Section 3: Research Studies
Increasing the Number of Substrates in Natural Gas will Diversify the Microbial Community
Studies have shown that when a complex sample like activated sludge is inoculated with natural gas, methane, under aerobic conditions, methanotrophic bacteria dominate the microbial community . Selecting the desired class of methanotrophs (either Type I or Type II) could be constrained to several operating conditions such as the nitrogen source, dissolved oxygen concentration, copper concentration and pH. In the same microbial community, methanotrophs coexist with methylotrophs and heterotrophs, which are capable of surviving on the metabolites of methane oxidation such as methane, formaldehyde, formate and other decay products. Some observed organisms include Hyphomicrobium [39,40] – a facultative methylotrophic genus – known to grow on C1 and C2 compounds. Hyphomicrobium is also capable of accumulating intracellular PHAs. Other organisms observed in the community include Burkholderia (known to contain several formaldehyde oxidation/detoxification pathways)  and Hydrotalea (know to assimilate formate) . Emphasis has been placed primarily on selecting for methane-utilizing bacteria capable of producing polyhydroxalkanoate (PHAs) from pure methane gas. Based on the selection strategies employed, a consistent stable community of Type II methanotrophs can be maintained throughout the process. However, there is no evidence in the literature on natural gas enrichments or PHA studies with natural gas as a feedstock.
In this study, we will explore the possibilities of using natural gas as a feedstock to produce diverse microbial enrichments. We hypothesize that a microbial community enriched with natural gas as a carbon source would encourage the selection of organisms capable of oxidizing subsets of the natural gas substrate and/or metabolites produced from its oxidation. In the presence of such a diverse substrate, the selected microbial community could prevent toxicity, and encourage cross feeding in the community. Based on the number of carbon substrate present, we hypothesize that diversity and richness will correlate with the number of carbon substrates present. This correlation could either be exponential, linear or logarithmic. Figure 8 demonstrates the possible diversity progressions that we expect to observe in the community. Therefore, in this study we will analyze the microbial communities by evaluating the community structures produced from different natural gas compositions.
Figure 8: Plausible Microbial Diversity Changes Based on Number of Gaseous Substrates Present
#1B Pure PHA Polymer Mixtures and Copolymer Production is Possible with Natural Gas as a Feedstock
When microbial communities are selected for PHA production using methane gas as sole feedstock, these communities, mostly dominated by Type II methanotrophs, are only capable of producing polyhydroxybutyrate (PHB). A recent study from the Criddle lab has shown that microbial communities dominated by Type II methanotrophs (Methylocystis) can incorporate volatile fatty acids like valeric acid during polymer production process to produce a copolymer of PHB and polyhydroxyvalerate (PHV) . This study went further to prove that the fraction of PHV incorporated into the polymer could be tailored based on the quantity of valerate added. Based on this study and on the concept that metabolic specialization can drive diversity in microbial communities , we hypothesize that a community enriched with different compositions of natural gas will include several other PHA-accumulating bacteria. These bacteria would be capable of producing a variety of pure PHA polymer mixtures and/or PHA copolymers. We will therefore investigate these possibilities.
#2 Natural Gas could be an Environmentally Viable Feedstock for PHA Production
Evaluating the environmental impact of PHA production from natural gas is important to establish a justification for its preference as a raw material source over currently available agricultural feedstock. Life cycle assessments (LCA) are valuable tools used to quantify and assess how materials used in a production process impact the environment. A study from the Criddle lab developed a cradle-to-gate LCA model to evaluate the environmental impacts of PHB production from biogas methane up to the point of the extracted polymer resin . This study showed that PHB production from biogas methane was energetically favorable compared to production from corn-based carbon sources (37.4 MJ/kg of PHB for biogas methane and 41.9 MJ/kg of PHB for corn). The use of corn-based materials requires large inputs of land and energy for growth of the crop, which makes its production process less environmentally benign. In this study however, PHB recovery from the cell material was a major contributor to it environmental impacts mostly due to the huge energy and chemical input required for PHB extraction. The study suggested using biogas methane to reduce the energy requirement and employing more environmentally suitable methods for extraction of the PHB resin. Given that natural gas contains a mixture of alkanes, it would, therefore, be valuable to assess its environmental footprint as a potential PHA production feedstock.
Natural gas – which is primarily methane – is known to have a lower carbon footprint compared to other fossil fuels. This classifies the gas as a clean-burning fuel due to its lower life cycle greenhouse gas emissions. Studies have reinforced this fact by showing that the increased use of natural gas in 2012 significantly reduced carbon dioxide levels to the lowest level since 1992 . Additionally, the price of natural gas has been on a steep decline in the past few years. Given these advantages, we thereby hypothesize that natural gas is a very promising feedstock for the production of value-added products like PHAs. Therefore, a life cycle analysis would provide justification for its use in the production of these products.
Section 4: Research Plan
Figure 9: Natural Gas Research Outline
4.1 Proposed Work
4.1.1 Inoculum Selection
4.1.2 Impacts of Multiple Gaseous Substrates on Community Diversity and PHA Accumulation
Molecular Analysis Studies:
To assess the microbial community structures, I will prepare enrichments using methane gas alone (as a baseline control) along with synthetic natural gas (SNG) created with different combinations of three gaseous substrates (methane, ethane, propane), and pipeline natural gas (PNG). SNG will mimic natural gas compositions from different sources where methane is the solvent. Stanford University’s PNG is supplied by the Pacific Gas and Electric (PG&E) Company. Samples would be cultured with ammonium as the nitrogen source, a modified W1 growth media used for culturing methanotrophs and oxygen as the electron acceptor. All experiments would be carried out at 30oC and sampling would be performed in replicates. Figure 10 demonstrates a proposed execution plan for the investigation with multiple gaseous substrates.
Figure 10: Investigations on the Impacts of Multiple Gaseous Substrates on Community Diversity
After cultures have grown to an optical density (OD670)>1, the selected enrichments would be harvested for further analysis. I will extract DNA from the harvested samples, amplify and sequence the 16S genes, either by performing Illumina or Sanger sequencing analysis for all instances. These sequencing analyses would allow us to identify the different organisms present at the community level and detect the key players. Throughout the continuous growth of the cultures, samples will be collected frequently based on a fixed time-series. These samples will be analyzed using a Terminal Restriction Fragment Length Polymorphism (T-RFLP) method to profile the microbial communities and measure the species’ richness and evenness in the community. The T-RFLP analysis will also enable us to determine the similarities and differences between enrichments growing on different gaseous substrate combinations. These results would give us insights into the effects of the different gaseous substrate compositions on the self-assembled microbial communities. They would also enable the detection of shifts in the microbial community structures and the stability in the community over time. Moreover, these molecular analysis results would provide information about organisms specialized at degrading the gaseous substrates, which would enable us to identify possible strategies for selecting desired communities. Figure 11 the experimental plan for the molecular analysis investigations.
Figure 11: Molecular Analysis Flow Chart
Growth Kinetics Studies
Substrate utilization and oxygen uptake rates would be used to assess the levels of gaseous substrate consumption and to monitor substrate degradation patterns in the different microbial communities. To carry out this assessment, gas samples would be drawn from the headspace of the reactor periodically throughout the growth cycle and analyzed on a Gas Chromatograph (GC) with a thermal conductivity detector (TCD). These results would provide information on the amount of oxygen required to optimally metabolize the carbon substrate combinations in order to select for the desired microbial community. With these results, we would observe the different trends in gaseous substrates consumption and correlate it to the dominating organisms in the community, thereby giving us insight about the most favorable organisms.
Given the gaseous substrate mixtures provided, growth rates would vary in all the different scenarios. In order to analyze the growth rates under the different gaseous substrate mixture conditions, liquid samples will be drawn from the reactors periodically. Growth rate analyses would be performed using both Volatile Suspended Solids (VSS) and Total Suspended Solids (TSS) techniques. These results would demonstrate the different growth patterns of the cultures growing on the gaseous co-substrates, which would enable us to observe any disparities or anomalies influenced by these changes. In addition, the pH of the growth reactors would be monitored regularly since different pH conditions can influence the growth of various microorganisms.
PHA Production Studies
An ultimate goal of this project is to select microbial communities capable of effectively producing PHAs. Therefore, I will investigate the impacts different gaseous substrates combinations have on PHA accumulation. Previous studies from the Criddle lab have shown that non-growth substrates can be co-metabolized by methanotrophs and incorporated into their polymer to produce PHA co-polymers . In light of this finding, we expect that samples enriched with a combination of alkanes would behave the same way. Given that the microbial enrichment would contain a diverse array of microorganisms, this diversity would result in the accumulation of a variety of polymer combinations: both pure PHA polymer and/or PHA co-polymers. Therefore, in this study, these microbial enrichments would be deprived of nitrogen (ammonia) to impose PHA accumulation and harvested for further PHA analysis. The resulting PHA produced would be quantified using a GC with a flame ionization detector (FID). A Nuclear Magnetic Resonance (NMR) analysis would be carried out to differentiate between pure PHA polymers and PHA co-polymers. A VSS and/or TSS analysis would also be performed during the PHA accumulation phase to determine peak points of PHA accumulation.
4.1.3 Life Cycle Assessment of Natural Gas as a Feedstock
In this study, we would use Life Cycle Analyses (LCA) design tools to assess the environmental impacts of natural gas as a feedstock for PHA production. The LCA would be comprised of four components: the goals and scope definition, an inventory analysis, impact assessment and data interpretation. Initially, the goal and scope definition would identify the measurement parameters. Specific environmental impact categories would be examined based on standards determined by the U.S. Environmental Protection Agency. In this section, the SimaPro LCA software would be used to collect, analyze and monitor the sustainability performance data and carbon footprint of natural gas as a PHA feedstock. Secondly, the inventory analysis would highlight the processes and flow of materials for PHA production from natural gas. Here, the boundaries of the study would be set. Thirdly, the impact assessment would report the impacts resulting from the production of PHA from natural gas and specify values for each impact. Finally, the data would be interpreted and the environmental impacts compared to PHA production from corn. These results would enable us to identify areas for environmental pollution prevention, guide performance improvements and ways to reduce resource consumption.
4.2 Preliminary Results
4.2.1 Microbial Communities Structures for Cultures Grown on Pure Methane Gas.
Fresh activated sludge from the Palo Alto Regional Water Quality Control Plant (PARWQCP) was filtered through a 100 um nylon filter to remove large particles. The filtrate was centrifuged (4300 rpm) for 15mins and the pellet re-suspended in a modified W1 growth medium. The modified W1 medium contained the following chemicals per L of solution: 8.3 mM MgSO4.7H2O, 1.4 mM CaCl2.2H2O, 36 mM NaHCO3, 3.6 mM KH2PO4, 6.8 mM K2HPO4, 10.5 uM Na2MoO4.2H2O, 8.2 uM CuCl2.5H2O, 200 uM Fe-EDTA, 5 mL trace metal solution, and 20 mL vitamin solution. The trace stock solution contained the following chemicals per L of solution: 500 mg FeSO4.7H2O, 400 mg ZnSO4.7H2O, 20 mg MnCl2.7H2O, 50 mg CoCl2.6H2O, 10 mg NiCl2.6H2O, 15 mg H3BO3 and 250 mg EDTA. The vitamin stock solution contained the following chemicals per L of solution: 2.0 mg biotin, 2.0 mg folic acid, 5.0 mg thiamine HCl, 5.0 mg calcium pantothenate, 0.1 mg vitamin B12, 5.0 mg riboflavin and 5.0 mg nicotinamide. The suspension was used as the initial inoculum for the study. 5-mL aliquots of inoculum were added to serum vials containing 44.5 mL of the modified W1 medium and 0.5 ml of a 1 M ammonium stock solution. The samples were inoculated with a methane-to-oxygen ratio of 1:2, and incubated at 30 oC under continuous shaking conditions (160 rpm). Every 24 hrs, the headspace of each bottle was flushed with the same methane-to-oxygen ratio while every 48 hrs, each bottle was amended with 0.5 mL of the 1 M ammonium stock solution.
After approximately 2 weeks of incubation, the enriched cultures reached an optical density (OD670) greater than 1.5. The cultures were centrifuged (4300 rpm), re-suspended in a modified W1 medium and divided into 10-mL aliquots for inoculation of triplicate fed-batch serum bottle cultures. Each fed-batch culture initially contained 10 mL of inoculum, 39.5 mL of the modified W1 medium and 0.5 mL of ammonium stock (total volume 50 mL). After a 48-h incubation period, each of the triplicate enrichments was subjected to a cyclic feeding and wasting regime, with alternating pulses of methane/oxygen and ammonium.
In order to establish the different types of bacteria species present in the enrichments, DNA was extracted from the samples, amplified and sequenced to obtain a 16S rRNA clone library. Figure 13 shows a chart of the bacterial species observed from cultures enriched with either methane gas. Based on the results obtained from 26 sequenced clones, Hyphomicrobium species – a facultative methylotroph that grows on C1 and C2 compound and accumulates PHA intracellular – made up about a quarter of the population. Unexpectedly, Type II methanotroph – Methylocystis – was observed as a minority member in the community. Other genera present in the community included Pandorea and Simplicispira, both of which are PHA producers. In an attempt to replicate these results or observe a more favourable outcome, the experiment was repeated with the same activated sludge that had been preserved in a 4 oC fridge for about 3-4 weeks. The outcome can be seen in Figure 12. Based on results obtained from 38 sequenced clones, all the species observed were Type I methanotrophs (Methylobacillus, Methylosarcina, Methylomicrobium) and the community structure was completely different from the first analysis. These results reveal that the inoculum plays a key role in this study and the community structure in an inoculum like activated sludge can change over time. Therefore, in order to obtain replicable results, a stable and uniform inoculum would need to be established and maintained for all experiments. Further investigations using multiple gaseous substrates and a uniform inoculum would reveal the diversity and shifts in the community.
|Figure 13: Community Structure from First Methane Enrichment||Figure 12: Community Structure from Second Methane Enrichment|
4.2.2 PHA Composition from Selected Communities
In order to investigate the PHA accumulated by the resulting enrichment, a PHA analysis was performed following a similar protocol described by Myung et al. . After 48 hrs of cell growth, the cultures were centrifuged (4300 rpm) for 15 mins to obtain a pellet. The pellet was re-suspended in a modified W1 media with no nitrogen and incubated with a methane-to-oxygen ratio of 1:2 for 24 hrs. After 24 hrs, the cells were centrifuged, and the pellet was freeze-dried for a PHA analysis. PHA quantification was carried out using a GC with a flame ionization detector (FID). Results from this analysis can be seen in Figure 14 below. The first community with PHA producers yielded a PHB content of 25% dry weight while the second community with Type I methanotrophs produced little to no PHB (~2%). These results affirm the fact that Type I methanotrophs do not produce PHA and methane gas as the sole carbon source would produce only PHB. Further studies using more uniform and stable inoculum would look at the different PHA compositions produced with multiple gaseous substrates and the alkane concentrations that produce a significant amount of diversity in the PHA content.
Figure 14: % PHB Produced from Different Methane Enrichments
Section 5: Expected Outcomes and Contributions
The primary goal for the studies outlined in this proposal is to establish a fundamental understanding around substrate diversity and how it impacts and/or alters a microbial community. The information from these studies will provide insights necessary to develop strategies for selecting communities capable of producing PHAs of different compositions. The project will culminate with a life cycle assessment, which would justify the environmental impacts that result from the use of natural gas as a feedstock.
Impacts of Multiple Gaseous Substrates on Community Diversity and PHA Accumulation: Here, we will use molecular biology tools to monitor and compare the community patterns upon utilization of multiple gaseous substrate combination. Meanwhile, gas chromatographic tools would provide us with a quantitative analysis of the PHA produced. Some specific expected outcomes are as follows:
- Diversity assessments would increase our knowledge of the underlying causes of metabolic specialization. These assessments would provide information on the communities assembled under the different imposed growth conditions, including the dominant species present. Knowledge about the distribution of the different organisms across the community would be relevant to obtain insights about their roles and significance in the community alongside enabling us to test the Johnson Theory . This information would enable us to detect community shifts, determine ways to maintain stable community structures, and propose possible causes of vulnerabilities (if any) in the community. Additionally, information from these studies would be instrumental when trying to identify approaches on producing tailor-made PHAs from a diverse community.
- Comparing the possibly different types of PHA produced from these diverse communities would provide vital information necessary to understand the roles the different organisms play in the PHA accumulation process. This could potentially lead to identification of certain organisms or community structures to target for the production of specific PHA types. Furthermore, the quantities of PHAs produced under the different conditions will help us detect the conditions needed to obtain a variety of desirable PHA compositions.
- Growth kinetic studies would give us information about the effective consumption of substrates, how fast (or slow) the organisms are growing and also how oxygen is being utilized under different conditions. This assessment would give us the insight needed to optimize the biomass yields in a population and provide parameters necessary for a life cycle assessment.
Life Cycle Analysis of Natural Gas as a Feedstock for PHA Production:
This assessment would be the first analysis to quantify the environmental impacts of natural gas as a feedstock for value-added products like PHA. We would focus on the natural gas mixtures that give rise to PHA polymers. The parameters obtained from the laboratory experiments would enable us to identify the environmental viability of the range of PHA products produced from the different natural gas mixtures. Finally, we would compare the results to the impacts of PHAs from agricultural feedstock, and determine areas for long-term improvements.
Overall, these studies would provide a basic understanding of the factors vital in creating diversity in microbial communities. All together we would employ very fundamental molecular biology and growth analysis techniques to answer questions in the field of environmental biotechnology and microbial ecology. These answers would benefit research that explores the future of biodegradable plastics and the positive implication of microbial diversity.
 Jasna Djonlagic and Marija S. Nikolic, "Biodegradable Polyester: Synthesis and Physical Properties," in A Handbook of Applied Biopolymers Technology: Synthesis, Degradation and Applications, Sanjay K Sharma and Ackmez Mudhoo, Eds. Cambridge, UK: Royal Society of Chemistry, 2011.
 Bernd H. A. Rehm, "Biogenesis of Microbial Polyhydroxyalkanoate Granules: A Platfrom Technology for the Production of Tailor-made Bioparticles," Curr. Issues Mol. Biol, vol. 9, no. 1, pp. 41-62, 2007.
 S. Philip, T. Keshavarz, and I. Roy, "Review - Polyhydroxyalkanoates: Biodegradable Polymers with a Range of Applications," Journal of Chemical Technology and Biotechnology, vol. 82, 2007.
 Peter De Schryver et al., "Poly-β-hydroxybutyrate (PHB) Increases Growth Performance and Intestinal Bacterial Range-Weighted Richness in Juvenile European Sea Bass, Dicentrarchus labrax," Applied Microbial and Cell Physiology, vol. 86, pp. 1535–1541, January 2010.
 Yiying Liu et al., "PHB-degrading Bacteria Isolated from the Gastrointestinal Tract of Aquatic Animals as Protective Actors Against Luminescent vibriosis," FEMS Microbiology Ecology, vol. 74, pp. 196–204, June 2010.
 Craig S. Criddle, Sarah L. Billington, and Curtis W. Frank, "Renewable Bioplastics and Biocomposites From Biogas Methane and Waste-Derived Feedstock: Development of Enabling Technology, Life Cycle Assessment, and Analysis of Costs," California Department of Resources Recycling and Recovery , Stanford University, Contract 2014.
 Allison J. Pieja, Katherine H. Rostkowski, and Craig S. Criddle, "Distribution and Selection of Poly-3-Hydroxybutyrate Production Capacity in Methanotrophic Proteobacteria," Microbial Ecology, vol. 62, pp. 564–573, May 2011.
 Allison J. Pieja, Eric R. Sundstrom, and Craig S. Criddle, "Poly-3-Hydroxybutyrate Metabolism in the Type II Methanotroph Methylocystis parvus OBBP," Applied and Environmental Microbiology, vol. 77, no. 17, pp. 6012–6019, September 2011.
 Jaewook Myung et al., "Long-term Cultivation of a Stable Methylocystis-dominated Methanotrophic Enrichment Enabling Tailored Production of Poly(3-hydroxybutyrate-co-3-hydroxyvalerate)," Bioresource Technology, vol. 198, pp. 811–818, 2015.
 David R Johnson, Felix Goldschmidt, Elin E Lilja, and Martin Ackermann, "Metabolic Specialization and The Assembly of Microbial Communities," The ISME Journal, vol. 6, pp. 1985-1991, May 2012.
 MIT Energy Initiative, "The Future of Natural Gas," MIT Study on The Future of Natural Gas, 2011.
 US Energy Development Corporation. US Energy Development Corporation. [Online]. http://www.usenergydevcorp.com/media_downloads/Natural%20Gas%20Dry%20Vs%20Wet_050913.pdf
 US Energy Information Administration. (2014, May) US Energy Information Administration. [Online]. http://www.eia.gov/todayinenergy/detail.cfm?id=16191
 Ayhan Demirbas, "Natural Gas," in Methane Gas Hydrates. London: Springer London, 2010.
 Nasdaq. (2015, July) Natural Gas. [Online]. http://www.nasdaq.com/markets/natural-gas.aspx?timeframe=2y
 U.S. Energy Information Administration. (2012, January) www.eia.gov. [Online]. http://www.eia.gov/todayinenergy/detail.cfm?id=4790
 American Gas Association. (2013) http://playbook.aga.org/#p=80. [Online]. https://www.aga.org/about-natural-gas
 Ching-Yee Loo and Kumar Sudesh, "Polyhydroxyalkanoates: Bio-bases Microbial Plastics And Their Properties," Malaysian Polymer Journal, vol. 2, 2007.
 Eric Pollet and Luc Averous, "Production, Chemistry and Properties of Polyhydroxyalkanoates," in Biopolymers: New Materials for Sustainable Films and Coatings, David Plackett, Ed. Chichester, UK: John Wiley & Sons, Ltd, 2011.
 Ranjana Rai and Ipsita Roy, "Polyhydroxyalkanoates: The Emerging New Green Polymers of Choice," in A Handbook of Applied Biopolymer Technology : Synthesis, Degradation and Applications, Ackmez Mudhoo Sanjay K Sharma, Ed. Cambridge, UK: Royal Society of Chemistry.
 Katherine H. Rostkowski, Craig S. Criddle, and Michael D. Lepech, "Cradle-to-Gate Life Cycle Assessment for a Cradle-to-Cradle Cycle: Biogas-to-Bioplastic (and Back)," Environ. Sci. Technol., vol. 46, p. 9822−9829, 2012 July.
 Leda R. Castilho, David A. Mitchell, and Denise M.G. Freire, "Production of polyhydroxyalkanoates (PHAs) from waste materials and by-products by submerged and solid-state fermentation," Bioresource Technology, vol. 100, pp. 5996–6009, July 2009.
 U.S. Energy Information Administration. (2015, November) eia.gov. [Online]. http://www.eia.gov/todayinenergy/detail.cfm?id=23752
 Richard S. Hanson and Thomas E. Hanson, "Methanotrophic Bacteria," Microbiological Reviews, June 1996.
 Jeremy D. Semrau, Alan A. DiSpirito, and Sukhwan Yoon, "Methanotrophs & Copper," Microbiological Reviews, p. 36, March 2010.
 Amanda S. Hakemian and Amy C. Rosenzweig, "The Biochemistry of Methane Oxidation," Annual Review of Biochemistry, p. 22, 2007.
 Yuri A. Trotsenko and John Colin Murrell, "Metabolic Aspects of Aerobic Obligate Methanotrophy," in Advances in Applied Microbiology., 2008, vol. 63.
 Andrew R. Pfluger et al., "Selection of Type I and Type II methanotrophic proteobacteria in a fluidized bed reactor under non-sterile conditions," Bioresource Technology, vol. 102, pp. 9919–9926, August 2011.
 Eric Sundstrom and Craig Criddle, "Alternating Ammonium and Nitrate as Nitrogen Sources Selects for Polyhydroxybutyrate (PHB) - Producing Methanotrophic Communities," 2013, Dissertation Thesis; In preparation for publication.
 Bernhard Schink and Alfons J. M. Stams, "Syntrophism among Prokaryotes," in Prokaryotes., 2006.
 Adrian Ho et al., "The More, The Merrier: Heterotroph Richness Stimulates Methanotrophic Activity," The ISME Journal, May 2014.
 Michiel Stock et al., "Exploration and Prediction of Interactions Between Methanotrophs and Heterotrophs," Research in Microbiology, vol. 164, September 2013.
 F. Rojo, "Enzymes for Aerobic Degradation of Alkanes," in Handbook of Hydrocarbon and Lipid Microbiology, K. N. Timmis, Ed. Berlin Heidelberg: Springer-Verlag, 2010, pp. 781-797.
 Alexander Wentzel, Trond E. Ellingsen, Hans-Kristian Kotlar, Sergey B. Zotchev, and Mimmi Throne-Holst, "Bacterial Metabolism of Long-chain n-alkanes," Appl. Microbiol. Biotechnol., vol. 76, pp. 1209-1221, August 2009.
 J.B. van-Beilen, Z. Li, W.A. Duetz, T.H.M. Smits, and B. Witholt, "Diversity of Alkane Hydroxylase Systems in the Environment," Oil & Gas Science and Technology, vol. 58, no. 4, pp. 427-440, 2003.
 Md. Fakruddin and Khanjada Shahnewaj Bin Mannan, "Methods for Analyzing Diversity of Microbial Communities in Natural Environments," Ceylon Journal of Science, vol. 42, no. 1, pp. 19-33, March 2013.
 Ian R. McDonald, Nina V. Doronina, Yuri A. Trotsenko, Craig McAnulla, and J. Colin Murrell, "Hyphomicrobium chloromethanicum sp. nov. and Methylobacterium chloromethanicum sp. nov., Chloromethane-utilizing bacteria Isolated from a Polluted Environment," International Journal of Systematic and Evolutionary Microbiology, vol. 51, pp. 119–122, 2001.
 Margaret M. Attwood and W Harder, "The Oxidation and Assimilation of C2 Compounds by Hyphomicrobium sp," Journal of General Microbiology, vol. 84, no. 2, pp. 350-356, 1974.
 Christopher J. Marx, Jonathan A. Miller, Ludmila Chistoserdova, and Mary E. Lidstrom, "Multiple Formaldehyde Oxidation/Detoxification Pathways in Burkholderia fungorum LB400," Journal of Bacteriology, vol. 186, no. 7, pp. 2173–2178, December 2003.
 P. Kampfer, N. Lodders, and E. Falsen, "Hydrotalea flava gen. nov., sp. nov., a new member of the phylum Bacteroidetes and allocation of the genera Chitinophaga, Sediminibacterium, Lacibacter, Flavihumibacter, Flavisolibacter, Niabella, Niastella, Segetibacter, Parasegetibacter, Terrimonas, Ferruginibacter, Filimonas and Hydrotalea to the family Chitinophagaceae fam. nov," International Journal of Systematic and Evolutionary Microbiology, vol. 61, pp. 518–523, 2011.
 Natural Gas Supply Association. (2014) ngsa.org. [Online]. http://www.ngsa.org/download