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POLYHYDROXYALKANOATE (PHA) PRODUCTION FROM METHANE & NATURAL GAS - LITERATURE REVIEW PART 3

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POLYHYDROXYALKANOATE (PHA) PRODUCTION FROM METHANE & NATURAL GAS - LITERATURE REVIEW PART 3

Wed, 04/03/2019 - 17:20
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Methane Siberia
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Section 3: Methane-Consuming Organisms

Methane-oxidizing bacteria, also called methanotrophic bacteria, can fortunately consume methane gas in aerobic environments, hence decreasing their accumulation in the atmosphere. These organisms play a major role in the global carbon cycle and have the potential of contributing to the reduction of global methane emissions [38]. They belong to a large group of bacteria called methylotrophs, and are capable of metabolizing C1 compounds like methane, methanol and other methylated compounds as their only source of carbon and energy [39]. The field of methylotrophy has been fast growing and a number of well-know organisms have been used to shape the major concepts and assumptions in the field. Methylomonas methanica, Methylosinus trichosporium and Methylococcus capsulatus are the most studied group of organisms, which represent the two most well known classes (Type I & II) of obligate methane utilizers or methanotrophs. The basic concepts behind methane oxidation have helped scientists classify these methylotrophs into both functional and phylogenetic groups, placing them into a smaller number of genera within Alpha-, Beta-, Gamma-proteobacteria and Actinobacteria [39]. More extensive studies have brought to light the ability of some methane-utilizers to consume a number of multi-carbon compounds in addition to methane and methanol. These organisms belong to the family of Methylocella [39].

 

Methanotrophic bacteria have become very attractive in recent years since they play a significant role in mitigating greenhouse gases by sequestering methane gas from the Earth’s atmosphere [40]. Aerobic methanotrophic bacteria in particular utilize methane as their sole carbon and energy source [41]. These methanotrophs are shown to experience optimal growth condition at moderate pH (5-8) and temperature ranges (20-35oC). However, a number of methanotrophs with extreme growth conditions exist such as psychrophilic (growth at temperature <15oC), thermophilic (growth at temperature >40oC), alkaliphilic (growth at pH >9.0) and acidophilic (growth at pH <5.0) methanotrophs [42]. 

 

The subsequent paragraphs of this section will provide more detail about the process of methane oxidation, carbon assimilation strategies, compare the two major classes of methanotrophs (Type I & II) and discuss their habitats.

3.1 Methane Oxidation and Assimilation

Methane monooxygenase (MMO) enzymes play a key role in initiating the oxidation of methane to methanol in the methanotrophic bacteria. These MMOs exhibit an unusual lack of substrate specificity and are therefore able to metabolize a large number of compounds including xenobiotic chemicals – chemical compounds foreign to living organisms [41]. Due to this ability to partake in a large number of biotransformations, methanotrophs have become attractive over the past years 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 with a high commercial value [41]. There are two forms of MMO found in methanotrophic bacteria. One of them is called soluble MMO (sMMO) and the other particulate MMO (pMMO). pMMO is reported to be present on almost all methanotrophic bacteria. This membrane-bound enzyme is located in the cytoplasmic membrane. sMMO on the other hand is found only in a few methanotrophs and is located in the cytoplasm. In those methanotrophs where both enzymes (sMMO & pMMO) are present, their expression 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 [42,38]. 

 

3.1.1 Structures of Methane Monooxygenases (MMOs)

Methane monoxygenase (MMO) as earlier mentioned is crucial enzyme in the methane oxidation process. MMO requires two reducing equivalents (NADH + H+) to split the di-oxygen bond. One of the oxygen atoms is reduced to form water (H2O) while the other oxygen atom is incorporated into methane to form methanol (CH3OH) [41]. 

 

One of these MMO enzymes, sMMO, particularly has wide substrate specificity and contains three components: a hydroxylase (MMOH), a reductase (MMOR), and a regulatory protein or the B component (MMOB). MMOH houses the active site with a 25kDa size. This component contains a non-heme iron and is an oligomer of three different subunits. MMOR shuttles electrons from NADH to the active site of MMOH and is 38.4kDa in size. This second component contains flavin adenine dinucleotide and an iron sulfide (Fe2S2) cluster. Finally, MMOB is required for activity and has a size of 15.8kDa. This final component is the smallest in size, and is a colorless protein with no cofactors. Because sMMO is capable of oxidizing alkanes, alkenes and aromatic compounds, it has been a favorable target for bioremediation. However, copper in both whole-cell and cell-free fractions inhibits the activity of sMMO [41,38,42]. 

 

Unlike sMMO, which is only present in a few methanotrophs, all known methanotrophs are capable of expressing pMMO. Even though pMMO is more prevalent, very little is known about its biochemistry due to the difficulty of working with a central protein. pMMO, however, consists of three subunits: pmoA (the alpha unit), pmoB (the beta units) and pmoC (the gamma unit). The metal content of pMMO’s active site is believed to contain copper ions and therefore requires copper for activity. pMMO has a limited substrate specificity but cells that contain pMMO have higher growth yields and greater affinities for methane than those that contain sMMO. This happens because the reaction that catalyzes sMMO requires NADH + H+ as an electron donor while pMMO uses a higher-potential electron donor. Therefore, the expression of sMMO genes by a number of methanotrophs could be a method of survival in environments where copper is limited and growth of methanotrophs with only pMMO is impossible [41,38]. 

 

3.1.2 Copper Regulation of MMO

Copper plays an important role in the metabolic switching between the expression of sMMO and pMMO genes in methanotrophs. After a series of studies using model organisms like Methylosinus trichosporium OB3b, it was discovered that cells under copper starvation conditions excreted small copper binding complexes. A decade later, a crystal structure of a copper-binding compound was discovered and named methanobactin [43]. Copper methanobactin is a small chromopeptide that contains one Cu+ per molecule. This compound has a pyramid-like shape with the copper ion bound at the base of the structure and spectroscopic studies have shown that methanobactin only contains Cu+. However, methanobactin can bind Cu+ without changing the oxidation state of Cu2+, which is eventually reduced to Cu+. The ability for methanobactin to bind other metals (such as silver, gold, nickel, cadmium, cobalt, iron, mercury, manganese, lead, zinc and uranium) has been investigated and studies have shown that methanobactin had a higher affinity for copper than it does for those metals listed. Methanobactin has the ability to facilitate copper uptake by making copper more available when it is only present as an insoluble material. Methanobactin interacts directly with pMMO to increase the enzyme activity and may have the potential to reduce copper toxicity. It is still unclear whether acidiphilic methanotrophs like Methylocella and Methylocapsa as well as Verrucomicrobia strains make methanobactin. It is possible that due to their extreme conditions, a high affinity Cu uptake system is unnecessary since Cu solubility increases with decreasing pH [43,42]. 

3.1.3 Pathways for Methane Oxidation and Assimilation

Aerobic methanotrophs contain specialized pathways for the complete oxidation of methane to CO2 and the assimilation of methane into cell biomass. Fig. (3) below shows a summary of two of these pathways.

 

Figure 3: Methane Metabolism

Methane Figure 3

Note 1: [1]-Methane monooxygenase (mmo); [2] -Methanol dehydrogenase; [3]-Formaldehyde dehydrogenase; [4]-Formate dehydrogenase; [5]-H4F linked C1 transfer

In the first step of this process, methane is oxidized to methanol by the MMO enzyme, as previously described. In an energy-conserving step that generates reduced cytochromes or reduced pyridine nucleotides [44], methanol is then further oxidized to formaldehyde by methanol dehydrogenase (MDH) enzyme. However, formaldehyde is very toxic to cell growth and in order to keep the intracellular levels low, formaldehyde has to be oxidized further immediately. Fortunately, methanotrophs have a number of formaldehyde detoxification strategies. Formaldehyde could either be oxidized directly to formate using the formaldehyde dehydrogenase (FaDH) enzyme or assimilated into biomass. Biomass can be assimilated through two routes: the ribulose monophose (RuMP) pathway by Type I methanotrophs or the serine pathways by Type II methanotrophs. Formate can also be oxidized further to CO2 via formate dehydrogenase (FDH), which concludes the complete oxidation of methane [45] [44].

 

 

Ribulose Monophosphate (RuMP) Pathway

Type I methanotrophs use the RuMP pathway for biomass assimilation. A molecule of formaldehyde is fed into the RuMP pathway and with the aid of hexulose phosphate synthase (Hps), where ribulose 5-phosphate (RuMP) condenses with formaldehyde to produce hexulose 6-phosphate. Hexulose 6-phosphate is very unstable and is therefore rapidly isomerized to fructose 6-phosphate by phosphohexose isomerase (Hpi) [40] [44]. Fructose 6-phosphate undergoes a series of rearrangements to generate glyceraldehyde 3-P which can then be used in cell biosynthesis [46]. Fig. 4 shows an overview of RuMP pathway.

 

Figure 4: Ribulose Monophosphate Pathway (RuMP)

Methane Figure 4

Serine Pathway

On the other hand, Type II methanotrophs assimilate formaldehyde through the serine pathway. Formaldehyde reacts spontaneously with a tetrahydrofolate (H4F) co-factor to produce methylene tetrahydrofolate (methylene-H4F). Methylene-H4F condenses with glycine to form serine and releases the cofactor. Serine is then deaminated and reduced through a series of steps to form glycerate, which is later converted to phosphoenolpyruvate (PEP). PEP reacts with CO2 to produce oxaloacetate and after a series of conversions, acetyl-CoA and glyoxylate are produced via malate. While glyoxylate is aminated to glycine and fed back to the pathway, acetyl-CoA can then be used in cell biosynthesis [46]. An overview of the serine pathway is shown in in Fig. 5.

Figure 5: Serine Pathway

Methane Figure 5

3.2    Type I & Type II Methanotrophs

 

As described previously, Type I methanotrophs utilize the ribulose monophosphate (RuMP) pathway for C1 assimilation while Type IIs on the other hand use the serine pathway. This disparity in their pathways for carbon (C1) assimilation is believed to correlate with their intracellular arrangements across the methanotrophic classes. These methanotrophs possess well-developed intracytoplasmic membranes arranged differently between them. The cell arrangements in Type I methanotrophs contain bundles of vesicular discs distributed through the cell while Type IIs contain paired peripheral layers. A major reason for this structural correlation with the pathways for carbon assimilation is assumed to be as a result of each processes difference in energy-yielding efficiencies since each pathway has varied energy requirements [40]. Table (4) below summarizes some major difference between Type I & Type II methanotrophs [47] [41] [42].

 

Table 4: Major Differences Between Type I & Type II Methanotrophs

Characteristics

Type I

Type II

Cell Morphology

Short rods usually occurring singly

Pear shaped cell, semicircular-shaped rods

Phylum

Gamma-Proteobacteria

Alpha-Proteobacteria

Pathway for Carbon Assimilation

Riboluse Monophoshate (RuMP) pathway for formaldehyde assimilation

Serine Pathway for formaldehyde assimilation

G+C content of DNA (mol%)

49-60

62-67

Signature Phospholipid Fatty Acid

14 – 16 carbons in length

18 carbons in length

Growth above 45oC

No

No

Common Genera

Methylococcus, Methylomonas, Methylobacter, Methylomicrobium

Methylosinus, Methylocystis

PHA production

No

Yes

 

 

3.2.1 Selection Mechanisms for Methanotrophic Bacteria

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. In this section, the main focus is on selecting methanotrophs from mixed culture communities for the purpose of PHA production. In this case it is imperative to select Type II methanotrophs over Type I methanotrophs, given that they are the only strain known to date to be solely capable of producing PHAs through the serine pathway.

 

A few approaches have been used successfully to restrict the growth of Type I methanotrophs in mixed cultures. Some of these approaches include the use of a low copper medium, cultivating the cultures under low pH conditions and growing the cells on gaseous nitrogen. Growth in low copper media activates the use of the sMMO enzyme, which is only expressed under low copper conditions. Fortunately, most Type I methanotrophs lack the sMMO enzyme, hence low copper concentrations will encourage the growth of Type II methanotrophs. However, methane oxidation using sMMO is very slow compared to that on pMMO [42]. Secondly, implementing low pH growth conditions seemed to yield very reliable results for selecting Type II methanotrophs. However, extremely low pH limits the growth of the methanotrophic biomass [48]. Another well-known selector for Type II methanotrophs is their ability for fix nitrogen. Unfortunately, nitrogen fixation is very energy intensive imposing a growth penalty on the organism and gas diffusion in the liquid phase tends be an issue of great concern when growing organism in a liquid culture [42] [49].

 

A more promising approach is alternating the nitrogen source between ammonium and nitrate during the growth phase [49]. Methane and ammonia are structurally alike and microorganisms capable of oxidizing these two compounds share several similarities. A major similarity is the close resemblance between the methane monoxygenase (mmo) and ammonia monooxygenase (amo) enzymes. These two enzymes are capable of catalyzing the co-oxidation of the alternative substrates where mmo can catalyze the oxidation of ammonia to produce hydroxylamine and amo can catalyze the oxidation of methane to methanol) [50]. The equations below show the basic step in the ammonia and methane oxidation.

 

An implication of this co-oxidation capability of methanotrophs indicates that high concentrations of ammonia could pose a growth inhibition problem where ammonia blocks the active site for methane on the mmo enzyme. This could result in slowing down methane oxidation or potentially producing toxic by-products like hydroxylamine and nitrite, hence affecting the growth of methanotrophs. Some methanotrophs possess the hydroxylamine oxidoreductase (hao) enzyme that detoxifies the hydroxylamine produced. These strains are able to convert ammonia at higher rates and are resistant to nitrite toxicity unlike those strains that lack hao genes. Therefore, in nitrogen-saturated communities, the methanotrophic community does tend to shift with the amount and type of nitrogen source provided. Studies have shown that Type II methanotrophs have a high tolerance to ammonia compared to their more competitive counterparts, Type I methanotrophs, which have a higher tolerance for nitrite [50,51,52]. Therefore, growth on ammonia could be a more efficient approach to effectively select and maintain Type II methanotrophs in a microbial community. However, when ammonia is the sole nitrogen source, the growth of Type II methanotrophs is significantly slow, possibly due to oxygen availability and co-metabolism of ammonia. A recent study has shown that, in order to boost the growth rates of Type II methanotrophs in a mixed culture, the nitrogen source could be alternated between ammonia (for selecting a Type II community) and nitrate (for accelerated growth) during the growth phase. This strategy resulted in higher growth rates [53].

 

3.2.2 Methanotrophic Biofilms - An Alternative Cultivation Option

Growing organisms in biofilms is another promising option that could yield high cell biomass and reduce the need for handling large amounts of water during the cell growth process. However, very little is known about methanotrophic biofilms and to date only a limited amount of research has been carried out in this area. Most of the work done so far has focused on the co-metabolism of chlorinated aliphatic hydrocarbons (CAHs). Nonetheless, suspended cell culturing (or growth in liquid cultures) continues to be used as the primary cultivation method for most microorganisms including methanotrophic bacteria. This method of growing cells in liquid cultures (or suspended cultures) has several drawbacks such as low gas transfer rates, huge water requirements, and even large energy inputs for mixing the cultures and pumping [54]. Recently, researchers have begun exploring the possibilities of growing microorganisms in attached growth systems to address these problems. This alternative method is referred to as “solid-state fermentation (SSF)” or “dry fermentation”. SSF occurs when microorganisms grow on a moist porous surface that is permeable enough for gas exchange in the presence of a minimum amount of free water, just enough to allow the organisms to have access to the necessary nutrients needed for growth. Growth on the permeable membrane surface is confined to a biofilm with its surface area in direct contact with the gas phase, encouraging an enhanced gas transfer rate [28,54].

 

In an attempt to create models and design reactors using methanotrophic biofilms for biodegradation of aliphatic hydrocarbons, a number of studies have successfully shown that the accumulation and maintenance of large amounts of methanotrophic biofilm is possible. These studies reported high oxygen-to-methane utilization rates especially in thicker biofilm, which could potentially become a problem with scaled up systems [55,56]. One of these studies indicated that the biofilm community consisted of methanotrophs along with nitrifiers and heterotrophs, even though methane was the sole carbon source. These researchers also discovered that the proportion of methanotrophs in younger biofilms (10 days old) was much larger than that in older biofilms (70 days old). This results suggested that the oxygen requirement in the thicker biofilms was much higher and hence encouraged the development of nitrifiers and heterotroph in the older biofilm community, which were able to evolve as a result of the oxygen limitation [55]. In an effort to address the issue of oxygen limitation, another study designed a membrane-aerated biofilm reactor (MABR) where oxygen was supplied at elevated pressure rates, hence easing the penetration of the gas into thicker biomass [56]. This study saw rapid biofilm growth at an average rate of 300 μm/day over the first 2-3 days of growth, and when flow rates were increased during biofilm development, the biofilm accumulation rate increased. Unfortunately, severe sloughing events were observed with the biofilm detaching completely from the membrane over a short period of time especially at elevated accumulation rates. Mathematical models to predict the oxygen uptake rates demonstrated that at a biofilm thickness below 250 μm, there was full penetration of gases and no instance of oxygen limitation [57]. Finally, a recent study demonstrated the feasibility of accumulating methanotrophic biofilm in a fluidized bed reactor (FBR) with granular activated carbon (GAC) carriers for PHB production [57]. These researchers showed that, under conditions of low dissolved oxygen (DO) concentration of approximately 2mg/L and the use of nitrogen gas (N2) as the nitrogen source, Type II methanotrophic biofilms capable of PHB production were successfully accumulated [58].

 

Biofilm systems and SSF technologies present several opportunities both economically and operationally in terms of their projected low capital and energy investment, and their potential to produce high cost value added products. However, it is important to be cognizant of the shortcomings of this process when designing systems of this nature [59]. 

 

  1. Some microorganism fail to attach onto biofilms and it is therefore essential to screen and select the most suitable microorganism(s) for the desired process. 
  2. A large amount of heat is generated during SSF due to the metabolic activity of the microorganism and very high temperatures might be unfavorable to support the growth of the microorganisms. It is important to properly aerate the system to avoid heat build-up but at the same time maintain the temperature desired for growth conditions.
  3. It is also common to experience varied rates of moisture on the membrane surface on which the biofilm grows. High moisture content reduces oxygen penetration while low moisture content results in poor nutrient availability and growth of the microorganism. It is therefore imperative to monitor and maintain an optimal amount of moisture in the system throughout the process.

During the design process, further work needs to be done to improve upon these obstacles for successful operation of the reactors.

3.3 The Origin & Habit of Methanotrophs

Methanotrophs are prevalent in the environment and can now be isolated from many environments containing both methane and oxygen. It was not until 1906 when a Dutch microbiologist first reported the discovery of Bacillus methanicus that was isolated from plants and pond water, and capable of growth on methane. Half a century later, Bacillus methanicus was renamed Pseudomonas methanica and a second methane oxidizer, Methylococcus capsulatus, was discovered [60]. The real turning point in methanotrophic microbiology began when Whittenbury and his colleagues isolated and characterized over 100 methane-oxidizing bacteria. Following this finding, a similar number of strains from different habitats were isolated and this strengthened the notion of their ubiquity in nature [60].

 

Methanotrophic activity has been detected in a wide range of habitats and isolated from several environments. In freshwater and sediment samples, Type I methanotrophs are reported to be the predominant species. Meanwhile in upland and forest soil samples, Type II methanotrophs happen to be more prevalent [47]. Rice fields, which are a major source of atmospheric methane, have a highly diverse methanotroph population [47]. In wastewater treatment plants (WWTPs), where methane is emitted during handling through the anaerobic decomposition of organic material and reported to contribute to about 7% of total global methane emission [33], several studies have detected diverse methanotrophic communities particularly in the activated sludge [61] [48]. 

Methanotrophs have also been discovered in extreme environments. Thermophilic methanotrophs have been isolated from hot springs [62], psychrophilic methanotrophs from ground water [63], halophilic and alkaliphillic methanotrophs from soda lakes [64], and acidophilic and acid tolerant methanotrophs from peatlands, acid forests [65] and volcanic mud [66]. Methanotrophic activity has also been detected in the glacier forefields of the Antarctic [67], at landfill sites [68], and in seawater [69].

 

Methanotrophic bacteria are also known to form great symbiotic relationships with a wide range of organisms, allowing these host organisms to live indirectly and sustainably from methane gas. Environments suitable for methanotrophic symbiosis include ones with the presence of both methane and oxygen, as the symbionts utilize methane both as an electron donor and carbon source, and oxygen as an electron acceptor to provide the animal host with nutrients [70]. In marine environments, the hosts position themselves in a mixing zone where there is direct access to the methane-rich fluids and oxidized seawater, placing the symbionts and the bacteria in a stable environment with simultaneous access to methane and oxygen. Reports have shown that Type I methanotrophs are the only class of methanotrophs mostly known to form symbiotic associations, especially in marine environments [69]. The reason for the absence of Type II methanotrophs in these symbiotic relations could be due to their low energy efficiency, since their carbon assimilation via the serine pathway requires more energy compared to that of Type Is through the ribulose monophosphate pathway (RuMP). Marine invertebrates particularly rely on their symbionts for energy and carbon needs and therefore it is more advantageous for them to associate with Type I methanotrophs. However, plant hosts capable of providing their own carbon and energy needs through photoautrophy can associate with Type II methanotrophs, which can provide a certain fraction of their carbon needs [70]. Methane concentration and flux play a critical role in sustaining methanotrophic symbiosis. A study has shown that methane concentration as low as 5 μM is enough to sustain the methanotrophic symbiosis, if the methane flux is high enough [70]. However, methane concentration above 300 μM can limit the methanotrophic symbiosis, possibly due to inhibitory effects caused by the build up of toxic metabolites such as methanol, formaldehyde and formate. Finally, the study concluded that low oxygen concentrations (<50 μM) could limit methane metabolism in these environments [71]. To date, the cultivation of methanotrophic symbionts has been unsuccessful.

 

Even though several methanotroph strains still remain uncultivated, methanotrophs are almost everywhere and their distribution and abundance in any environment is dependent upon a large number of factors such as oxygen availability, nutrient variations and growth conditions.

3.4 Diversity in the Microbial Population

Pure culture studies have been and continue to be hugely vital in establishing the basic fundamentals for some key transformational processes and metabolic activities for individual species. However, in nature, microorganisms constantly cooperate or compete with each other for resources, and therefore understanding these interactions and mutual relationships is crucial [72]. The wide array of different types of organisms and their relative abundance in a community is often referred to as biodiversity [73]. This 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 [74]. Studies have reported cases where microorganisms excrete metabolites such as vitamin precursors or amino acids that are beneficial to another organism, which lacks the synthesis pathway to handle the initial substrate. Interactions of this manner tend to be extremely profitable [75]. However, there is still a lack of knowledge about how many microbial species exist in the world and how useful they could be. Therefore, microbial diversity analyses are necessary to broaden our understanding of the diversity of genetic resources among the different distributions of microorganisms, the functional role of microbial diversity, and the regulation and consequences of biodiversity [73]. The governing factors affecting microbial diversity are either abiotic, such as physical and chemical factors, or biotic, such as genetically associated factors [73]. A proper understanding of these factors is needed to correctly assess the metabolic activities at play and every microbes’ role in a mixed culture. 

 

Methanotrophic bacteria are quite capable of forming interactions with a wide variety of organisms ranging from marine invertebrates to plants and several heterotrophic bacteria, where they are mostly provided carbon compounds derived from methane [76]. Many of these interactions are mutually beneficial, such as the removal of formaldehyde – a toxic by-product of methane oxidation. Hypomicrobium strains are often observed in methanotrophic co-enrichments and they are believed to oxidize methanol, which is leaked out of or excreted by the methanotroph during methane oxidation. This interaction could helps in potentially preventing inhibition of the methanotrophic growth caused by the build of methanol production. It has also been suggested that Hypomicrobium might also be capable of removing toxic formaldehyde during growth [77]. Cobalamin (a form of Vitamin B12) produced by rhizobia (a heterotrophic soil bacteria capable of fixing nitrogen) was also found to stimulate the growth of some methanotrophs [78]. In several cases, heterotrophic bacteria were found to increase the biomass of the methanotrophic co-cultures and the richness of the heterotrophic bacteria was important in stimulating methanotrophic activity [76,74]. Meanwhile vitamins and organic acids are known to induce the population growth of methanotrophs, another study has proven that other extracellular substrates from non-methanotrophic bacteria are capable of enhancing the methane oxidation activity in a density dependent manner [79]. These studies are a clear indication that methanotrophs are able to thrive and survive in diverse communities. Diversity studies can give us a better understanding of some of those roles methanotrophs play in such complex communities and how these interactions can be leveraged for favorable purposes. 

3.4.1 What Controls Diversity of a Microbial Community?

In order to understand the dynamics between interacting microorganisms, a few fundamental questions would need to be addressed:

  1. How do microbial communities react to physical, biological and biochemical changes, and external disturbances?
  2. What role does metabolic specialization in microbial communities play in tackling biochemical conflicts such as substrate inhibition, enzyme specificity or resource competition?
  3. What factors influence the assembly of a microbial community structure? 
  4. Do new species evolve gradually as a result of reproductive isolation?
  5. How can microbial communities be constructed to obtain an optimum biotransformation of desired products?

 

Answers to all these questions will produce a new understanding of how microbial communities can be assembled to be beneficial for the environment and mankind.

 

3.4.2 Metabolic Specialization Due to Substrate Diversity

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 at 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 believed to shape the assembly of microbial communities and is promoted by the biochemical conflicts between different metabolic processes [80].

 

A number of biochemical conflicts could promote metabolic specialization. One of these conflicts results from competition for a wide variety of intracellular resources. For example, the solvent capacity of a cell determines the amount of enzymes or macromolecules that a cell can handle. Once this capacity is exceed, properties of the macromolecules could potentially change detrimentally. Therefore cells will tend to metabolize only those substrates that are most productive to them. Biochemical conflicts could also result from the production of inhibitory metabolites or end products, which could potentially be growth inhibiting. In this situation, it is possible for cross feeding cell types to evolve which are capable of degrading these toxic substances. Another biochemical conflict could occur due to enzyme specificity where the same enzyme interacts with a variety of substrates. As a result, metabolic specialization could be promoted by improving the specificity of only one substrate, which in turn reduces the specificity for the other substrates present [80]. The consequence of biochemical conflicts mostly influences a genetic change, which could be beneficial for some metabolic processes and unfavorable for others. It is therefore important to understand the constraints and interactions between the metabolic processes involved. However, there are several challenges related with measuring biochemical conflicts. Biochemical conflicts often affects multiple metabolic processes that are connected through complex interaction and it is problematic to entirely place restrictions between only two metabolic processes. Even though concurrent improvements can be made in both processes, these improvements might occur at a cost to other metabolic processes. Furthermore, different environmental conditions greatly affect the interactions between metabolic processes given that each process in a cell is connected to a large network of other processes [80]. Knowledge of the biochemical causes of metabolic specializations would be important to build a basic framework of design principles to establish useful strategies for constructing microbial communities capable of maximizing the performance of desired biological transformations like PHA production. 

 

This section has examined the role of methane-utilizing bacteria specifically methanotrophs in mitigating methane emission, their methane oxidation mechanisms, how the preferred methanotrophic class can be selected particularly for PHA accumulation, and the impacts of biodiversity in microbial community. From this review, it is evident that methanotrophs are able to form strong networks with a wide variety of microorganisms. These sustainable interactions could potentially become beneficial for the production of PHA polymer, which is a high value product. In the following section, we will take a closer look at the structure, composition and production process of the PHAs.

 

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