microorganisms and life at high temperatures. Springer Science & Business Media. 

Charlier, D., & Droogmans, L. (2005). Microbial life at high temperature, the challenges, the strategies. Cellular & Molecular Life Sciences, 62(24). 

De Vera, J. P., Schulze-Makuch, D., Khan, A., Lorek, A., Koncz, A., Möhlmann, D., & Spohn, T. (2012, April). The adaptation potential of extremophiles to Martian surface conditions and its implication for the habitability of Mars. In EGU General Assembly Conference Abstracts (p. 2113). 

Kabir, S., Rajendran, N., Amemiya, T., & Itoh, K. (2003). Real-time quantitative PCR assay on bacterial DNA: In a model soil system and environmental samples. The Journal of general and applied microbiology, 49(2), 101-109 

Kim, J. S., & Crowley, D. E. (2007). Microbial diversity in natural asphalts of the Rancho La Brea Tar Pits. Applied and environmental microbiology, 73(14), 4579-4591 

Luz, A. P., Pellizari, V. H., Whyte, L. G., & Greer, C. W. (2004). A survey of indigenous microbial hydrocarbon degradation genes in soils from Antarctica and Brazil. Canadian journal of microbiology, 50(5), 323-333 

Magot, M., Ollivier, B., & Patel, B. K. (2000). Microbiology of petroleum reservoirs. Antonie van Leeuwenhoek, 77(2), 103-116 

Mo Bio Laboritiries Inc. PowerSoil DNA Isolation Kit. Version 07272016 

Palabiyik, Y., & Ozdemir, A. (2019, November). Oil and gas seeps in Turkey: A review. In 7th International Symposium on Academic Studies in Science, Engineering and Architecture Sciences, November (pp. 15-17). 

Röling, W. F. M., Ortega‐Lucach, S., Larter, S. R., & Head, I. M. (2006). Acidophilic microbial communities associated with a natural, biodegraded hydrocarbon seepage. Journal of applied microbiology, 101(2), 290-299. 

Rothschild, L. J., & Mancinelli, R. L. (2001). Life in extreme environments. Nature, 409(6823), 1092-1101. 

Reicosky, D., Reeves, J., Schuman, J., Gollany, H., Potter, K., Allmaras, R., & Honeycutt, W. GRACEnet Sampling Protocols I. Soil Sampling Guidelines 

Schulze-Makuch, D., Haque, S., de Sousa Antonio, M. R., Ali, D., Hosein, R., Song, Y. C., ... & Hallam, S. J. (2011). Microbial life in a liquid asphalt desert. Astrobiology, 11(3), 241-258 

Stackebrandt, E., Liesack, W., & Goebel, B. M. (1993). Bacterial diversity in a soil sample from a subtropical Australian environment as determined by 16S rDNA analysis. The FASEB Journal, 7(1), 232-236. 

Teske, A. (2019). Hydrocarbon-degrading microbial communities in natural oil seeps. Microbial communities utilizing hydrocarbons and lipids: Members, metagenomics and ecophysiology, 1-31. 

USDA-NRCS. 2004. Compliant Cavity (3B3). p. 98-100. In: R. Burt (ed.) Soil survey laboratory methods manual. Soil survey investigations report no. 42, version 4.0. USDA-NRCS National Soil Survey Laboratory, Lincoln, NE 

Van Hamme, J. D., Singh, A., & Ward, O. P. (2003). Recent advances in petroleum microbiology. Microbiology and molecular biology reviews, 67(4), 503-549. 

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Cayla Martin is from South Africa who graduated from Westminster University in December 2022 as a Biology major. She is interested in nature as a springboard for innovative climate change solutions.