Response of methanotroph-heterotroph consortia to different environmental factors

Chidambaram Padmavathy, Karthigeyan (2017) Response of methanotroph-heterotroph consortia to different environmental factors. PhD thesis, James Cook University.

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Methane (CH₄) is a potent greenhouse gas with a global warming potential of 28-34 times that of carbon dioxide (CO₂) over a 100-year period according to IPCC 2014. Globally, landfills are the third largest anthropogenic source of CH₄ emissions (~760 MMTCO₂eq), accounting for 11 % of total emissions (6,875 MMTCO₂eq). Landfill soils are shown to be the active sink for CH₄ due to the presence of CH₄ oxidising bacteria (methanotrophs), which act as natural bio-filters. Theoretically, landfill CH₄ can be re-routed through commercial bio-filters for CH₄ remediation. The microbial biomass generated could then be utilised for valuable co-product production, such as fatty acids, polyhydroxybutyrate (PHB, a key ingredient in the production of biodegradable plastics). Re-routing CH₄ for PHB production has significant advantages compared to fossil fuel-derived plastics or sugar-derived bioplastics due to the simple concept of killing two birds with one stone; i.e. reducing the carbon footprint and producing renewable biodegradable PHB-based plastics.

Research based on the bio-chemistry and the mechanism involved in the CH₄ oxidation process, bioremediation potential and biofuel/bioplastic production utilising axenic mono-species methanotroph cultures has been widely studied in the last decades. Little is however, known about the feasibility of utilising mixed cultures (methanotrophs and non-methanotrophs) for such purposes, as responses to environmental conditions are poorly studied. This knowledge is vital, as mono-species cultures are unlikely to be maintained in industrial outdoor/landfill scenarios. Therefore, characterising the community composition of methanotrophs and the co-habiting nonmethanotrophs in landfill cover soils and their resilience and responses to different nutrient-governed environmental conditions was the primary focus of this thesis. Specifically, responses of mixed consortia to factors such as variable CH₄ and oxygen (O₂) concentrations, as these vary with time and age in landfills, potentially affecting methanotroph diversity, and variable concentrations of essential trace elements, primarily copper (Cu²⁺) and iron (Fe²⁺), as levels of these govern expression and activity of the key enzymes of the CH₄ oxidation pathway, affecting CH₄ oxidation capacity (MOC) of methanotrophs.

In this Ph.D, I aimed to

1) establish methanotroph-heterotroph consortia (mixed consortia) from different landfill cover soils,

2) examine the robustness of community composition of these consortia to variable CH₄:O₂ (air) and Cu²⁺/Fe²⁺ ratios and investigate the flow on impacts on CH₄ removal efficiency, oxidation capacity, fatty acid and PHB content.

To achieve these aims, four different landfill cover soils (vegetated cover, mulch cover, fresh and compost soils) were collected from Stuart landfill, Townsville, Australia (chapter 2). Initially landfill soil slurries were subjected to high CH₄ environments (50:50 and 20:80 (v/v) of CH₄: air) to measure MOC and total microbial/ methanotrophic diversity using next-generation sequencing. Mulch soil showed higher MOC and was rich in methanotroph diversity compared to other soils and compost soil, the most commonly used landfill cover, had acceptable levels of both type -I and - II methanotrophs, as well as MOC. Therefore, based on MOC and methanotrophic diversity of the soil slurries, mulch - (LB) and compost soil (CB) were selected to establish a mixed consortium. While the enriched consortia (LB and CB) showed similar methanotrophic community profiles (dominated by Methylosarcina, a type -I methanotroph), the co-habiting non-methanotrophs differed. Irrespective of varying microbial structure in both LB and CB, the established consortia showed more or less similar MOC and CH₄ removal efficiencies. These findings suggest that such microbial consortia could be used for biological landfill CH₄ emission abatement.

The robustness and responses of these established mixed consortia to variable CH₄:O₂ ratios (10-50 % CH₄ in air) was then further investigated to assess potential for biological CH₄ mitigation, as these gases change in proportion in landfill environments (chapter 3). PHB-content was also examined to evaluate potential for co-production of biodegradable plastics. Interestingly, CH₄/O₂ ratios did not affect the dominance profile of the mixed consortia, which were dominated by Methylosarcina (50-70 %) and Chryseobacterium (10-30 %) and an average removal efficiency of ~50 % was achieved. Due to the low abundance of type –II methanotrophs PHB content of both LB (~2.5 %) and CB (~1.4 %) was lower than expected. The sustained co-dominance of Methylosarcina and Chryseobacterium suggests that there may be advantageous biological interactions between these microbes, such as assistance in CH₄ oxidation by Chryseobacterium through potential horizontal gene transfer.

In an attempt to increase PHB accumulation, cultivation conditions known to favour growth of type -II methanotrophs (nutrient-limitation, high CH₄ concentration, low pH), the established mixed consortia (LB and CB) were sequentially enriched in nutrient-deficient conditions under high CH₄ concentrations. These further enriched consortia were then then cultivated as sequentially varying Cu²⁺/Fe²⁺ ratios (chapter 4). Irrespective of the similar methanotroph structure (LB and CB) and the same cultivation conditions, the responses of the mixed community to changing Cu²⁺/Fe²⁺ ratios differed. Methylosarcina and Chryseobacterium was initially dominant in both LB and CB, but increased in Cu²⁺/Fe²⁺ ratios led to dominance of Azospirillum and Sphingopyxis over methanotrophs in LB and dominance of Sphingopyxis and Methylosinus (a type -II methanotroph) in CB. Irrespective of the reduced presence of methanotrophs, MOCs and CH₄ removal efficiencies were maintained in LB. Overall, the dominance of type -II methanotrophs and, consequently PHB content, was negatively affected by increasing Cu²⁺/Fe²⁺ ratios in both LB and CB. The outcome of this research highlights that single-species responses differ significantly to those of mixed consortia and that more in depths research is warranted to understand allelopathic, metabolic and molecular interactions in mixed microbial assemblages, particularly when subjected to variable environmental conditions.

In conclusion, my research has increased knowledge on microbial community structure of mixed consortia and their responses to variable CH₄/O₂ (air) and Cu²⁺/Fe²⁺ ratios. Results suggest that the non-methanotrophs co-dominated with methanotrophs may offer beneficial services to methanotrophs in reducing toxic metabolite concentrations and potentially thrive through utilisation of secreted complex carbohydrates (extracellular polymeric substances (EPS)), as no complex carbon sources were provided in the growth medium. Future enzymatic/physiological studies are required to further unravel metabolic interactions and functional relationships in mixed microbial communities. Such studies will help to develop community models to evaluate potential for CH₄ emission abatement a priori. The case-scenario on PHB production potential from landfill CH₄, presented in the general discussion, shows that the cost of PHB production can be reduced to 1.5–2.0 AUD meeting the market value of synthetic plastics by increasing production volumes through building a centralised extraction and refinement facility suitable for large metropolitan cities. However, the scenario is based on optimal bio-filter performance in terms of CH₄ abatement and PHB contents, which had not been reached in the above mixed consortia environmental variable response studies. This demonstrates that future studies need to couple biochemical profiling with optimised bio-filter designs and microbial networks to ascertain the true potential for co-development of renewable products like bioplastics (PHB). Such work will lead to establishing techno-economic models for optimised bio-filter performance with regards to CH₄ abatement in general and coproduct potential specifically.

Item ID: 51748
Item Type: Thesis (PhD)
Keywords: bio-plastics, biopolymer, bioreactor, bioreactors and techno-economics, bioremediation, Chryseobacterium, compost soil, copper, greenhouse gas emission abatement, iron, landfill cover soil, methane mono-oxygenase (MMO), methane, methanotrophs, Methylosarcina, polyhydroxyalkanoates (PHA), polyhydroxybutyrate (PHB)
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Additional Information:

Publications arising from this thesis are available from the Related URLs field. The publications are:

Chapter 1 and Chapter 5: Chidambarampadmavathy, Karthigeyan, Karthikeyan, Obulisamy Parthiba, and Heimann, Kirsten (2017) Sustainable bio-plastic production through landfill methane recycling. Renewable and Sustainable Energy Reviews, 71. pp. 555-562.

Chapter 1: Chidambarampadmavathy, Karthigeyan, Kartikeyan, Obulisamy P., and Heimann, Kirsten (2015) Role of copper and iron in methane oxidation and bacterial biopolymer accumulation. Engineering in Life Sciences, 15. pp. 387-399.

Chapter 1: Karthikeyan, O.P., Karthigeyan, C.P., Cirés, Samuel, and Heimann, Kirsten (2015) Review of sustainable methane mitigation and bio-polymer production. Critical Reviews in Environmental Science and Technology, 45. pp. 1579-1610.

Chapter 3: Chidambarampadmavathy, K., Karthikeyan, O.P., Huerlimann, R., Maes, G.E., and Heimann, K. (2017) Response of mixed methanotrophic consortia to different methane to oxygen ratios. Waste Management, 61. pp. 220-228.

Chapter 4: Chidambarampadmavathy, Karthigeyan, Obulisamy, Kartik P., and Heimann, Kirsten (2015) Biopolymers made from methane in bioreactors. Engineering in Life Sciences, 15 (7). pp. 689-699.

Date Deposited: 11 Dec 2017 23:37
FoR Codes: 10 TECHNOLOGY > 1002 Environmental Biotechnology > 100203 Bioremediation @ 35%
06 BIOLOGICAL SCIENCES > 0605 Microbiology > 060501 Bacteriology @ 30%
10 TECHNOLOGY > 1003 Industrial Biotechnology > 100302 Bioprocessing, Bioproduction and Bioproducts @ 35%
SEO Codes: 85 ENERGY > 8505 Renewable Energy > 850501 Biofuel (Biomass) Energy @ 50%
85 ENERGY > 8598 Environmentally Sustainable Energy Activities > 859802 Management of Greenhouse Gas Emissions from Electricity Generation @ 25%
85 ENERGY > 8598 Environmentally Sustainable Energy Activities > 859805 Management of Solid Waste from Energy Activities @ 25%
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