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MICROBIAL DEGRADATION OF NATURAL GAS - EFFECTS OF MULTIPLE COMPETING SUBSTRATES - PART 1

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MICROBIAL DEGRADATION OF NATURAL GAS - EFFECTS OF MULTIPLE COMPETING SUBSTRATES - PART 1

Sun, 11/17/2019 - 12:57
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Natural Gas

Section 1: Proposal Summary

Natural Gas Background

The role of natural gas in the emerging economy is currently a matter of great debate. Advances in technologies for extraction and recovery of natural gas from subsurface deposits have drawn attention to this resource as prices have fallen noticeably. Of greater significance from an environmental perspective is the fact that natural gas is the cleanest burning fossil fuel with a significantly lower carbon footprint compared to other fossil fuels. This is because complete combustion of one mole of methane – the major component of natural gas – yields one molecule of CO2, whereas complete combustion of longer chain hydrocarbon molecules yields multiple molecules of CO2. Oxidation of longer chain alkanes also has a higher potential for releasing hazardous gaseous pollutants. Moreover, much less energy is required to transport natural gas over a given distance compared to liquid or solid fuels [1]. Given these benefits, it is not surprising that natural gas distribution systems are virtually ubiquitous.

 

The low cost of natural gas and ubiquity of natural gas distribution systems could potentially enable its use as a low-cost carbon feedstock for chemical syntheses, such as microbial fermentations. A serious concern for its use in any such applications, however, is the high global warming potential of methane, which is 25 times that of CO2 [2]. Leaks from natural gas pipelines and the drilling process or losses during use could thus pose a significant threat to the environment, and undo the benefits. Management of such leaks needs strategies, and enabling use of natural gas for chemical synthesis operations will require insights into the rates and effects of microbial degradation of natural gas. At present, there is a surprising lack of knowledge on this subject, perhaps because researchers are focused on methane alone – the major component of natural gas – with scant attention to other components. In reality, the situation is complex. Natural gas is a mixture of alkanes consisting of methane (the solvent or primary component), and other gas solutes such as ethane, propane and butane. In pipeline applications, tert-butylthiol or t-butyl-mercaptan – a substance that smells like rotten eggs – may be added to enable detection of a leak.

 

A potential attractive application for natural gas would be its use as feedstock for production of polyhydroxyalkanoates (PHAs). PHAs are naturally-occurring biodegradable polymers produced by a wide variety of bacteria through the fermentation of several carbon substrates when grown with excess carbon feedstock under nutrient-limiting conditions. The PHA polymers that accumulate as intracellular granules within the bacteria can be extracted and purified for use in a range of applications. Different feedstocks give rise to different PHA copolymers with different elasticity and strength [3,4]. Because these polymers are moldable, they can be extracted and purified for use as biodegradable plastics. All of these properties make PHAs promising replacements for persistent plastics such as polypropylene. PHAs can also serve as prebiotics (a substance that when ingested, alters gut microflora and promotes disease resistance) for aquatic animals and as an insecticide carrier for agricultural purposes [5,6,7]. At present, PHAs are made from harvested feedstock (sugar from corn; oil from soy bean or palm nuts), but use of methane is potentially more viable, both economically and environmentally [8]. 

 

Type II methanotrophic bacteria can produce pure polyhydroxybutyrate (PHB) of high quality under nutrient-limited conditions [9,10]. Recently, Myung et al. [11] demonstrated that the range of PHAs produced by Type II methanotrophs can be expanded by adding co-substrates. This discovery opens the door for broader use of both biogas methane and natural gas. For use of natural gas, however, the diversity of solutes presents a challenge. A major concern is the potential for inhibition or toxicity due to co-metabolism, and the production and accumulation of partial oxidation products. This is particularly the case for pure cultures. Enrichments could potentially address this issue through diversification and metabolic specialization, but the impacts of such activity on the rates of degradation and on the products produced are unclear. In theory, a more diverse natural gas composition would yield a more diverse community, containing organisms capable of co-existing in the same environment and working syntrophically to prevent accumulation of toxic intermediates [12]. The underlying hypothesis of this proposal is that increasing the amount and diversity of solutes in natural gas will increase community diversity, generate new pathways for PHA synthesis, and result in co-polymers or polymer blends that reflect the diversity of substrates in the feedstock.

 

Research Objectives and Proposed Studies

The overarching goal of this project is to gain a basic understanding of the interactions that take place in mixed cultures enriched under different conditions and how these interactions could be targeted for designing PHA-producing systems. This proposal focuses on the following areas:

  1. Impacts of Multiple Gaseous Substrates on Community Diversity and PHA Accumulation: Here we hypothesize that increasing the number of substrates in natural gas will diversify the microbial community. Therefore, a more diverse natural gas feedstock could produce a mixture of pure PHA polymers and a wide variety of co-polymers.
  2. Life Cycle Assessment of Natural Gas as a Feedstock: A life cycle analysis would be carried out to justify the environmental feasibility of natural gas as a substrate for PHA production.

 

These studies would employ a series of molecular techniques to test and monitor the microbial community, growth analysis and PHA quantification methods to observe growth rates in the community and measure PHA accumulation. Life cycle analysis software – SimaPro – would be used to carryout the environmental impact assessments.

 

Natural Gas - Section 2: Background

2.1 Natural Gas as a Resource

Natural gas is undergoing a revolution. Advances in drilling technologies have significantly lowered its prices and accelerated its use as a fuel. Natural gas is attractive because it is clean-burning and releases less CO2 per unit of energy than other fossil carbon fuels. In less than half a century, the global market for natural gas has increased more than fourfold (104 trillion cubic feet (Tcf) in 2009) since 1965 [13]. The U.S. alone consumed 23.4 Tcf of natural gas in 2009, accounting for slightly under a quarter of the total energy supply. Of the total energy required by the residential and commercial sectors in 2009, 76% came from natural gas alone [13]. Natural gas could potentially be a bridge to a low-carbon future because of its low-carbon intensity compared to other supplies of carbon.

 

Natural gas is formed when subsurface deposits of burned biomass undergo methanogenesis – anaerobic microbial decomposition to methane and CO2 – and catagenesis – thermal decomposition due to sustained application of intense heat and pressure over tens of millions of years. The major chemical component of natural gas is methane (CH4) accompanied by lesser portions of heavier hydrocarbons such as ethane (C2H6), propane (C3H8), butane (C4H10), and pentane (C5H12). When natural gas is extracted from the ground as “wet” natural gas, it contains less than 85% of methane as a solvent with natural gas liquids (NGL) solutes (ethane, propane, butane, pentane) [14,15]. Typically, the NGLs are extracted to make “dry” natural gas, a purified product that is almost all methane and the NGLs are sold separately. The composition of dry natural gas varies depending on its source and procedures used to processing it [16]. As shown in Table 1, dry natural gas includes trace amounts of impurities such as nitrogen, oxygen and carbon dioxide [16]. Additionally, as a safety precaution, a sulfur-like odorant, usually called mercaptan (mixtures of t-butyl mercaptan, isopropyl mercaptan, tetrahydrothiophene, dimethyl sulfide and other sulfur compounds), is deliberately added to the gas pipeline to help detect leaks. 

 

Table 1: A Typical Composition of Natural Gas. Adapted from A. Demirbas (2010)

Chemical Component

Range (vol. %)

Methane (CH4)

87 – 96

Ethane (C2H6)

1.8 – 5.1

Propane (C3H8)

0.1 – 1.5

Butane (C4H10)

0.01 – 0.3

Pentane (C5H12)

Trace to 0.14

Nitrogen (N2)

1.3 – 5.6

Carbon dioxide (CO2)

0.1 – 1.0

Oxygen (O2)

0.01 – 0.1

Hydrogen sulfide (H2S), Ammonia (NH3), Hydrogen (H2)

Trace Amounts

 

The quality of natural gas varies based on the type of resource. Conventional resources are of higher quality because they have high permeability, require less technology for drilling and production, and yield higher recovery factors than non-conventional resources such as shale gas, tight gas sands, methane hydrates, etc. [13]. As seen in Figure 1, natural gas production is global and its delivery systems are ubiquitous (Figure 2). In the U.S., natural gas supplies are abundant, and its transportation to end-users is straightforward and more energy efficient than transporting other fuels. U.S. natural gas infrastructure contains 300,000miles of transmission lines, numerous natural gas systems, storage facilities, processing plants, distribution pipelines, and import terminals [13]. Distribution lines are wired directly into the majority of homes, buildings and industries. As demand and supply of natural gas increases, expansion of infrastructure will be required to accommodate this need. Moreover, as technologies have improved and markets expanded, natural gas prices have plummeted from about $5/MMBtu in 2014 to less than $3/MMBtu in 2015 [17].

 

Figure 1: Global Natural Gas Production

Fig 1 - Natural Gas

Note 1: Image from U.S. Energy Information Administration, 2012 [18]

 

Figure 2: Natural Gas Delivery Systems in the U.S.

Fig 2 - Natural Gas

Note 2: Image from American Gas Association, 2015 [19]

With the current appealing nature of natural gas, its potentials could be explored in other avenues other than the energy industry. Natural gas could be exploited in the material and agricultural industry as a potential feedstock to make high-value products like biodegradable polymers. Most recently, methane gas has been used as a cheap feedstock to make biodegradable polymers called polyhydroxyalkanoates (PHAs) [9,10]. Therefore, given the high methane content of natural gas, it could be used to make PHA products and PHA animal feed supplements. 

 

2.2    Benefits and Concerns of Polyhydroxyalkanotes (PHAs)

Polyhydroxyalkanoates (PHAs) are polymers that are biodegradable, biocompatible and recyclable. PHAs exhibit excellent mechanical properties in terms of elasticity and strength, and are therefore a great replacement for synthetic polymers [20,21]. PHAs are a family of naturally-occurring polymers synthesized by a wide variety of bacteria when grown with excess carbon feedstock and under nutrient-limiting conditions. PHAs occur as intracellular storage compound and accumulate as granules within the bacteria [22]. When extracted and purified, PHAs can be used for a wide range of applications such as packaging material, cosmetic products, medical implants and drug delivery. PHAs can also serve as prebiotic for aquatic animals and for agricultural purposes. In the agricultural sector, poly(3-hydroxybutrate-co-3-hydroxyvalerate) or PHBV – a PHA copolymer – has been used to administer insecticides when crops are sown [5]. PHAs can also be used as carriers for bacterial inoculants to improve nitrogen fixation. PHAs help the bacterial cells used to prepare long-term inoculants to withstand harsh environmental conditions [5]. In the aquatic sector, PHAs have been proven to sustain aquaculture. Studies have shown that short chain fatty acids (SCFA) can increase the growth rate of aquatic animals [6,7]. Since SCFA are highly soluble in water and result in low uptake efficiency, PHAs, which are biodegradable polymers of fatty acids and insoluble in water, can be a supplement to the animal feed. The biodegradation of the PHA in the animal’s intestinal tracts would produce their fatty acid derivatives and improve the growth of the aquatic animal. In a recent study, the weight of European sea bass juveniles increased approximately 3-fold after a 6-week period of growing on PHB [6]. In another study, bacteria isolated from the gut of 3 aquatic animals, Siberian sturgeon, European sea bass and Giant River prawn, degraded PHB efficiently [7]. In the same study, brine shrimp larvae’s survival rate increased when incubated in the medium containing these bacterial isolates, PHB and a virulent pathogenic strain. These studies are proof that PHAs can affect crop yield, improve the performance of fish growth and also protect fish gut microbes from pathogenic infections. Therefore, PHAs are able to replace synthetic plastics and also contribute to the well-being of fish and agricultural crops.

 

PHA polymers contain carbon, hydrogen and oxygen, which have a general structure as seen in Figure 3. Approximately 150 different PHA polymers have been identified so far [20]. This number continues to increase as new PHA discoveries occur across the globe. 

 

Figure 3: General PHA Structure

Natural Gas Fig x

PHAs have attracted much research and commercial interest due to these beneficial properties. However, manufacturing of PHA products still encounters several challenges, such as the use of unsustainable raw materials and the high cost of production. These challenges prevent its progress towards commercialization. Most commercially available PHA products today use food crops such as corn, sugar cane, palm oil, etc. for raw materials. The use of food crops has detrimental implications on food security. Therefore this approach would significantly increase the prices of staple foods since supply will become diverted towards polymer production. Furthermore, huge amounts of land will be required to grow these crops in order to match the feedstock demand [23]. This would also involve a substantial amount of water on a regular basis to irrigate these crops. Additionally, these raw materials are expensive which results in a major increase in the cost of the biopolymer product compared to its synthetic polymer counterparts [24]. Raw materials like corn are known to account for 50% of the total production cost. Therefore, these raw material choices alongside the price disparity not only places PHAs at a main disadvantage but also threaten to cause the global population to shy away from using PHA products or investing into its benefits. 

 

An effective way to address some of the issues related to the current production of PHA polymers is to utilize inexpensive carbon substrates like natural gas. Fortunately, the current prices of natural gas have dropped significantly compared to that of food crops like corn (Fig. 4). Therefore using natural gas as a feedstock will address the expensive production of PHAs by providing a cost advantage through large-scale centralized production, especially with its current delivery infrastructure being ubiquitous. Additionally, natural gas as a feedstock will create avenues to build PHA production industrialization systems distributed around the country and the world at large. Therefore, natural gas could become a bridge to the production of renewable PHAs and other high-value products.

 

Figure 4: Trends Comparing Corn Prices in ¢/Bushel (units on the left) and Natural Gas Prices in $/MMBTUs (units on the right) Over a 1-year Period. (Reprinted from tradingeconomic.com)

Fig 4

 

2.3 Specific Implications of PHAs from Natural Gas

PHAs from natural gas could have potential rewards in terms of their environmental impact, biodegradability and their economic advantage.

 

Primarily, PHAs from methane gas are environmentally preferable compared to those produced from agricultural feedstock stock like corn. Rostkowski et al. recently performed a life cycle assessment evaluating the environmental impacts of the polyhydroxybutyrate (PHB) from biogas methane [23]. This study showed that PHB from biogas (global warming potential of -1.94 kg CO2 equiv.) was environmentally and energetically favourable compared to PHB from corn (global warming potential of -0.1 kg CO2 equiv.). The less-favourable impact from corn resulted from the large amounts of land and energy required to cultivate the agricultural feedstocks. Energy and chemicals required during PHB recovery, and the energy requirement for aeration and agitation, were the largest drawbacks to the production of PHB from biogas methane. This study proposed mitigating these impacts by using 18-26% of the incoming biogas methane to accommodate the energy requirements. The study proposed that using methane for energy generation could shrink the global warming potential of PHA from biogas to -6.06 kg CO2 Equiv. [23]. Interestingly, PHAs required for animal feed supplements do not need to be extracted out of the cell. Therefore, this application would not only reduce the processing time but also significantly decrease the energy and chemicals required during PHA extraction, and its equivalent environmental impact. 

 

Secondly, PHAs from methane gas have a unique property in that they can be degraded anaerobically, making them an even more attractive alternative for synthetic polymers. During anaerobic biodegradation, microbes break down the PHA polymer to produce methane and carbon dioxide, in the absence of oxygen. This process usually occurs in landfills or anaerobic digesters. The resulting biogas (a mixture of methane and carbon dioxide) from the process can be burned to produce heat and electricity, or re-captured to produce more PHA [8]. 

 

Finally, PHAs from methane are more economically viable compared to those produced from food crops like corn and palm oil. The cost of food crops can vary greatly and is driven by the availability of food. Meanwhile, methane is generated from landfills and anaerobic digesters, and its prices are less sensitive to change. Since biogas methane has no available market price, its value can be related to natural gas whose current prices have fallen well below corn prices (see Fig. 4). However, biogas methane prices can be assumed to be less than that of natural gas, given that they are produced freely and available abundantly in the landfill, livestock cultivation and sewage treatment sites. This, therefore, makes the methane feedstock even cheaper. Table 2 compares the PHA conversion yields and feedstock prices for glucose, palm oil and methane gas.

 

Table 2: Yield Values and Feedstock Costs for PHA production from glucose, palm oil and methane gas. (CalRecycle Report 2014)

Substrate

Value

Feedstock Cost

Glucose

0.25 – 0.34 g PHA/g glucose

$0.94 – 1.46 per kg PHA

Palm oil

0.61 g PHA/g palm oil

$0.45 per kg PHA

Methane gas

0.49 – 0.5 g PHB/ g methane

$0.28 per kg PHB

 

All of the aforementioned benefits from methane gas are also possible with natural gas as a feedstock since it has become increasingly accessible with new extraction technologies. The PHA production cycle from natural gas would now look like that shown in Figure 5. Even with natural gas being a fossil carbon source, a somewhat closed-loop production process is possible. Additionally, the low price projections of natural gas and its already present infrastructure provide a cost-effective solution to accommodate the energy demands required for PHA production. Due to the abundance of natural gas supplies in the U.S., recent reports state that a huge amount of gas is still flared rather than marketed [25]. Additionally, leaks from natural gas pipelines and the drilling process or losses during use could pose a huge threat to the environment, and undo the benefits. Management of such leaks needs strategies, and enabling use of natural gas for PHA production will require insights into the rates and effects of microbial degradation of natural gas. At present, there is a surprising lack of information on this subject. Therefore, with the possibility of natural gas as an alternative feedstock to make PHAs, natural gas entities would be encouraged to improve their capture targets, and upgrade their delivery systems in order to direct some of the gas for the production of valuable products like PHAs. 

 

Figure 5: PHA Production from Natural Gas

Natural Gas Fig

 

2.4    Methanotrophs

Methane is the predominant component of natural gas. Methane is a very potent greenhouse gas and a major contributor to the global climate change. Therefore mitigating its emissions into the atmosphere is crucial. Fortunately, methane-oxidizing bacteria called methanotrophs can consume methane in aerobic environments to produce CO2. These methanotrophs use methane as their source of carbon and energy, and they can be classified into two well-known categories: Type I methanotrophs and Type II methanotrophs [26]. Methanotrophs possess methane monooxygenase (mmo) enzymes, which play a key role in initiating the oxidation of methane to methanol. These MMOs exhibit an unusual lack of substrate specificity and are therefore able to partake in a large number of biological transformations. This ability has made methanotrophs attractive to scientists who now use these capabilities to develop bioremediation technologies, and also use these bacteria for the production of value-added chemicals/products like PHAs [26]. MMOs exist in two forms: 1) the soluble MMO (sMMO) found only in a few methanotrophs and 2) the particulate MMO (pMMO) present in all methanotrophic bacteria. The expression of the different MMOs is controlled by the regulation of copper in the growth media. sMMO is expressed in the presence of low copper concentration while pMMO is expressed in the presence of high copper concentrations [27,28]. Aerobic methanotrophs contain specialized pathways to completely oxidize methane to CO2 and to assimilate methane into the cell biomass. Type I methanotrophs utilize the ribulose monophose (RuMP) pathway while Type II methanotrophs use the serine pathway, and each of these pathways have different energy requirements [29]. Therefore, methane assimilation across different methanotrophic strains varies based on the pathways and MMOs they possess.

 

Methanotrophs are ubiquitous in the environment and the methanotrophic class of interest can be selected by applying a number of culture methods. Understanding and implementing strategies for selecting one methanotrophic strain over the other is important to streamline or eliminate organisms of least importance for the biotransformation required. Type II methanotrophs are the only strains known to date to be solely capable of producing PHAs through the serine pathway [9]. Therefore, a number of developed strategies have been proven successful to restrict the growth of Type I methanotrophs in enriched cultures. One of these approaches includes the use of low copper medium, which activates sMMO that is expressed under low copper conditions. Since most Type I methanotrophs lack the sMMO genes, this method encourages the growth of Type II methanotrophs. However, methane oxidation using sMMO is very slow compared to that on pMMO [27]. Other approaches have included cultivating the cultures under low pH conditions and growing the cells on gaseous nitrogen. However, Type II methanotrophs are typically slow growers compared to Type I methanotrophs [26,9,30]. This slow growth is possibly due to the inefficiency of the serine pathway and achieving high cell densities is usually challenging. Therefore, establishing growth strategies to accelerate and maintain the steady growth of Type II methanotrophs is imperative. It has recently been discovered that alternating the nitrogen source between ammonium and nitrate during the growth phase can help achieve this goal [31]. 

 

Methanotrophic bacteria are also very capable of producing PHAs. The production of PHB has been linked to the serine cycle through which acetyl-CoA is produced [9]. A study to test for PHA production in methanotrophic bacteria showed that Type I methanotrophs did not produce PHB and therefore did not possess the phaC gene needed for PHB production [9]. Meanwhile, Type IIs were capable of producing PHB from methane gas via the serine pathway and hence possessed the phaC gene. Downstream of the serine pathway, three main enzymes (β-ketothiolase, Acetoacetyl-CoA reductase & PHA synthase) are essential for PHB synthesis. β-ketothiolase encoded by phaA, condenses two acetyl-CoA molecules to form acetoacetyl-CoA. Acetoacetyl-CoA reductase encoded by phaB, then reduces acetoacetyl-CoA to (R)-3-hydroxybutryl-CoA which finally gets polymerized by an esterification process. With the aid of the PHA synthase enzyme (phaC), poly(3-hydroxybutrate), PHB is produced as a result [9] [22]. A schematic of this biological pathway can be seen in Figure 6.

 

Figure 6: PHB Production from Methane via the Serine Cycle

Natural Gas Fig

Note 3: [1]-Methane monooxygenase (mmo); [2]-Methanol dehydrogenase; [3]-Formaldehyde dehydrogenase; [4]-Formate dehydrogenase; [5]-3-ketothiolase; [6]-Acetoacetyl-CoA reductase; [7]-PHA Synthase

Given the high methane content of natural gas, it is a potential feedstock candidate for PHA production. However, the diverse nature of the natural gas mixture presents a number of opportunities as well as challenges. Growth inhibition is one major challenge. Growth inhibition could be caused by the presence of toxic compounds or degradation metabolites from the gaseous substrate. Growth inhibition is possible if the natural gas is fed directly to pure cultures of methanotrophs. To address this concern, mixed-culture enrichments – which contain majority methanotrophs together with other alkane oxidizers – will be capable of oxidizing natural gas concurrently. Other members in the consortium capable of growth on their metabolites and impurities – which would otherwise be toxic to these pure cultures – could consume these metabolites and hence hinder the possibility of inhibition. Furthermore, natural gas could be degraded to synthesize a variety of PHA copolymers and mixtures of pure PHA polymers with methane as the majority feedstock. The nature and the percentage of the co-polymer composition would reflect the percentage of higher alkanes present in the natural gas. However, regulating the composition of polymer mixtures is uncertain. Nevertheless, adjusting the alkane fraction could lead to a modification or diversity in the resulting co-polymer or pure polymer produced, conferring numerous useful properties such as flexibility, toughness and impact resistance. A recent invention has been able to capture a similar concept. Myung et al. used mixed culture enrichments dominated by a methanotrophic population to produce poly(3-hydroxybutyrate-co-3-hydroxy valerate) (PHBV) [11]. The results demonstrated that the addition of soluble volatile fatty acid (VFA) co-substrates (like valerate) during polymer synthesis resulted in the production of the PHBV copolymer. Methane gas was the primary growth substrate. Therefore, natural gas as a feedstock has capabilities of diversifying the PHA produced. 

 

2.5 Impacts of Substrate Diversity

In nature, microorganisms coexist and constantly cooperate or compete with each other for resources [32]. A diverse substrate like natural gas tends to encourage biodiversity in a microbial community. Biodiversity plays an important role in boosting the ecosystem’s productivity where all species are able to benefit from one another in a sustainable manner. Even though microorganisms are sensitive to environmental changes, they are able to form complex community interactions with organisms at different trophic levels, and hence adapt to the environmental perturbation [33]. Methanotrophic bacteria themselves are quite capable of forming interactions with a wide variety of microorganisms [34]. Many of these interactions are mutually beneficial, such as the removal of formaldehyde – a toxic by-product of methane oxidation. Understanding these interactions and mutual relationships when natural gas is provided as a feedstock is important. These dynamics between interacting microorganisms can be affected by a number of physical, biological and biochemical changes. Furthermore, external disturbance from their environment and the availability of diverse substrates like natural gas can influence these interactions. Therefore understanding how species adapt and assemble themselves in microbial communities is crucial in order to perform targeted biotransformation.

 

The concept of metabolic specialization can further our understanding of this ideology. Metabolic specialization is a biological principle that defines the collaborative effort between consortia of species to collectively consume a variety of substrates. In this case, individual species specialize in consuming a subset of these substrates, or a metabolite produced from the oxidation of these substrates, to collectively generate a desirable product. This principle is supposed to shape the assembly of microbial communities and therefore, it is promoted by a number of biochemical conflicts between different metabolic processes [12]. Three biochemical conflicts have been identified and hypothesized to influence metabolic specialization. 

  1. Conflicts resulting from competition for intracellular resources could occur where the solvent capacity of a cell is exceeded, hence causing the cell to metabolize only the most productive substrates. 
  2. Conflicts due to growth-inhibitory metabolites or end products are also possible. This encourages the evolution of cross-feeding cells types capable of degrading the toxic substances.
  3. Conflicts due to enzyme specificity could occur where the same enzyme interacts with a variety of substrates. This could cause the enzyme to improve its specificity for only one substrate and reduce it specificity for the others. 

 

These consequences of biochemical conflicts mostly influence a genetic change, which could be beneficial for some metabolic processes and unfavourable for others. It is therefore important to understand the constraints and interactions between the metabolic processes involved. However, measuring biochemical conflicts has several challenges. Biochemical conflicts often affect multiple metabolic processes that are connected through complex interaction and it is problematic to entirely place restrictions between only a few metabolic processes. Even though concurrent improvements can be made in a number of processes, these improvements are achievable at a cost to other metabolic processes. Furthermore, different environmental conditions greatly affect the interactions between metabolic processes since each process in a cell is connected to a large network of other processes [12]. A framework of design principles could thereby be built from obtaining knowledge of the biochemical causes of metabolic specializations. Therefore, scientists can use these principles to establish strategies for constructing microbial communities capable of performing desired biological transformations like PHA production. 

 

Natural gas as a growth substrate would thereby provide some background into the dynamics occurring between interacting microorganisms. Figure 7 shows a schematic of some potential pathways involved in the natural gas degradation process by certain specialized microorganisms. The initial step in the degradation of each alkane present can be carried out by a wide variety of enzymes specific to certain specialized microorganisms. However, it is not uncommon to have coexistence of degradation systems in one microorganism. Therefore, it is imperative to understand how alkanes are incorporated in the cell and how the organisms work together for beneficial purposes. 

 

Figure 7: PHA Biosynthesis Pathways from Natural Gas Substrates [35,3,4,36,37]

Natural Gas Fig

Note 4: [1]-Alkane monooxygenase; [2]-Subterminal alkane monooxygenase; [3]-Alcohol dehydrogenase; [4]-Baeyer Villiger monoxygensae; [5]-Aldehyde dehydrogenase; [6]-Esterase; [7]-Acyl-CoA Synthase; [8]-PHA Synthase 

 

2.6 Project Objectives

Broadening our understanding of the factors affecting microbial diversity will allow us to assess the metabolic activities at play and every microbe’s role in a mixed culture. Microbial diversity analyses would provide the tools to evaluate the functional role of microbial diversity and the consequences of biodiversity [38]. Therefore, the ultimate goal of this project is to gain a basic understanding of the interactions that take place in mixed cultures enriched under different conditions and how these interactions could be targeted for designing PHA-producing systems. 

 

The major objectives of this project are as follows:

  1. To understand the implication of substrate diversity on a microbial community structure, specifically when natural gas of different compositions is supplied as the carbon substrate and to quantify the resulting PHA produced.
  2. To justify the environmental impacts of natural gas as a sustainable PHA feedstock through a life cycle assessment.

 

Continue to Section 3: Research Studies

 


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