Biosynthesis of Polyhydroxyalkanoates (Phas) in Methane-Utilizing Mixed Cultures

Biosynthesis of Polyhydroxyalkanoates (PHAs) in Methane-utilizing Mixed Cultures

Pawarisa Luangthongkam

BSc (Hons) Biotechnology MSc Food Engineering and Bioprocess Technology

A thesis submitted for the degree of Doctor of Philosophy at The University of Queensland in 2018 School of Chemical Engineering

Abstract

Methane-to-polyhydroxyalkanoates (PHAs) has been studied for more than a decade, but interest and investment has increased greatly in recent years; there are now even some commercialization activities in this space [1]. This interest and investment has been driven by both the abundance and relatively low price of methane [2] and the growing recognition of the value of PHA as a truly biodegradable material. However, methane-derived PHA is still expensive [2, 3]. In this thesis, the application of mixed culture and thermophilic biotechnology [3-6] are explored as means to potentially reduce production costs [3, 7]. Further, the material properties and narrow processing window of poly(3-hydroxybutyrate) (PHB) call for polymer modifications via incorporation of, for example, 3-hydroxyvalerate (3HV) [8]. In this work, the addition of odd-fatty acids, which is a conventional approach for poly(3-hydroxybutyrate- co-3-hydroxyvalerate) (PHBV) synthesis [2], along with a novel approach of indirect 3HV generation from methane, are considered.

Methanotrophic mixed cultures, enriched from environmental samples, were established. The cultures produced PHBV via: (i) direct synthesis from 3HV precursors – in mesophilic and thermophilic conditions, and (ii) indirect synthesis via microbial symbiosis – in mesophilic conditions. Regarding synthesis by supplementing with 3HV precursors, it had previously been concluded that methane oxidation was necessarily for 3HV production [9, 10]. However, in this work, 3HV synthesis in a methanotrophic community – in both mesophilic and thermophilic conditions - in the absence of methane was achieved. Regarding indirect accumulation, 3HV production without organic acid supplementation was achieved, due to symbiotic processes occurring within the methane-fed consortia. The mesophilic community was characterized using shotgun metagenomic sequencing, which revealed the possible interactions supporting mixed culture PHA accumulation.

Most significantly, this thesis presents a comprehensive enrichment of a thermophilic community capable of PHA production from methane. Twelve selection conditions with a combination of three variables (pH, nitrogen source, and media concentration) were tested. The potential for PHA production in the selected thermophilic community was characterized via shotgun metagenomic sequencing and PHA induction experiments. PHA content of the thermophilic community, which reported up to 10±3 wt% when supplemented with odd-fatty acids, may be considered low compared to mesophilic methanotrophic production. But the community was able to generate PHA with up to 100 mol% of 3HV, which is a higher HV ii fraction than that reported for any other methanotrophic study. The genome directed metabolic analysis of metagenome-recovered genomes revealed high phylogenetic diversity in the thermophilic community. The investigation of PHA synthase (phaC) gene showed several phaC high-quality sequences and interestingly one of these sequences was belonging to a thermophilic methanotroph from Methylocaldum genus. That organism was most prevalent, accounting for 40% of the community that carried the phaC gene.

The Methylocaldum sp. was therefore targeted for isolation. By using metagenomics, the metabolic pathways of the methanotrophic organisms including the Methylocaldum sp. were reconstructed, and revealed putative functions for PHAs synthesis. The genome directed metabolic analysis of metagenome-recovered genomes disclosed the presence of β-oxidation- linked PHA polymerase genes (phaCE) and β-oxidation pathway (fadABDG) in the Methylocaldum sp., which explained the assimilation of odd-carbon fatty acids for intracellular PHA synthesis. It appears that the axenic culture of Methylocaldum was able to synthesize 3HB units only without a trace of 3HV monomer, regardless of the substrate supplied. Nevertheless, this study demonstrates that the thermophilic Methylocaldum sp. can accumulate PHA, highlighting the potential for thermophilic fermentation for future methane-to-PHA production.

Overall, this thesis has investigated mixed culture methane-to-PHA, with the view to developing new strategies and platforms for PHA copolymer synthesis. The main outcomes are the success in the selection and isolation of the first thermophilic PHA-producing methanotroph and the demonstration of PHBV production - with high 3HV fractions - by the thermophilic community. One additional advance is the demonstration of microbial interaction within mesophilic consortia for PHBV synthesis directly from methane. It is envisaged that these outcomes will help to provide alternative and promising ways to drive future PHA commercialization.

iii Declaration by author

This thesis is composed of my original work, and contains no material previously published or written by another person except where due reference has been made in the text. I have clearly stated the contribution by others to jointly-authored works that I have included in my thesis.

I have clearly stated the contribution of others to my thesis as a whole, including statistical assistance, survey design, data analysis, significant technical procedures, professional editorial advice, and any other original research work used or reported in my thesis. The content of my thesis is the result of work I have carried out since the commencement of my research higher degree candidature and does not include a substantial part of work that has been submitted to qualify for the award of any other degree or diploma in any university or other tertiary institution. I have clearly stated which parts of my thesis, if any, have been submitted to qualify for another award.

I acknowledge that an electronic copy of my thesis must be lodged with the University

Library and, subject to the policy and procedures of The University of Queensland, the thesis be made available for research and study in accordance with the Copyright Act 1968 unless a period of embargo has been approved by the Dean of the Graduate School.

I acknowledge that copyright of all material contained in my thesis resides with the copyright holder(s) of that material. Where appropriate I have obtained copyright permission from the copyright holder to reproduce material in this thesis.

iv Publications included in this thesis

No publications included

Submitted manuscripts included in this thesis

Luangthongkam P., Strong J., Evans P., Jensen P., Tyson G., Laycock B., Lant P., Pratt S. The effect of methane and odd-chain fatty acids on 3-hydroxybutyrate (3HB) and 3- hydroxyvalerate (3HV) synthesis by a Methylosinus-dominated mixed culture. Submitted for publication in New Biotechnology and incorporated as Chapter 4. Contributor Statement of contribution Luangthongkam, Pawarisa (Candidate) Conceived the research (50%) Designed and performed experiments (80%) Analysed and interpreted data (80%) Wrote and edited paper (60%) Strong, James Conceived the research (20%) Designed and performed experiments (20%) Evans, Paul Analysed and interpreted data (20%) Jensen, Paul Wrote and edited paper (5%) Tyson, Gene Wrote and edited paper (5%) Laycock, Bronwyn Conceived the research (10%) Wrote and edited paper (10%) Lant, Paul Conceived the research (10%) Wrote and edited paper (10%) Pratt, Steven Conceived the research (10%) Wrote and edited paper (10%)

Luangthongkam P., Evans P., Syed Mahamud S.N., Jensen P., Tyson G., Laycock B., Lant P., Pratt S. Methanotrophic-heterotrophic symbiosis for 3-hydroxybutyrate and 3-hydroxyvalerate production from methane. Submitted for publication in New Biotechnology and incorporated as Chapter 5. Contributor Statement of contribution Luangthongkam, Pawarisa (Candidate) Designed and performed experiments (40%) Analysed and interpreted data (60%) Wrote and edited paper (40%)

v Contributor Statement of contribution Evans, Paul Conceived the research (20%) Designed and performed experiments (20%) Analysed and interpreted data (40%) Wrote and edited paper (20%) Syed Mahamud, Syarifah Nuraqmar Conceived the research (50%) Designed and performed experiments (40%) Jensen, Paul Wrote and edited paper (5%) Tyson, Gene Wrote and edited paper (5%) Laycock, Bronwyn Conceived the research (10%) Wrote and edited paper (10%) Lant, Paul Conceived the research (10%) Wrote and edited paper (10%) Pratt, Steven Conceived the research (10%) Wrote and edited paper (10%)

Luangthongkam P., Evans P., MacDonald S., Jensen P., Tyson G., Laycock B., Lant P., Pratt S. Non-conventional polyhydroxyalkonate (PHA) biosynthesis by a thermophilic methanotroph. Submitted for publication in ISME Journal and a modified version of this publication was incorporated in Chapters 6.

Contributor Statement of contribution Luangthongkam, Pawarisa (Candidate) Conceived the research (40%) Designed and performed experiments (50%) Analysed and interpreted data (40%) Wrote and edited paper (30%) Evans, Paul Conceived the research (20%) Designed and performed experiments (30%) Analysed and interpreted data (30%) Wrote and edited paper (30%) MacDonald, Samantha Designed and performed experiments (20%) Analysed and interpreted data (30%) Jensen, Paul Wrote and edited paper (5%)

vi Contributor Statement of contribution Tyson, Gene Conceived the research (10%) Wrote and edited paper (5%) Laycock, Bronwyn Conceived the research (10%) Wrote and edited paper (10%) Lant, Paul Conceived the research (10%) Wrote and edited paper (10%) Pratt, Steven Conceived the research (10%) Wrote and edited paper (10%)

Luangthongkam P., Laycock B., Evans P., Lant P., Pratt S. High-level production of poly(3-hydroxybutyrate-co-3-hydrovalerate) by thermophilic culture grown on methane. Submitted for publication in New Biotechnology and incorporated as Chapter 7.

Contributor Statement of contribution Luangthongkam, Pawarisa (Candidate) Conceived the research (70%) Designed and performed experiments (100%) Analysed and interpreted data (95%) Wrote and edited paper (60%) Laycock, Bronwyn Conceived the research (10%) Wrote and edited paper (15%) Evans, Paul Analysed and interpreted data (5%) Wrote and edited paper (5%) Lant, Paul Conceived the research (10%) Wrote and edited paper (10%) Pratt, Steven Conceived the research (10%) Wrote and edited paper (10%)

Other publications during candidature

No other publications

vii Contributions by others to the thesis

This thesis included contributions made by others. All proper technical acknowledgements are expressed in this section.

Syarifah Nuraqmar Syed Mahamud and Christine Wegener (Beuth University of Applied Sciences, Germany) provided inoculums and assisted in bioreactor operation for Chapters 4 and Chapter 5.

Dr. James Strong from Queensland University of Technology (QUT) provided valuable assistance in experimental design and sample analysis for Chapter 4.

Dr. Paul Evans and Samantha MacDonald contributed with a community profiling using shotgun metagenomics DNA sequencing, an assembly and binning of metagenome-assembled genomes (MAGs), a reconstruction of metabolic pathway, and isolation of methanotrophic culture for Chapter 6.

Dr. Beatrice Keller-Lehmann, Nathan Clayton, and Jianguang Li contributed with analytical analysis of gas chromatography, Flow Injection Analyser (FIA) for ammonia, nitrate, nitrite, and phosphate detection, Volatile Fatty Acids (VFAs) for propionic acid and valeric acid for Chapters 4-8.

Statement of parts of the thesis submitted to qualify for the award of another degree

Chapter 5 was a collaborative work with Syarifah Nuraqmar Syed Mahamud whose results were also submitted as part of her dissertation for the degree of Doctor in Philosophy in Chemical Engineering submitted in September 2018 in UQ.

Chapter 6 was a collaborative work with Samantha MacDonald whose results were also submitted as part of her bachelor degree in Chemistry and Molecular Biosciences submitted in May 2018 in UQ.

Research involving human or animal subjects

No animal or human subjects were involved in this research

viii Acknowledgements

I would like to express my sincere gratitude to my advisors Asso. Prof. Bronwyn Laycock, Prof. Paul Lant, and Asso. Prof. Steven Pratt who have been mentors behind my success pursuing this PhD. I am really grateful and thankful for all their insightful comments and suggestions, for time and patience reviewing my documents, and for their kindness during challenging times. PhD is a rough journey nevertheless I am appreciated to have this incredible advisory team.

My sincere thanks to Dr. Paul Evans, Samantha MacDonald, and Prof. Gene Tyson from Australian Centre for Ecogenomics (ACE), and Dr. Paul Jensen from Centre of Advanced Water Management (AWMC) for their interest in my work, for helps and supports, and for valuable exchange that refine the completion of this thesis.

I would like to thank my reviewers Dr. Esteban Marcellin Saldana and Asso. Prof. Stefano Freguia for their time assessing three milestones during my candidature and for their valuable comments, suggestions, and feedbacks.

I also extend my gratitude to Dr. James Strong, Dr. Sergi Astals-Garcia, Syarifah Nuraqmar Syed Mahamud, Dr. Shao Dong Yap, Mike Meng, and Catherine Macintosh for wonderful vibes in labs, incredible friendships, and constant encouragements.

Heartfelt thanks go to Boon Beng Lee and Nurhafizah Ab Hamid for being my oversea brother and sister, Fabio Terzini for genuinely humors and funs, Fei Ren for love and kindness. Thanks to Siti Nurehan Ballinger, Johnathan Tan, and Luisa Prasetyo for warm and memorable welcome to Australia.

Special thanks to all Chemical Engineering post-graduate students and UQ friends for sharing these memories together.

Finally and most importantly, my deepest love and gratefulness goes to my mother Wipawan Luangthongkam who absolutely stays behind all my success and achievement, my brother Pavarit Luangthongkam who always has no doubt in me, my sister Pawarisara Luangthongkam who always be my motivation and inspiration. Thank you for your fully trusts, loves and supports, and sacrifices you have done.

ix Financial support

This research was supported by an Australian Research Council (DP15010306).

Keywords

Polyhydroxyalkanoates, methane, methanotrophs, mixed microbial cultures, bioprocess.

Australian and New Zealand Standard Research Classifications (ANZSRC)

091006 Manufacturing Processes and Technologies (excl. Textiles), 30%

060405 Gene Expression (incl. Microarray and other genome-wide approaches), 30%

100302 Bioprocessing, Bioproduction and Bioproducts, 40%

Fields of research (FoR) Classification

0904 Chemical Engineering – 100%

x Table of Contents Abstract ...... ii Declaration by author ...... iv Publications included in this thesis ...... v Submitted manuscripts included in this thesis ...... v Other publications during candidature ...... vii Contributions by others to the thesis ...... viii Statement of parts of the thesis submitted to qualify for the award of another degree . viii Research involving human or animal subjects ...... viii Acknowledgements ...... ix Financial support ...... x Keywords ...... x Australian and New Zealand Standard Research Classifications (ANZSRC) ...... x Fields of research (FoR) Classification ...... x Table of Contents ...... xi List of Figures ...... xv List of Tables ...... xx List of Abbreviations ...... xxi Chapter 1 Introduction ...... 1 1.1 Thesis objectives ...... 2 1.2 Thesis organization ...... 3 Chapter 2 Literature Review ...... 5 2.1 Introduction to microbial polyhydroxyalkanoates (PHAs) ...... 5 2.1.1 Polyhydroxyalkanoates background ...... 5 2.1.2 Properties of PHA ...... 6 2.1.3 Microbial PHA production ...... 6 2.1.4 Challenges in PHA production ...... 10 2.2 PHA production from methane ...... 11 2.2.1 Methane ...... 11 2.2.2 Methanotrophs ...... 12 2.2.3 Thermophilic and thermotolerant methanotrophs ...... 13 2.2.4 Strategies for methanotrophic production of PHBV copolymer ...... 15 2.3 Conclusion and recommendation ...... 17 Chapter 3 Materials and Methods ...... 18 xi 3.1 Sample collection and selection for mesophilic PHA production ...... 19 3.2 Reactor operation and PHA accumulation ...... 20 3.3 PHA analysis ...... 21 3.3.1 PHA content and composition via gas chromatography ...... 21 3.3.2 PHA determination by fourier-transform infrared spectroscopy (FTIR) ...... 22 3.3.3 Nile blue A staining ...... 22 3.4 Microbial community analysis ...... 22 3.5 Analytical methods ...... 23 3.5.1 Gas concentration analysis ...... 23 3.5.2 Total suspended solids (TSS) measurement ...... 23 3.5.3 Nutrients analysis ...... 23 3.5.4 Volatile fatty acids (VFAs) analysis ...... 24 3.6 Calculation ...... 24 Chapter 4 The effect of methane and odd-chain fatty acids on 3-hydroxybutyrate (3HB) and 3-hydroxyvalerate (3HV) synthesis by a Methylosinus-dominated mixed culture ...... 26 4.1 Introduction ...... 26 4.2 Materials and methods ...... 28 4.2.1 Semi-continuous enrichment reactor ...... 28 4.2.2 PHA Accumulation ...... 29 4.3 Results and discussion ...... 29 4.3.1 Community growth characteristic in the presence of methane and nitrogen . 29 4.3.2 Community flux in the semi-continuous reactor ...... 29 4.3.3 PHA accumulation ...... 33 4.4 Conclusion ...... 35 Chapter 5 Methanotrophic-heterotrophic symbiosis for 3-hydroxybutyrate and 3- hydroxyvalerate production from methane ...... 36 5.1 Introduction ...... 37 5.2 Materials and methods ...... 38 5.2.1 Methane-fed microbial enrichment culture ...... 38 5.2.2 PHA Production ...... 39 5.2.3 Community metagenome sequencing ...... 39 5.3 Results and discussion ...... 40 5.3.1 Enrichment of a methane-consuming community using a fed-batch process 40 xii 5.3.2 Methane-fed PHA production ...... 41 5.3.3 Community dynamics in semi-continuous-batch reactor ...... 43 5.3.4 Metabolic capacity of the bioreactor microbial community ...... 45 5.3.5 Possible interactions within the methane-driven community for 3HB and 3HV generation ...... 48 5.4 Conclusion ...... 51 Chapter 6 Biosynthesis of poly-3-hydroxybutyrate (PHB) in a thermophilic gammaproteobacterial methanotroph ...... 52 6.1 Introduction ...... 54 6.2 Materials and methods ...... 55 6.2.1 Sample collection ...... 55 6.2.2 Selection experiment ...... 55 6.2.3 Methanotrophic enrichment in semi-continuous reactors ...... 57 6.2.4 Metabolic potential of the thermophilic bioreactor community ...... 57 6.2.5 Culture isolation and purity test ...... 58 6.2.6 Cultivation condition of the isolate ...... 58 6.2.7 Fluorescence in situ hybridization ...... 59 6.2.8 PHA accumulation in pure isolate ...... 59 6.3 Results and discussion ...... 60 6.3.1 Selection experiment ...... 60 6.3.2 Methanotrophic enrichment in semi-continuous reactors ...... 63 6.3.3 PHA production in the thermophilic community ...... 65 6.3.4 Monitoring of the microbial community structure in semi-continuous reactors ...... 65 6.3.5 Metabolic potential of the thermophilic bioreactor community ...... 67 6.3.6 Culture isolation and purity check ...... 73 6.3.7 PHA production in the Methylocaldum isolate ...... 74 6.4 Conclusion ...... 77 Chapter 7 Thermophilic biotechnology for production of PHA with high ratio of 3-hydroxyvalerate (3HV) to 3-hydroxybutyrate (3HB) ...... 78 7.1 Introduction ...... 78 7.2 Materials and methods ...... 79 7.2.1 Culture condition ...... 79 7.2.2 PHB and PHBV production ...... 79 xiii 7.2.1 Statistical analysis ...... 81 7.3 Results ...... 83 7.3.1 Microbial community in thermophilic enrichment ...... 83 7.3.2 Effect of temperature and odd-fatty acids on PHA production in the presence of methane ...... 83 7.3.3 PHA production in the absence of methane ...... 87 7.3.4 Effect of acetic acid on PHA production ...... 90 7.4 Discussion ...... 94 Chapter 8 Conclusion and future perspective ...... 97 References ...... 101 Appendix ...... 129

xiv List of Figures

Figure 1.1 Schematic representation of the overall thesis structure. Three work packages were presented covering (i) mesophilic sample collection and culture selection previously done by Syarifah Nuraqmar Syed Mahamud [23] (■), (ii) mesophilic PHA production (■), and (iii) thermophilic PHA production (■). The research objectives (■) relative to each thesis chapter (■) and significant outcome relative to each work packages (■) are provided...... 4

Figure 2.1 Poly(3-hydroxyalkanoate) (PHA) chemical structure [14]...... 5

Figure 2.2 Natural and engineered PHA pathways [57] ...... 8

Figure 3.1 Schematic representation of the overall thesis methodology. Three work packages were presented covering (i) mesophilic sample collection and culture selection previously done by Syarifah Nuraqmar Syed Mahamud [23] (■), (ii) mesophilic PHA production (■), and (iii) thermophilic PHA production (■). The research objectives (■) relative to each thesis chapter (■) and significant methodology relative to each work packages (■) are provided...... 18

Figure 3.2 Overall scheme of semi-continuous reactor operation and PHA production. A portion of culture discharged from reactor after t cultivation time is represented as Vw, while

Vm represents volume of fresh medium added at the end of each nutrient cycling. Vi represents the initial liquid volume or operating volume in serum bottle...... 20

Figure 4.1 Overall scheme of 2-L reactor operation and PHA production using methane as a single carbon source...... 28

Figure 4.2 The relative abundance of bacterial 16s rRNA genes at the genus level expressed as % of OTUs in the sample that were present at more than 1%...... 30

Figure 4.3 Maximum composite likelihood phylogenetic tree for 25 OTUs affiliated to Proteobacteria and Bacteroidetes. Bootstrap values ≥50% are represented as open (○) circles, ≥70% as grey (●) circles, and ≥90% as filled (●) circles...... 32

Figure 4.4 PHA content presented as fractions of 3HB (■) and 3HV (■) on a mass basis (wt%) obtained at the end of the PHA accumulation phase in the presence and absence of methane. A bold red dotted line ( - - ) represents the initial PHA content in the culture before being subjected to PHA accumulation...... 35

xv Figure 5.1 The pattern of methane consumption and PHA accumulation in methanotrophic– heterotrophic communities grown using different solids retention times (SRTs) over 400 days of a typical growth repeating the operation. Methane consumption rate (μmol day-1) (o), 3HB content on mole basis (mol%) (▲), and 3HV content on mole basis (mol%) (•) in the PHA produced are shown. Dash line (- - -) shows median methane consumption rate for the various SRTs...... 41

Figure 5.2 Methane consumption and production of carbon dioxide, biomass, and intracellular PHA on a C-mmole basis during a typical 72-h of growth phase in the presence of nitrogen (ammonium) (A) and a 72-h accumulation phase in the absence of nitrogen (B), using cells harvested from the 2-L reactor on Day 382. C-mmole of methane (○), carbon dioxide (x), total biomass (■), and total PHA (¨) are shown...... 42

Figure 5.3 Bar graph representing relative community compositions of 18 bacterial genera which represented > 1% of total sequence counts, comparing each biomass sampled on Day 0, 45 and 289. Data on Day 0 (inoculum) and Day 45 obtained from 16s rRNA sequencing, while Day 289 retrieved from metagenomic sequencing. Left and right braces highlight total population of C1-substrates and methane utilizers, respectively. (*) represents microorganisms with PHA production capability in literature...... 44

Figure 5.4 Phylogenetic representation of bacterial phaC genes from class I, II, III and IV. Bold phaC sequences corresponding to high-quality MAGs have been recovered from the methanotrophic-heterotrophic community. Bootstrap values ≥50% are represented as open (○) circles, ≥70% as grey (●) circles, and ≥90% as filled (●) circles...... 47

Figure 5.5 Illustration of the likely interactions within the methanotrophic-heterotrophic community when methane serves as the sole carbon source and polyhydroxyalkanoates (PHAs) with 3-hydroxybutyrate (3HB) and 3-hydroxyvalerate (3HV) monomer units are being generated. (*) indicates the community members with the phaC gene. ( ) shows carbon substrates utilization associated with growth and PHB production. ( ) shows the cometabolic transformation of 3HV precursor to PHAs with 3HV composition...... 50

Figure 6.1 Schematic diagram represents the workflow of Chapter 6 ...... 53

Figure 6.2 FTIR Scans for selected thermophilic enrichment series after the 72-h PHA accumulation phase under nitrogen-deficient condition. Grey area indicates a peak area of the

xvi carbonyl group of PHA. (A) H1 sample in enrichment H series. (B, C, and D) are H2 sample in enrichment series of G, B, and H, respectively. See Table 6.1 for specific details of each enrichment series...... 62

Figure 6.3 Total methane consumption rate and specific consumption rate of thermophilic enrichment in semi-continuous reactors. (ᴏ), (∆), (□) represents total methane consumption -1 (μmol CH4 day ), and (●), (▲), (■) represents specific consumption of methane (mg CH4 mg TSS-1 day-1) of Phase 1 (Day 0-220), Phase 2 (Day 220 -475, and Phase 3 (Day 475-750). The trendlines represent a direction of specific consumption rates. Error bar represents the standard error of three replicates...... 64

Figure 6.4 Bright-field photomicrograph of PHA-accumulating thermophilic biomass overlaid with Nile Blue A stain for PHA viewed under fluorescence...... 65

Figure 6.5 The relative abundances of bacterial 16S rRNA genes expressed as % of OTUs in methanotrophic enrichment grown in semi-continuous reactors at 55°C (Table A3)...... 67

Figure 6.6 Phylogenetic representation of the 22 high-quality recovered MAGs from the thermophilic community based on the concatenated alignments of the 120 bacterial marker genes used in the GTDB [240]. Recovered MAGs are highlighted in red. This phylogenetic tree was generated by Samantha McDonald (2018) [241]...... 70

Figure 6.7 Taxonomic tree of the phaC sequences identified from the thermophilic parent reactor MAGs. A taxonomic tree of the representative database and MAG derived phaC sequences was generated using Maximum-Likelihood treeing methods with bootstrap values from 100 iterations. Numbers assigned to bioreactor phaC sequences refer to the MAGs from which these sequences were derived in Table A4...... 71

Figure 6.8 Metabolic reconstruction of methanotroph and Methyloceanibacter MAGs from the thermophilic community ...... 72

Figure 6.9 Taxonomic tree of the 16s rRNA sequences identified from the Methylocaldum sp. isolate. A taxonomic tree was generated using Maximum-Likelihood treeing methods with bootstrap values from 100 iterations. Staphylococcus epidermidis was used as an outgroup. 74

Figure 6.10 Images of the isolate Methylocaldum organism after serial dilutions and erythromycin treatment (left) and a Fluorescent in situ hybridization (FISH) image of the

xvii isolate stained with the Cy3-labelled control general EUB338 bacterial probe and FITC- labelled specific gammaproteobacterial GAM42a probe (right). The image was taken by Samantha McDonald (2018) [241]. Scale bars represent 5µm...... 74

Figure 6.11 Nile Blue A stain of the Methylocaldum isolate culture forming PHA. Methylocaldum cells with the addition of carbon sources of methane only (Top Images) or methane and even-fatty acids (Bottom Images). Cells stained with Nile Blue A (Bottom Images) using the methods of Ostle and Holt (1982) are shown with unstained cells [250]. All cells were visualised under UV light (550nm) and were overlaid with phase contrast microscopy images. Ovals indicate the positions of likely PHA granules...... 76

Figure 7.1 A schematic diagram represents the overall operation of thermophilic culture growth using methane as primary substrate and PHA production under various conditions. Steps 1-4 refer to operation of the parent reactor described in Chapter 6. Step 5 refers to boosting the amount of biomass available. Step 6 refers to the experiment series for this chapter...... 80

Figure 7.2 PHA (♦), 3HB (●), and 3HV (▲) content presented on a mass basis (wt%) and 3HB (■) and 3HV (■) fraction on a mol basis (mol%) obtained during PHA accumulation phase in the presence of methane at 55 and 58°C. Error bars are standard deviations (n=3). Statistic letters denote significant differences within each analyte for 3HV and 3HB content with p<0.05 were derived in Table A5...... 84

Figure 7.3 Specific consumption and production of propionic acid (■), valeric acid (■), PHA (■), 3HB (■), and 3HV (■) on carbon mole basis during PHA accumulation phase in the presence of methane at 55 and 58°C: (A) Traditional PHA accumulation, (B) 100-ppm propionic acid addition, (C) 100-ppm, and (D) 200-ppm valeric acid addition. Error bars are standard deviations (n=3). All data were derived in Table A8...... 86

Figure 7.4 PHA (♦), 3HB (●), and 3HV (▲) content presented on a mass basis (wt%) and 3HB (■) and 3HV (■) fraction on a mol basis (mol%) obtained during PHA accumulation phase in the presence and absence of methane at 55°C. Error bars are standard deviations (n=3). Letters denote statistically significant differences at the 95% confidence level within each analyte for 3HV and 3HB contents with derivation given in Table A6...... 87

xviii Figure 7.5 The concentration of (A) propionic acid and (B) valeric acid expressed as percentage concentration (ppm) presented in cell suspension during 48-h PHA accumulation phase in the presence and absence of methane at 55°C. Error bars are standard deviations (n=3)...... 88

Figure 7.6 Specific consumption and production rates of propionic acid (■), valeric acid (■), PHA (■), 3HB (■), and 3HV (■) on a carbon mole basis during the PHA accumulation phase in the presence and absence of methane at 55°C: (A) Normal PHA accumulation, (B) 100-ppm propionic acid addition, (C) 100-ppm, and (D) 200-ppm valeric acid addition. Error bars are standard deviations (n=3). The data were derived in Table A8 and Table A9...... 89

Figure 7.7 PHA (♦), 3HB (●), and 3HV (▲) content presented on a mass basis (wt%) and 3HB (■) and 3HV (■) fraction on a mol basis (mol%) obtained during PHA accumulation phase in the presence and absence of methane, acetic acid, and propionic acid at 55°C. Error bars are standard deviations (n=3). Letters denote statistically significant differences at the 95% confidence level within each analyte for 3HV and 3HB contents with derivation given in Table A7...... 91

Figure 7.8 The concentration of (A) acetic acid and (B) propionic acid expressed as percentage concentration (ppm) presented in cell suspension at the given time during PHA accumulation phase in the presence and absence of methane, acetic acid, and propionic acid at 55°C. Error bars are standard deviations (n=3)...... 92

Figure 7.9 Specific consumption and production of acetic acid (■), propionic acid (■), valeric acid (■), PHA (■), 3HB (■), and 3HV (■) on carbon mole basis in the presence and absence of methane at 55°C: (A) traditional PHA accumulation, (B) 100-ppm acetic acid, (C) 100-ppm propionic acid, and (D) 100-acetic acid and 100-ppm propionic acid. Error bars are standard deviations (n=3). Additional data were derived in Table A8 and Table A9...... 93

xix List of Tables

Table 2.1 List of known enzymes involved in PHA pathways in Figure 2.2 ...... 8

Table 2.2 PHA synthase (PhaC) classification [64] ...... 10

Table 2.3 Main characteristics of the thermophilic and thermotolerant methanotrophs of the phylum proteobacteria [106] ...... 14

Table 2.4 PHA production using various co-substrates by methanotrophs [9, 10, 278]………16

Table 4.1 PHA content and composition produced by the Methylosinus-dominated culture after 72 h of incubation in the presence and absence of methane with supplementation of odd-chain fatty acids ...... 34

Table 6.1 Selection conditions for thermophilic methanotrophs ...... 55

Table 6.2 Specific methane consumption rate of 24 methanotrophic enrichment series after a 20-day of incubation at 55°C...... 61

Table 6.3 PHB production by the Methylocaldum isolate after 48 h incubation at 45°C in the presence and absence of methane with or without co-substrate addition...... 76

Table 7.1 A summary of PHA accumulation conditions at 55°C and 58°C with and without methane availability and different supplementation of organic acids...... 82

xx List of Abbreviations

%3HB 3HB fraction in total PHA (mol 3HB mol PHA-1) %3HV 3HV fraction in total PHA (mol 3HV mol PHA-1) %PHA PHA intracellular content (g PHA g TSS-1) %PHB PHB intracellular content (g PHB g TSS-1) 3HB 3-Hydroxybutyrate 3HV 3-Hydroxyvalerate Ac Acetic acid CBB Calvin-Benson-Bassham

CH4 Methane CoA Coenzyme A FIA Flow Injection Analyser FTIR Fourier transform infrared MAGs Metagenomic assembled genomes MMC Mixed microbial community Pr Propionic acid Val Valeric acid PHA Poly(3-hydroxyalkanoate)

PHA0 Initial PHA concentration

PHAt PHA concentration at time t PHB Poly(3-hydroxybutyrate) PHBV Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) RuBP Ribulose bisphosphate RuMP Ribulose monophosphate SRT Solid retention time TSS Total suspended solids VFA Volatile fatty acid

X0 Initial active biomass

Xt Active biomass at time t

xxi Chapter 1 Introduction

Polyhydroxyalkanoates (PHAs) are biodegradable polyesters synthesized by many microorganisms including bacteria and archaea [11]. Their properties are competitive with conventional plastics such as polyethylene (PE) and polypropylene (PP) [8], and, therefore, have been used in diverse applications in the range from packaging to medical functions [12]. Many companies such as Tianjin Green Bioscience Co., Ltd. (China), Metabolix (USA), and Biocycle PHB Industrial SA (Brazil) are currently producing PHAs by using pure cultures and plant-derived carbon sources such as crops, sugar cane, and oils as the feedstock [1, 3]. The pure culture production using plant-derived feedstocks has raised concerns about feed-food competition and high production cost, due to a requirement of refined substrates and aseptic operation, limiting widespread capitalization [1, 3, 7].

There has been renewed interest in the use of methane for PHA production because of its abundance and the opportunity to use a cheap non-food feedstock. Methane can be converted naturally by methanotrophic bacteria into poly(3-hydroxybutyrate) (PHB) in the excess of carbon source with nitrogen limitation [2, 13, 14]. Traditionally, methane-to-PHA production involves the use of pure methanotrophic cultures [1]. However, employment of robust and self-regulating methanotrophic mixed cultures, which operate under non-aseptic conditions, has offered a promising alternative [1, 10, 15, 16], thereby reducing operating costs [17, 18]. Mango Materials, a start-up company from USA, has achieved a pilot-scale methane-to-PHA production for commercialization [1].

Nevertheless, challenges limiting commercialization of PHA from methane include the relatively poor mechanical properties of the PHB homopolymer, such as relatively high crystallinity, stiffness, and brittleness, and the narrow windows for processing [8, 19]. One solution for improving material properties is the production of poly(3-hydroxybutyrate-co-3- hydroxyvalerate) (PHBV), which can be achieved by adding external 3-hydroxyvalerate (3HV) precursors (i.e. levulinic acid, propionate, and valerate) [2, 8]. This strategy has been demonstrated widely in many methanotrophic cultures; however, their fatty acid metabolism have not been revealed and it should be noted that the addition of 3HV precursors represents an additional cost into the process [2].

Mixed microbial culture knowledge offers a potential opportunity to obtain a diverse composition of PHA from methane without external substrate addition owing to genetic

1 diversity and diverse metabolic pathways [20]. Kalyuzhnaya et al (2013) and Bothe et al (2002) demonstrated that it is possible for methanotrophs to interact with non-methanotrophic PHA- producing microorganisms making indirect production of other hydroxyalkanoate (HA) monomers feasible [21, 22]. However, the production of PHA via microbial interaction is not simply a function of microbial composition. It requires an understanding of symbiotic mechanisms between microorganisms in order to obtain particular biopolymers, which has not yet been achieved.

Besides the needed investigation on symbiosis, the application of thermophiles for next generation PHA production is gaining interest given that it is beneficial for production cost reduction and microbial contamination control [3-6]. Thermophilic bioprocess is considered energy-efficient because less energy efforts are required for maintaining bioreactor-operating temperature, evidenced by a comprehensive techno-economic analysis for a large-scale production of PHA from methane [3]. To date, some thermophilic bacteria have shown potential capacity to accumulate PHB, but there is quantitative evidence of PHA production in thermophilic methanotrophs.

In short, many challenges in commercialization of methanotrophic PHA processes still exist. Further study and development in both operation and material properties is essential in particular for introducing competitive diversifications for the production, which as a result will increase value and demand for PHAs.

1.1 Thesis objectives

The ultimate goal of this study is to develop new strategies for a low-cost production of PHB and PHBV from methane when the gas serves as the main carbon source in mixed microbial cultures.

The research objectives of this study are listed as follows: 1. To assess different strategies for PHBV synthesis from methane in mixed microbial cultures (MMC) 2. To enrich a thermophilic methanotrophic mixed culture capable of PHA production 3. To investigate the feasibility of PHB and PHBV production from methane in the thermophilic mixed culture 4. To isolate and characterize thermophilic methanotrophs with PHA producing capability 2 1.2 Thesis organization

This thesis consists of eight chapters including a brief introduction, detailed materials and methods, results and discussion, and lastly a summary of goals achieved. A description of each chapter is presented as follows:

Chapter 1 provides the overview, objectives, and motivations of this Ph.D. thesis.

Chapter 2 summarises current knowledge of PHA production. It starts with an introduction of microbial PHA production and challenges in a large-scale production of PHA. The focuses include: (i) challenges and limitation in microbial PHA biosynthesis, (ii) advantages of PHA production from methane, (iii) benefits of PHA production under high-temperature condition, (iv) current knowledge of thermophilic methanotrophs, and (v) strategies for PHB and PHBV production in methanotrophs.

Chapter 3 provides details of the experimental set-up, operation conditions, and analytical techniques (chemical, biological, and molecular analysis) used in this thesis. The method for calculation of the kinetic and stoichiometric parameters reported in Chapter 4-8 is shown in this chapter.

Chapters 4 and 5 demonstrate two different strategies for PHA copolymer production in mesophilic conditions. Chapter 4 describes a direct approach for PHA copolymer synthesis by supplementation of additional co-substrates. The key methanotroph is Methylosinus, which is known to be able to synthesise both 3HB and 3HV monomers. Chapter 5 describes an indirect approach via symbiosis between microorganisms in a mixed microbial culture (MMC). The key methanotroph is Methylomonas, which processes methane but does not accumulate PHA. These chapters are prepared for publication in peer-reviewed journals.

Chapter 6 presents a history of thermophilic methane-fed enrichment obtained by applying different selection and enrichment criteria. Evidence of capability to produce PHB in the mixed community is presented. The metabolic potential for PHA formation is confirmed by Metagenome-assembled genomes (MAGs). In addition, an isolation technique of a PHA-producing methanotroph from the mixed microbial culture (MMC) was performed in this chapter and revealed quantitative and qualitative evidence of PHA accumulation in the isolate.

3 Chapter 7 describes an extended capability of PHB and PHBV production in the thermophilic mixed culture. Carbon distribution and mass balance were carried out to demonstrate the impact of feeding strategies on PHA content and composition under nitrogen-limited conditions.

Chapter 8 gives the conclusions from this thesis and provides perspectives for future work.

A schematic representation of the thesis structure is presented in the following figure.

Biosynthesis of Polyhydroxyalkanoates (PHAs) in Methane-utilizing Mixed Cultures

Preliminary work Thermophilic PHA Production (This section was done prior to this thesis study)

Environmental sample collection Research objective 2 and 3: • To demonstrate selection and enrichment criteria for thermophilic methanotrophic mixed culture Culture selection capable of PHA production • To isolate and characterize thermophilic Ammonium Nitrogen gas methanotrophs with PHA producing capability as a nitrogen source as a nitrogen source

Biomass production using ammonia as a Chapter 6 nitrogen source

Environmental sample collection Mesophilic PHA Production

Culture selection Research objective 1: Methane as sole carbon source • To assess different strategies for PHBV synthesis from methane in mixed microbial cultures (MMC) Culture enrichment Methane as sole carbon source

Chapter 4 Chapter 5 Research objective 4: PHB production in mixed culture • To investigate the feasibility of PHB and PHBV Methane served as sole carbon source production from methane in the thermophilic mixed Culture enrichment culture Methane-fed semi-continuous reactors • with sufficient ammonium as nitrogen source A comprehensive enrichment of a thermophilic PHA-producing community solely fed with methane Chapter 7

Direct PHBV Indirect PHBV production production Pure culture isolation and cultivation PHB and PHBV production in mixed culture Methylosinus Methylomonas dominated culture dominated culture • An isolation of a thermophilic phaC-harbouring • The first PHA accumulation by thermophilic culture methanotroph grown on methane • A conventional • A novel approach • The first thermophilic production of PHAs that approach for of indirect 3HV contain repeating units beyond 3HB PHBV synthesis generation from by the addition methane PHB production in pure culture of odd-carbon- number fatty acids • The first quantitative evidence of PHA accumulation in a thermophilic methanotroph

4 Chapter 2 Literature Review

2.1 Introduction to microbial polyhydroxyalkanoates (PHAs)

2.1.1 Polyhydroxyalkanoates background

Polyhydroxyalkanoates (PHAs) are a family of biopolyesters synthesized by various microorganisms including bacteria and archaea [24, 25]. Microorganisms synthesize PHAs when there is a growth-limiting concentration of critical element, such as nitrogen (N), oxygen (O), phosphate (P), or trace elements (i.e. Cu or Fe), in the presence of excess carbon source [11, 26-29]. PHAs are typically made up of 600-35,000 (R)-hydroxy fatty acid monomer units [14]. Each monomer unit contains a side chain R group that is usually a saturated alkyl but can also take the form of unsaturated alkyl groups, branched alkyl groups, and substituted alkyl groups, although these forms are less common [14]. PHA can be classified as; (i) short-chain length PHA (scl-PHA), consisting of 3-5 carbon atoms in the monomer unit, (ii) medium-chain length PHA (mcl-PHA), ranging from 6 to 14 carbon atoms, and (iii) long-chain length PHA (lcl-PHA), which contains 15 or more carbon atoms per monomer [2, 8, 14, 30].

Figure 2.1 Poly(3-hydroxyalkanoate) (PHA) chemical structure [14].

PHAs are gaining attention within the family of biodegradable polymers due to their promising properties such as biodegradability and good thermomechanical characteristics [11, 12]. As a family they have become a candidate to reduce human dependency on fossil resources as they can be produced from diverse renewable carbon sources via microbial activities [11]. To date, there are more than 150 different PHA monomers have been identified and this number 5 continue to increase as new types of PHAs are being introduced through chemical or physical modification, or through the creation of genetically modified organisms (GMOs) to produce PHAs with specialized functional groups [14, 31]. These features have given rise to diverse PHA properties, which can be tailored for various applications ranging from biodegradable packaging materials to biomedical products [12, 32, 33].

2.1.2 Properties of PHA

PHAs are biodegradable, biocompatible, and non-toxic thermoplastics [12, 34, 35]. Their chemical and physical characteristics, such as degree of crystallinity, melting point (Tm), and glass transition temperature (Tg) depend on the monomer used. Poly(3-hydroxybutyrate) or PHB is the most studied of the PHAs. It has been considered as a potential candidate to replace petroleum-based plastics, as it possesses comparable thermal and mechanical properties to polypropylene (PP) [2]. However, commercial applications of PHB are limited because of its stiffness and brittleness, as a result of high crystallinity and a post-processing crystallization [8]. In addition, due to its thermal degradation at high temperature, PHB has a narrow processing window, which means it is difficult to retain the properties while processing [2, 8].

Many researches have been dedicated to manipulating the mechanical properties of PHB [8, 31]. The most common solution is to incorporate different secondary HA monomers other than 3HB, for example 3-hydroxyvalerate (3HV), 3-hydroxypropionate (3HP), 3-hydroxyhexanoate (3HH), or 4-hydroxybutyrate (4HB), into the polymer chain to form copolymers. The most well-known copolymer of PHB is poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV). Compared to the homopolymer of 3HB, PHBV is more flexible due to increased co-polymer elongation, and has higher tensile strength. However, the properties of the copolymer depend on the mole percentage of 3HV [8]. A comparison of the physical properties of different PHAs was summarised by Laycock et al. (2013) [8] and Anjum et al. (2016) [31].

2.1.3 Microbial PHA production

The capability to undertake PHA accumulation is widespread among more than 300 bacterial and archaeal species [14, 31, 32]. Two groups of bacteria are classified based on the stress conditions required for PHA synthesis [31]. The first group comprises the majority of microorganisms, which synthesise PHA intracellularly under limitation of an essential nutrient such as nitrogen (N), phosphorous (P), copper (Cu), and sulphur (S) in the presence of excess carbon source [2, 14, 30, 32, 36-40]. Azohydromonas australica [41-43], Burkholderia sp. [44-

6 46], Cupriavidus necator [47], Methylosinus trichosporium [9, 24, 48, 49], Methylocystis parvus [9, 10], Pseudomonas putida [50-52], and Rhodobacter sphaeroides [53, 54] are examples of bacteria in this group. A second group of bacteria are known to produce PHA during growth favorable conditions without nutrient limitation, such as Alcaligenes eutrophus, Alcaligenes latus, and mutant strain of Azotobacter vinelandii, and recombinant E.coli [55, 56]. To date, an extensive range of substrates has been used for the PHA accumulation including industrial by-products, fats, oils, lignocellulosic materials, agricultural wastes, glycerol, sugars, and wastewater. Tan et al. (2014) [14] and Anjum et al. (2016) [31] present a summary of bacterial strains and carbon sources used for the synthesis of PHAs.

2.1.3.1 Biosynthetic PHA pathways

Figure 2.2 summarises a total of 14 pathways for microbial PHA formation and a list of enzymes involved the formation is given in Table 2.1 [57]. Most of these pathways have been developed in recombinant bacterial strains leading to the formation of diverse PHAs [14, 57]. Only pathway I, II, and III are natural PHA biosynthetic pathways that depend on carbon source and bacterial genetic capacity [14, 57].

Pathway I is the most common PHA pathway used by many well-known PHA producers such as Methylosinus trichosporium, Methylocystis parvus, Cupriavidus necator, Pseudomonas aeruginosa, and Chromatium vinosum [14, 31, 58, 59]. The metabolic flux of this pathway is critically dependent on nutrient available, particularly on, a pool of coenzyme A from the tricarboxylic acid (TCA) cycle [12, 14, 30, 31, 40, 55, 58, 60]. Under nutrient-rich conditions, acetyl-CoA molecules are channeled to the TCA cycle for energy production and cell growth, and consequently, release large amounts of coenzyme A and block PHA synthesis by inhibiting 3-ketothiolase (phaA). In contrast, the coenzyme A levels are non-inhibitory in the presence of excess carbon when an essential nutrient such as nitrogen and phosphorus is limiting, allowing acetyl-CoA to be directed towards PHA synthetic pathways for PHA accumulation. A monomer of 3HB is generated by a condensation of two molecules of acetyl-CoA to form a molecule of acetoacetyl-CoA by 3-ketothiolase (phaA). The acetoacetyl-CoA is then converted to 3-hydroxybutyryl-CoA by acetoacetyl-CoA reductase (phaB), before PHA synthase (phaC) catalyzes the polymerization of 3-hydroxybutyryl-CoA into poly(3-hydroxybutyrate) (PHB).

Pathway II is commonly found in microorganisms capable of fatty acids utilization such as Pseudomonas aeruginosa and Aeromonas caviae [31, 59]. They generate intermediates via the

7 β-oxidation of fatty acids. The enoyl-CoA is converted to (R)-hydroxyacyl-CoA by an (R)-specific enoyl-CoA hydratase (phaJ). Pathway III is an alternative pathway for medium- chain length PHA (mcl-PHA) synthesis [14, 31, 61]. It can generate monomers from a diverse carbon source including fatty acids, oils, and sugars. A key enzyme for this pathway is acyl-ACP-CoA transacylase (phaG). It generates (R)-3-hydroxyacyl intermediates from the fatty acid biosynthetic pathway by converting their acyl carrier protein (ACP) form to the CoA form [62].

Figure 2.2 Natural and engineered PHA pathways [57]

Table 2.1 List of known enzymes involved in PHA pathways in Figure 2.2 No. Pathway Abbreviation Enzyme 1 PhaA Beta-ketothiolase 2 Pathway 1 PhaB Acetoacetyl-CoA reductase 3 PhaC PHA synthase 4 PhaZ PHA depolymerize 5 Dimer hydrolase Associated Pathway 6 (R)-3-hydroxybutyrate dehydrogenase 7 Acetoacetyl-CoA synthase 8 PhaJ (R)-Enoyl-CoA hydratase 9 Epimerase 10 Pathway II FabG 3-ketoacyl-CoA reductase 11 Acyl-CoA oxidase 12 Enoyl-CoA hydratase 8 Table 2.1 List of known enzymes involved in PHA pathways in Figure 2.2 (Cont.)

PhaG 3-hydroxyacyl-ACP CoA transferase 13 Pathway III FadD Malonyl-CoA-ACP transacylase 14 Pathway IV Acetoacetyl-CoA reductase 15 SucD Succinic dehydrogenase 16 Pathway V 4hbD 4-Hydroxybutyrate dehydrogenase 17 OrfZ 4-Hydroxybutyrate-CoA transferase ThrA Aspartokinase 1 18 ThrB Homoserine kinase Pathway VI ThrAC Threonine synthase 19 IlvA Threonine deaminase 20 BktB (PhaA) Beta-Ketothiolase DhaT Alcohol Dehydrogenase 21 Pathway VII AldD Aldehyde dehydrogenase 22 Hydroxyacyl-CoA synthase 23 Pathway VIII Lactonase 24 ChnA Cyclohexanol dehydrogenase 25 ChnB Cyclohexanone monooxygenase 26 ChnC Caprolactone hydrolase 27 ChnD 6-Hydroxyhexanoate dehydrogenase Pathway IX 28 ChnE 6-Oxohexanoate dehydrogenase 29 Semialdehyde dehydrogenase 30 6-Hydroxyhexanoate dehydrogenase 31 Hydroxyacyl-CoA synthase FadA 3-Ketoacyl-CoA thiolase 32 Pathway X FadB 3-Hydroxyacyl-CoA dehydrogenase 33 IdhA Lactate dehydrogenase 34 Pathway XI Pct Propionate CoA transferase 35 PhaC1 Type II PHA synthase 36 CimA Alpha-Isopropylmalate synthase 37 LeuCD 3-Isopropylmalate dehydratase 38 LeuB 3-Isopropylmalate dehydrogenase Pathway XII 39 PanE 2-Hydroxybutyrate dehydrogenase 40 Pct540 Propionate CoA transferase 41 PhaC1437 Type II PHA synthase 42 Pcs Propionyl-CoA synthase 43 Pathway XIII DhaB Glycerol dehydrogenases 44 PduP Propionaldehyde dehydrogenase 2.1.3.2 PHA marker gene

PHA synthase (phaC) is a key enzyme that functions by polymerizing available monomer units and catalyzing the terminal step of PHA biosynthesis [57, 63, 64]. It is ubiquitous in bacterial and archaeal PHA synthesis [65]. Therefore, it has been used as a genetic marker to identify and taxonomically classify PHA-producing microorganisms [63]. The enzyme consists of four distinct classes according to its sequence and substrate specificity [63, 64, 66]. Classes I and II comprise enzymes consisting of a single unit synthase of PhaC, while Classes III and IV require an additional subunit of PhaE and PhaR, respectively, for their functions [14, 57, 64, 66].

9 Class I, III, and IV synthases are found in many PHA-forming microorganisms including members from the Chloroflexi, Firmicutes, Euryarchaeota, Proteobacteria and Thaumarchaeota phyla [67-72]. These organisms tend to produce a range of short chain length monomers such as (R)-3-hydroxybutyrate (3HB). On the other hand, Class II synthase favors medium chain length monomers such as 3-hydroxyhexanoate (3HHx) derived from fatty acids [73, 74].

Table 2.2 PHA synthase (PhaC) classification [64] Class Subunits Example Organisms Substrate 3HA -CoA (C -C ), 4HA -CoA, I PhaC Ralstonia eutropha scl 3 5 scl 5HAscl-CoA II PhaC Pseudomonas aeruginosa 3HAmcl-CoA (>C5) 3HA -CoA (C -C ), 4HA-CoA, III PhaC/PhaE Allochromatium vinosum mcl 6 8 5HA-CoA IV PhaC/PhaR Bacillus megaterium 3HAscl-CoA (C6-C8)

2.1.4 Challenges in PHA production

Many companies have established a large-scale production of PHB, such as Imperial Chemical Industries, Mango Materials, Newlight Technology, Tianjin Biogreen, and TianAn Biologic Material Co [3]. However, widespread industrial use of PHA has been less successful to date due to the challenges of high production cost and material properties.

2.1.4.1 Economic situation

According to techno-economic studies, there are two major obstacles preventing a success in the large-scale production of PHA; (i) use of plant-derived feedstocks and (ii) use of pure cultures. A major cost of large-scale PHA production is the carbon feedstock, estimated to be up to 40% of the product cost [3]. Therefore, many research groups are investigating the potential for using waste streams, such as dairy waste, sugar waste streams, agricultural crop residues, and petrochemical wastes [75, 76]; however, these waste streams are limited by their abundance and distribution in nature [77]. Therefore, an alternative cheap feedstock with high abundance, that is widely available and unrelated to food markets, is desirable.

Pure cultures have been widely used in many biotechnological processes for high-value products. However, a major risk for running production batches is microbial contamination [78]. Therefore, these production facilities require sterilization and aseptic techniques to prevent any microbial invasion, which can significantly increase the process complexity and

10 cost [79]. To date, there are two alternative strategies gaining interests for lower cost production of PHAs (i) use of a mixed microbial culture (MMC) and (ii) use of extremophile production strains. Either approaches offer robustness and contamination resistance for biotechnology processes include PHA production [4, 6, 75, 80].

The first PHA synthesis using MMC was reported by Wallen and Rohwedder in 1974 in wastewater treatment plants designed for biological phosphorus removal (EBPR), when PHA was extracted from the activated sewage sludge [81]. Until now, there are many types of research that have been conducted using MMC for PHA production [82-89]. Meanwhile, only limited research has been done on the use of extremophiles for PHA production. For example, Hermann-Krauss et al. (2013) [75] and Ibrahim et al. (2010) [80] have reported on the use of fermentation batches for PHA production operated under non-aseptic high-salinity and high- temperature conditions when biodiesel by-products and glucose were used as feedstock.

2.1.4.2 Material properties

Microbial PHAs can be tailored to have wide range of material properties in the same way as petrochemical plastics, by varying the monomer composition and macromolecular structure of PHA, depending on the producer organism and the carbon source used for the synthesis [90]. The PHA structure can be controlled by adjusting the carbon substrates to achieve desired monomer content, engineering metabolic pathways, or feeding the culture with carbon substrates containing functional side chains that can undertake chemical modifications [91-93].

2.2 PHA production from methane

2.2.1 Methane Methane gas is formed because of biological degradation of organic matter in engineered processes and natural environments under oxygen-limited conditions. Overall, 40% of global methane emissions come from natural sources, whereas 60% is released from anthropogenic sources such as a production and transportation of coal, natural gas, and oil, livestock and agricultural practices and landfilling of wastes [94]. For a decade, the accessibility of methane on the global market has rapidly increased as a result of the rapid expansion of methane extraction in the form of natural gas and the generation of methane-rich biogas [77]. This marks methane as a potential feedstock for novel processes including PHA production. The use of methane as a carbon source for PHA production offers a range of advantages such as the potential approach for GHG emission reduction and the lack of competition with food [95].

11 Levett et al. (2016) demonstrated that the raw material cost for a large-scale production of PHA from methane can be reduced to only 22% of production for methane [3].

2.2.2 Methanotrophs Methanotrophs are a subset of a physiological group of bacteria known as methylotrophs [39]. They have the unique ability to utilize methane as a sole carbon and energy source [39]. Certain methanotrophs are able to produce PHB from methane under growth-unfavorable conditions [38]. The ability of methanotrophic bacteria to produce PHB was discovered in 1906 [96] and has been studied for decades [9, 24, 28, 97-104].

Traditionally, methanotrophic bacteria were classified as Type I (γ-proteobacteria) and Type II (α-proteobacteria), based on their use of the ribulose monophosphate (RuMP) pathway (Type I) or serine cycle (Type II) for formaldehyde assimilation [105]. Type X (γ-proteobacteria) was further subdivided as it utilized the RuMP pathway as the primary pathway for formaldehyde assimilation, yet expressed low levels of enzymes from the serine pathway and Calvin-Benson- Bassham (CBB) Cycle [106]. Type X methanotrophs were usually found in extreme conditions such as high-temperature [106]. Karthikeyan et al. (2014) summarized the important characteristics used for type differentiation of methanotrophs, based on methane utilization pathways [30].

Methanotrophic PHB formation starts from acetyl-CoA and process via enzyme-mediated reactions, described as pathway I in section 2.1.3.1. The primary enzymes involved in the biosynthesis of methanotrophic PHB are β-ketothiolase (phaA), acetoacetyl-CoA reductase (phaB), and PHA synthase (phaC). In general, the α-proteobacteria (Type II) that typically assimilate formaldehyde using the serine pathway are the target of PHA studies, because they convert oxidized methane into acetyl-CoA, which has a critical function in the TCA cycle and PHA cycle [107]. However, there are conflicts in the literature on which methanotrophs produce PHB and which do not. Although many quantitative reports of PHB production have been carried out in type II methanotrophs, proving their capability for PHB formation [9, 60, 97, 98, 100, 104, 108, 109], there are also reports of PHB production in type I methanotrophs [60, 100, 102, 106, 110-114]. Pieja et al. (2011) evaluated the genetic data of twelve methanotrophic strains from six genera, which showed that all type I strains tested negative for the phaC gene crucial for PHB production while all Type II strains tested positive for phaC and PHB production [38].

12 2.2.3 Thermophilic and thermotolerant methanotrophs

The thermophilic production of PHA from methane has been highlighted as an economical platform for PHA production. A recent techno-economic assessment of PHA production by Levett et al. (2016) demonstrated that the large-scale cultivation processes of thermophilic microorganisms (100,000 tonne per annual) are considered to be energy-efficient because of the removal of a refrigerant needed in the cooling system for lower temperature production[3]. The process itself can generate high thermal energy during high cell density cultivation, along with heat input generated by the bioreactor ́s stirring system, this contributes to the heating of the process [6]. As a result, both heating and cooling costs can be reduced and overall it can reduce the production cost of PHA from $4.1-$6.8 per kg PHA under the mesophilic conditions to $3.2-$5.4 per kg PHA when methane serves as carbon feedstock [3].

Current knowledge of thermophilic and thermotolerant methanotrophs is limited. Methylococcus capsulatus is the first thermotolerant methanotroph discovered in 1966 [115]. The collection of thermophilic and thermotolerant methanotrophs had been limited to the Methylococcus genera until the discovery of Methylocaldum and Methylothermus from hot springs in Hungary and Japan [116-118]. Table 2.3 summarises the thermophilic and thermotolerant methanotrophs reported to date in the literature. Most discovered thermophilic and thermotolerant methanotrophs belong to the Methylococcus, Methylocaldum and Methylothermus genera, which are all gammaproteobacteria [106, 113, 116]. Thermophilic methanotrophs from Methylococcus and Methylocaldum genera can assimilate methane using either the ribulose monophosphate (RuMP) pathway, serine cycle, or CBB pathways, while the Methylothermus possess only the RuMP pathway [106]. Methylocystis parvus strain Se48 is the first isolate of a thermotolerant methanotroph assimilated to the alphaproteobacteria. In contrast to the typical alphaproteobacterial methanotrophs, Methylocystis parvus Se48 relies on a serine cycle for carbon assimilation and shows a likely capacity to produce PHB under the microscope. However, this organism has been lost and never been taxonomically validated [116].

13 Table 2.3 Main characteristics of the thermophilic and thermotolerant methanotrophs of the phylum proteobacteria [106]

Methylococcus Methylocaldum Methylocaldum Methylocaldum Methylothermus Methylocystis sp. Characteristic capsulatus gracile tepidum szegediense thermalis Se48 Morphology Cocci Pleomorphic Pleomorphic Pleomorphic Rounded rods Vibrioids Cell size (μm) 0.7-0.9 x 0.7-0.9 0.4-0.5 x 1.0-1.5 1.0-1.2 x 1.0-1.5 0.6-0.8 x 1.0-1.5 0.6-0.8 x 1.0-1.2 0.6-1.0 x 1.2-1.7 Motility - + + + - - Brownish black to Brownish black to Colony color White to creamy Brownish black Light-brown White brown light-brown Temperature range 30-55 (37-42) 20-47 (42) 30-47 (42) 37-62 (55) 37-65 (55-57) 15-53 (37) (optimum) (°C) pH range (optimum) 6.0-8.0 (6.5-7.5) - - 6.0-8.5 (7.2) 6.5-8.0 (6.5) 5.0-7.5 (5.5-6.8) Salinity (%NaCl) - - - 0-2 (0.5) 0-3 (0.5) 0-0.5 (0) RuMP, Serine, RuMP, Serine, RuMP, Serine, RuMP, Serine, C1 assimilation pathway RuMP Serine CBB CBB CBB CBB Bottom deposits Thermal spring, Source of isolation of water bodies Activated sludge Soil Thermal spring Thermal spring silage, manure activated sludge Reference [119] [120] [120] [113, 120] [121] [116]

*NA = data not available

14 2.2.4 Strategies for methanotrophic production of PHBV copolymer

As mentioned in section 2.1.2, the applications of PHB are limited because of its brittleness and stiffness because of its high crystallinity. Hence, the copolymerization of HB with other monomers such as 3-hydroxyvalerate (HV) is needed to overcome those poor properties. There are many combinations of biological and chemical methods that could be combined to produce more attractive PHA copolymers. These strategies are summarised in this section.

2.2.4.1 Direct PHA synthesis by addition of co-substrates

This strategy relies on the supplementation of methane with co-substrates such as propionate

(C3) or valerate (C5). Many heterotrophic bacteria have a non-specific enzyme activity to metabolize these substrates via β-oxidation to form a fatty acid-CoA, such as propionyl-CoA or valeryl-CoA, which is later condensed with acetyl-CoA to form intermediates and enable the production of the poly(3-hydroxybutyrate-co-3-hydroxyvalerate) copolymer [9, 122-125]. In the case of propionyl-CoA, 3-ketovaleryl-CoA is formed via the condensation of propionyl-

CoA with acetyl-CoA. Meanwhile, the 3-ketovaleryl-CoA can be formed directly from C5 co- substrate [9].

The fatty acid uptake in methanotrophs have not been clarified. The methanotrophic

biosynthesis of PHBV by the addition of C3/C5 compounds, however, has been achieved [9, 10, 104]. One example demonstrated in Methylobacterium organophilum CZ-2 used the mixed feeding of methane and propionate [103, 126]. The accumulation of PHA with a HB:HV ratio of 70:30 at 88 wt% PHA content was obtained when a pulse of propionate was concurrently metabolized during methane consumption. This study is inconsistent with other studies in the obligate methanotrophs Methylocystis parvus OBBP and Methylosinus trichosporium OB3b [9, 10, 104]. The addition of propionate and valerate in this case led to PHBV synthesis at up to 50 wt% of total dried biomass with 22 mol% of HV fraction, and higher levels of co-substrate were associated with higher 3HV contents incorporated into the copolymer. The direct synthesis by addition of co-substrates has been proven successful in many methanotrophic pure cultures and mixed consortia; however, the addition of co-substrates is an additional cost. Table 2.4 summarises substrates used as a supplement for PHAs production in methanotrophs

15 Table 2.4 PHA production using various co-substrates by methanotrophs [9, 10, 278].

PHA monomer ratio (%) Co-substrates 3HB 3HV 4HB 5HV 6HHx None 100 0 0 0 0 Propionate 92 8 0 0 0 Butyrate 100 0 0 0 0 3-Hydroxybutyrate 100 0 0 0 0 4-Hydroxybutyrate 91.5 0 9.5 0 0 Valerate 75 25 0 0 0 5-Hydroxyvalerate 95 1.4 0 3.6 0 Hexanoate 100 0 0 0 0 6-Hydroxyhexanoate 97.6 0 1 0 1.4 Octanoate 100 0 0 0 0 3HB: 3-hydroxybutyrate, 3HV: 3-hydroxyvalerate, 4HB: 4-hydroxybutyrate, 5HV: 5-hydrovalerate, 6HHx: 6-hydroxyhexanoate.

2.2.4.2 Indirect PHA synthesis in consortia

Methanotrophs exist in many ecosystems. They are considered as a primary food producer comprising a complex food web with many heterotrophs [127]. This multi-tiered microbial food web possibly offers another potential route for methanotrophic PHA production where non-methanotrophic strains capable of PHA generation survive by utilizing non-methane carbon sources when methane serves as an external feedstock.

Methanol, formaldehyde, and formate are common by-products produced during methane oxidation in methanotrophs [128]. In addition, Kalyuzhnaya et al. (2013) demonstrated that methanotrophs possessing the RuMP pathway for carbon assimilation, Methylomicrobium alcaliphilum 20Z, could produce additional by-products including lactate, acetate, succinate, lactate, and H2 under low oxygen condition. These products were excreted extracellularly and thereby consequently could be used by other bacteria when co-culturing with other microorganisms [21].

A study by Bothe et al. (2002) provided an example of this symbiotic strategy within the methanotrophic consortia of Methylococcus capsulatus Bath with Ralstonia species, Aneurinibacillus species, and a Brevibacillus agri strain [22] in continuous reactors used for single cell protein production [22]. Ralstonia is a well-known acetate-oxidizing bacterium capable of PHA production [129]. Ralstonia eutropha, for example, can produce various types of PHA when provided with appropriate substrates [130]. For example, the 3HV content in PHBV increased up to 41 mol%, when levulinic acid was added to a Ralstonia eutropha culture [130]. The presence of Ralstonia species, as well as another microorganisms, suggests that a

16 mixed microbial culture may serve as a feasible symbiotic strategy that allows PHBV generation from methane-derived carbon, with the methanotrophs providing carbons for the Ralstonia. Under favorable conditions, with HA supplementation, this culture also could facilitate copolymer production, although the PHA yield vary depending on the Ralstonia sp. fraction in the mixed population.

2.3 Conclusion and recommendation

Methane gas has emerged as a potential feedstock for the production of PHB and its copolymers. Compared to plant-based substrates, the biological production of PHA from methane through methanotrophs offers many advantages, for example, lower substrate cost, an appreciable decrease in environmental impact, and less vulnerability to change in the price of food crops and energy [77]. The current research into PHA biosynthesis from methane is limited to the use of pure cultures and known strains in mixed microbial cultures [28, 37-39]. The establishment of PHA production in a truly mixed culture could maintain a genetic diversity more effectively, allow the diverse production of PHA, and provide further benefit through long-term operation in non-aseptic conditions [131-133]. In order to maintain favorable conditions for a methane-utilizing community capable of PHB production, selection and enrichment criteria (e.g. media concentrations, nitrogen source, pH, and co-substrate supplements) need to be studied. In addition, current studies into PHB-producing methanotrophs are restricted to mesophilic strains [14, 30]. To date, cultivation processes of thermophilic microorganisms are considered more energy-efficient due to the removal of a refrigerant during processing, which can reduce one-fourth of the overall operational expenses [3]. However, it should be noted that the genetic pathways involving PHA biosynthesis are not widespread in thermophilic methanotrophs. A screening of such organisms, therefore, needs to be addressed by applying different selection strategies favorable for the thermophilic growth of methanotrophs and their PHA production. Besides the challenges in production, a study of biological and chemical strategies for the production of PHBV copolymer is necessary to offer extended commercial applications, which would increase its industrial relevance.

17 Chapter 3 Materials and Methods

In this chapter, an overview of the experimental setup and operating conditions of the bioreactors implemented in this thesis, and details of the analytical methods, rates, and yield calculations, are presented. Details of experimental conditions specific to each research objective are included in the relevant results chapters.

A schematic representation of the overall methodology is presented in the following figure.

Biosynthesis of Polyhydroxyalkanoates (PHAs) in Methane-utilizing Mixed Cultures

Preliminary work Thermophilic PHA Production (This section was done prior to this thesis study)

Biomass production using ammonia as a Chapter 6 nitrogen source

Environmental sample collection Mesophilic PHA Production Two samples were collected from natural hot spring Culture selection at 55°C Research objective 1: Twelve selection conditions • To assess different strategies for PHBV with methane as sole carbon source synthesis from methane in mixed microbial - PHA determination by FTIR cultures (MMC)

Culture enrichment at 55°C Nutrients cycling reactors with methane and nitrate Chapter 4 Chapter 5 Research objective 4: PHB production in mixed culture at 55°C • To investigate the feasibility of PHB and PHBV Using biomass removed from the semi-continuous Culture enrichment production from methane in the thermophilic mixed reactors and methane served as sole carbon source Methane-fed semi-continuous reactors culture with sufficient ammonium as nitrogen source - PHA determination by Nile blue A staining - PHA content and composition by GC - Microbial community analysis Chapter 7 Direct PHBV Indirect PHBV - High-throughput shotgun sequencing production production Addition of odd-carbon Methane served as sole fatty acids -carbonPHA sourcecontent and Culture isolation and cultivation at 45°C PHB and PHBV production in mixed composition by GC culture at 55°C and 58°C - PHA content and - Microbial - Serial dilution and antibiotic treatment Biomass removed from the semi-continuous reactors composition by GC community - Fluorescence in situ hybridization (FISH) was incubated under nitrogen deﬁcient conditions with - Microbial analysis various combinations of carbon sources community analysis - High-throughput shotgun sequencing PHB production in pure culture at 45°C - PHA content and composition by GC - Microbial Addition of even- and odd-carbon-number fatty acids interactions for PHA production - PHA content and composition by GC

18 3.1 Sample collection and selection for mesophilic PHA production Prior to commencing the current work presented in Chapter 4 and Chapter 5, preliminary work comprising sample collection, cultivation, and culture selection was undertaken by Syarifah Nuraqmar Syed Mahamud [23].

Overall, fourteen environmental samples were haphazardly collected from various locations around Brisbane (QLD, Australia), for example, sediments from Lake Macdonald and Lake Denwin (Noosa, QLD), and biomass from the Centre for Solid Waste Bioprocessing (The University of Queensland). These locations were selected as suggested by Chistoserdova et al (2015) [276], who reported that habitats for methanotrophs are not limited to landfill sites or anaerobic sludge. Instead, they can be found in fresh water, soil, and also plant-related samples. It should be noted that the sample collection has been done prior to the start of this current thesis and there is a limited information on sampling sites and sample characteristics. However, even though sample collection was repeated on the same locations, the community developed in this study is subject to local culture conditions and it is not necessarily able to be reproduced.

6 mg of each sample was inoculated into 160 mL serum bottles (Wheaton, Mealville, NJ, USA), containing 15 mL nitrate mineral salts (NMS) medium, a common media used for methanotrophic growth [9, 10, 104, 123], prepared according to Whittenbury et al. (1970)

[134]. The media composition contained the following chemicals: 4 mM MgSO4.7H2O, 1.6

mM CaCl2.H2O, 10.4 μM Fe-EDTA, 2.6 μM NaMO.4H2O, 10 μM CuCl2.2H2O, 1.8 μM

FeSO4.7H2O, 1.4 μM ZnSO4.7H2O, 0.1 μM MnCl2.7H2O, 0.4 μM CoCl2.6H2O, 0.1 μM

NiCl2.6H2O, 0.2 μM H3BO3, 0.9 μM EDTA, 0.1 μM Biotin, 0.045 μM folic acid, 0.15 μM thiamine hydrochloride, 0.22 μM D-pantothenic acid, 0.0007 μM vitamin B12, 0.13 μM riboflavin and 0.4 μM nicotinamide. The vessels were capped with butyl-rubber stoppers and

crimp-sealed. The headspace was filled with air plus 30 mL of a gas mixture (90% CH4, 10%

CO2). Samples were incubated at room temperature for 4 weeks. After that, 5 mL of each sample (fully suspended) was taken and combined. This biomass suspension was centrifuged at 10000g for 5 minutes (Eppendorf Multipurpose Centrifuge 5804R). Excess fluid was decanted off.

The collected biomass was then inoculated into two 250 mL serum bottles (Wheaton, Mealville, NJ, USA) containing 30 mL medium, with a headspace volume of 220 mL. These two bottles were provided with different nitrogen sources during this selection experiment: (S1) ammonium (14.8 M, 1.6 mL of 28% ammonia solution, Merck Millipore, USA per 1 L of the 19 media) was provided for the culture used in Chapter 4, while (S2) nitrogen gas was provided as the sole nitrogen source for the culture used in Chapter 5. These cultures were grown in

modified NMS media contained 4 mM MgSO4.7H2O, 2 mM KH2PO4, 0.4 mM

Na2HPO4.12H2O, 1.6 mM CaCl2.H2O, 0.72 μM FeSO4.7H2O, 0.03 μM ZnSO4.7H2O, 0.015

μM MnSO4.7H2O, 4.8 μM H3BO3, 0.2 μM CoCl2.6H2O, 5 μM CuCl2.2H2O, 0.008 μM

NiCl2.6H2O, 0.01 μM Na2MoO4.2H2O, 1.7 μM EDTA, while other components remain as

stated above. The headspace was then filled with air plus 50 mL of a gas mixture (90% CH4,

10% CO2), added through injection. The presence of CO2 is required for RuMP-employing methanotrophs growth [30] and for pH moderation. This explanation has been added into

Once the methane consumption was detectable in these cultures, an extinction-dilution technique was employed to minimise microbial diversity in the culture and the mixture was serially diluted to a final dilution of 10-7. Using the highest dilution factor displaying visual turbidity, two bioreactors were established using these cell suspensions as inoculums. Further details of each reactor operation and conditions are provided in Chapter 4 and Chapter 5.

3.2 Reactor operation and PHA accumulation The experimental data described in Chapters 4, 5, and 6 was produced in a semi-continuous bioreactor. A simple schematic representation of the operation, consisting of (i) a parent semi- continuous reactor and (ii) separate PHA production in a batch reactor, is illustrated in Figure 3.2.

Figure 3.2 Overall scheme of semi-continuous reactor operation and PHA production. A

portion of culture discharged from reactor after t cultivation time is represented as Vw, while 20 Vm represents volume of fresh medium added at the end of each nutrient cycling. Vi represents the initial liquid volume or operating volume in serum bottle.

The first stage (growth stage) was performed in a semi-continuous reactor under aerobic conditions. A nutrient cycling activity was adopted where in a portion of culture (Vw) was replaced by a certain amount of fresh medium (Vm) at desired time (t) to prevent nutrient depletion enabling continuous exponential growth. A culture was fully mixed before discharging. Operation of the bioreactor during this period was controlled by the solids retention time (SRT), which is an average time biomass spends in the system expressed in days [135].

%& %& !"# = = ; where %( = %0 '( %( )

Where Vi and Qw represents individual reactor volume (mL) and dilution rate (mL day-1), respectively

In the second stage (PHA production), a portion of the culture removed from the reactor in Step 4 was centrifuged to discard supernatant. The pellet was later washed twice with nitrogen-free medium to avoid transfer nitrogen before re-suspension in fresh media with no nitrogen to trigger PHA production. The liquid suspension was transferred into 250 mL serum bottles (Wheaton Science Products, Millville, NJ) and shaken to obtain a dispersed cell suspension. Serum bottles were sealed with butyl rubber stoppers and aluminum crimp seals. Cultures then were incubated from 48 or 72 h. Samples were collected and analyzed for nutrients (by flow injection analysis in Section 3.5.3), VFAs (volatile fatty acids in Section 3.5.4), TSS (total suspended solids in Section 3.5.2), and PHA content and composition in Section 3.3.

3.3 PHA analysis

3.3.1 PHA content and composition via gas chromatography

PHA content and composition was analyzed using a Perkin-Elmer gas chromatograph (DB-5 column) equipped with a flame ionization detector (FID) as previously described by Arcos- Hernandez et al. (2013) [136]. Samples were centrifuged at 6300g for 5 min. The supernatant was discarded. The pellet dried in an oven at 70°C overnight before addition of acidified methanol with benzoic acid (3% w/w of H2SO4) (2 mL) and chloroform (2mL). Samples were digested for 20 h at 100°C. After cooling to room temperature (2 h), 1 mL of MilliQ water was

21 added to allow phase separation. After settling for 1 h, the organic phase was transferred to a vial for GC analysis. Calibration was based on reference standards of a biologically sourced PHBV copolymer (70:30 mol% 3HB: 3HV, Sigma Chemicals). The calibration standards were prepared using the same method. PHA content was reported as a percentage on a dry weight basis (wt%). The oven temperature is 90°C (1 min) before increase to 121°C (10C min-1 for 0 min) and to 270°C (45°C min-1 3 min). The detector temperature and injection port temperature were 300 and 250°C.

3.3.2 PHA determination by fourier-transform infrared spectroscopy (FTIR)

Rapid qualitative determination of PHA in culture was done by using fourier-transform infrared spectroscopy (FTIR) as described by Randriamahefa et al (2003) [277]. 1-2 mL of wet solid biomass was smeared into a thin layer on a microscope slide. The samples were dried at 70°C overnight to obtain a solid sample for spectroscopy. The IR spectra were recorded using a Thermo Electron Nicolet 6700 with single bounce diamond attenuated total reflection (ATR) using a spectral range from 400 cm−1 to 4000 cm−1, 32 scans and a resolution of 4 cm-1. The baseline correction was processed using Omic software (version 7.4.174). Spectra were deconvoluted using Peakfit software (version 4.12) and the area calculated.

3.3.3 Nile blue A staining

Nile blue A staining is a phenoxazone dye used to show strong fluorescence visualization of PHA granules under microscope [250]. It is a common method used for rapid screening of PHA-producing culture. In short, 1% aqueous solution of Nile blue A was heated to 55°C in a water bath before use. 5-10 μL of culture was smeared onto a glass slide and heat-fixed using a Bunsen burner. The heat-fixed smears of cells were submerged into the Nile blue A solution at 55°C for 10 min. The slide was rinsed with tap water followed by 8% acetic acid and repeated twice to remove excess stain. The washed slide was covered with a coverslip, blotted dry with paper tissue, and examined at 1000-x magnification under a Nikon XQA microscope with an attached DS-Qi2 Monochrome Microscope Camera. A dual excitation interference of excitation filters: FITC (475-490 nm) and TRITC (540-565 nm) was used.

3.4 Microbial community analysis

Periodic monitoring of the microbial biomass from the parent reactor during nutrient-sufficient condition was performed using 16S rRNA gene sequencing. A total of 2 mL of biomass from

22 the bioreactor was centrifuged at 6300g for 5 minutes (Eppendorf Multipurpose Centrifuge 5804R). After discarding the supernatant, the cell pellet was stored at -20°C. DNA extraction was performed using the Fast DNA®SPIN Kit for Soil (MO BIO Laboratories, Inc.) according to the manufacturer’s protocols. Extracted DNA was sent to the Australian Centre for Ecogenomics (ACE) sequencing facility for Illumina iTag sequencing and data processing. Specific information regarding the processes has been published [183, 184, and 185]. Returned 16S rRNA gene sequences that met the quality threshold were clustered using a 95% cutoff and used for community composition analyses.

3.5 Analytical methods

3.5.1 Gas concentration analysis

5 mL gas samples were periodically withdrawn for gas-phase analysis using a gas-tight syringe (Restek, Bellefonte, PA). These samples were analysed for methane and carbon dioxide content using a Shimadzu GC-2014 gas chromatograph containing a Valco GC valve (1 mL sample loop), a HAYESEP Q 80/100 packed column (2.4 m length; 1/8” outside diameter, 2 mm inner diameter) and equipped with a thermal conductivity detector (TCD). The temperatures for chromatography injector, oven, and detector were set at 75, 45 and 100°C, respectively. Argon at 135.7 kPa was used as a carrier gas at 28 mL min-1.

3.5.2 Total suspended solids (TSS) measurement

Total suspended solids (TSS) were analysed by removing 5 mL of suspended cultures collected in replicates and centrifuging at 6300g for 5 min (Eppendorf Multipurpose Centrifuge 5804R). After discarding the supernatant, the cell pellet was dried in an oven at 70°C overnight and cooled in a desiccator for 24 h before weighing. Otherwise, approximately 2 mL of culture was used for the determination of optical density via a spectrophotometer at 670 nm.

3.5.3 Nutrients analysis

1-2 mL of sample was centrifuged at 10000g for 5 min (Sigma 1-14 Microcentrifuge) to separate liquid from biomass. The supernatant was filtered through 0.2 μm filters (Millex-GP Syringe Filter Unit SLGP033RS) and stored at 4°C until analysis. The filtered sample was diluted with + MilliQ water. The final volume of each sample was 5 mL. The concentration of ammonium (NH4 ), - 3- nitrate (NO3 ) and phosphate (PO4 ) were measured using a Lachat QuickChem800 Flow Injection

23 Analyser (FIA) (Lachat Instrument, Milwaukee, USA) by the Advanced Water Management Centre (AWMC) analytical services laboratory (ASL).

3.5.4 Volatile fatty acids (VFAs) analysis

The liquid suspensions were immediately placed in an icebox, and subsamples (1 to 2 mL) were centrifuged at 10000g for 5 min (Sigma 1-14 Microcentrifuge) to separate liquid from biomass. The supernatant was filtered through 0.2 μm filters (Millex-GP Syringe Filter Unit SLGP033RS) and stored at 4°C for the gas chromatograph analysis to assess the concentration of volatile fatty acids (VFAs) that was run by the Advanced Water Management Centre (AWMC) analytical services laboratory (ASL). The concentrations of acetate, propionate, butyrate, valerate, and ethanol in the supernatant were measured using a gas chromatograph (Agilent Technologies, Model 7890A, USA) with a flame ionization detector (FID) and a polar capillary column (DB-FFAP 30m x 0.53mm x 1.0um) on filtered samples following addition of an internal standard (1000 ppm stock of six VFAs, Sigma Aldrich, USA) and 1% formic acid. The detector and injection port temperature was 250 and 220°C. The oven temperature was hold at 60°C for 2 min before increased to 240°C (20°C min-1) and hold for 2 min. Total running time was 13 min. The pellets were stored at -20°C with 0.5 mL of bacterial glycerol stock solution for further analysis.

3.6 Calculation

Culture growth rates were determined from the changes in number or mass of cells (X) during exponential growth phase over a period of time (t). To calculate bacterial growth rates, the following equation was use: 12 = 32 1) Where X is the number or mass of cell measured by optical density at 600 nm (mg mL-1), t is time (h-1) and μ is specific growth rate (h-1). The equation can be rearranged and integrated into simple form as: 9: 452 = 3) + 4527 or 2 = 278

Where X0 is the initial number or mass of cell and Xt is the number or mass of cell at time t.

When the specific growth rate (μ) was obtained, it can be used to calculate the generation time or doubling time, which represents time require for the culture to double in mass, by using the following equation.

24 45 2 ;<=>?@5A )@B8 = 3 PHA concentration in biomass as obtained from GC analysis, expressed as mass per volume (g PHA L-1), was assessed by dividing total PHA (mg PHA L-1) by total suspended solids (TSS) concentration (mg TSS L-1) on a mass basis (wt%).

DEF (BA DEF KLM) DEF (H)%) = : × 100 #!! (BA #!! KLM) 3HB and 3HV content on mass basis were measured as the concentration of each monomer obtained from GC analysis divided by total PHA concentration. The mass concentration was converted to mole basis using the following molecular weights: 86 g 3HB mol-1 and 100 g 3HV mol-1.

- Active biomass (Xt) at time t was determined as the total concentration of biomass (mg TSS L 1), measured as total suspended solids (TSS), less the PHA concentration (mg PHA L-1).

LM LM LM 2:(BA 2: K ) = #!!: (BA #!! K ) − DEF: (BA DEF K )

The specific consumption of methane (RSTU), carbon dioxide (RSVW), acetic (RXY), propionic acid (R[\), and valeric acid (R]^_) and specific production of 3HB and 3HV (RT` and RT], respectively) were calculated with reference to active biomass (Xt) concentration:

(aEb, : − aEb,&) (acd, : − acd,&) RSTU = , RSVW = (): − )&) × 2& (): − )&) × 2&

(XY eLXYf) ([\eL[\f) (]^_ eL]^_f) RXY = , R[\ = , R]^_ = (:eL:f)×gf (:eL:f)×gf (:eL:f)×gf

(hT` eLhT`f) (hT] eLhT]f) RhT` = , RhT] = (:eL:f)×gf (:eL:f)×gf

R[TX = RhT` + RhT]

The specific consumption of carbon substrates and production rates of monomer 3HB and 3HV are reported based on carbon mole basis. Biomass was assumed to have an empirical composition of C4H8O2N [60]. The biomass was then converted to C-mol using a C-mol weight of 25.5 g biomass C-mol-1. The polymer was converted to C-mol basis using the following C-mol weights: 21.5 g 3HB C-mol-1 and 20 g 3HV C-mol-1.

Fraction of 3HV produce to total odd-fatty acid uptake was calculated as a percentage of total 3HV produce divided by total propionic acid or valeric acid consume on C-mol basis.

(3E%: − 3E%&) (3E%: − 3E%&) %ihT] = × 100 ; %ihT] = × 100 (Dk: − Dk&) (%l?: − %l?&) 25 Chapter 4 The effect of methane and odd-chain fatty acids on 3-hydroxybutyrate (3HB) and 3-hydroxyvalerate (3HV) synthesis by a Methylosinus-dominated mixed culture

This chapter is a modified version of a paper prepared for publication in New Biotechnology. This chapter partly addresses the research objective 1, which is to assess different strategies for PHBV synthesis from methane in mixed microbial cultures (MMC). The strategy considered here is the direct synthesis of a PHA copolymer from methane by adding supplementary odd- carbon-number fatty acids.

The culture used in this chapter was selected from diverse environmental samples and grown with methane and ammonium as a nitrogen source as discussed in Section 3.1. A microbial community analysis revealed that the culture was dominated by Methylosinus sp. The culture showed the capability of maintaining low levels of poly(3-hydroxybutyrate) (PHB) content under growth conditions. This culture then produced PHB when fed with methane under nitrogen starvation conditions and utilized the stored PHB slowly in the absence of methane. The production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) was observed when the culture was fed with odd-carbon-number fatty acids – in the presence and absence of methane.

The major outcomes of this chapter are: • A Methylosinus-dominated methane-fed culture enables PHA production with a high 3HV fraction up to 65 mol%; • Propionic acid and valeric acid can induce the 3HV synthesis in the methanotrophic community both in the presence and absence of methane; • Valeric acid addition produces a higher PHA content and higher 3HV fraction than propionic acid does; and • The production of PHBV and assimilation of odd-carbon-number fatty acid in the absence of methane seems to correlate with the degradation of stored PHB.

4.1 Introduction

Methane (CH4) gas, which is a cheap and abundant carbon source, has emerged as a potential feedstock for the production of poly-3-hydroxybutyrate (PHB), a biodegradable, biocompatible, and renewable bioplastic [2]. Techno-economic studies have demonstrated that

26 the use of methane for polyhydroxyalkanoate (PHA) production significantly reduces the overall cost and environmental impact [3, 11, 137]. In addition, a mixed microbial community of methane-utilizing bacteria (methanotrophs) has a robust and self-regulating nature [7], offering the opportunity to operate under non-aseptic conditions, thereby reducing the large- scale production operating costs [138].

Methanotrophs metabolize methane as their sole source of carbon and energy for growth under balanced growth conditions. Under nutrient limiting conditions, they can accumulate carbon as PHB [37, 39]. PHB is an attractive polymer, however, it has not made a significant impact in the market because of its narrow melt processing window, and because its high crystallinity and lack of flexibility limit its application [19]. One approach to the production of biopolymers with more desirable properties is an incorporation of 3-hydroxyvalerate (3HV) into the polymer chain [8]. This approach can improve the impact strength and flexibility of the polymer [139]. Hence, polymers with different mechanical properties can be made, ranging from hard and crystalline to elastic and rubbery [1, 10].

The feasibility of PHB production and incorporation of 3HV in methanotrophic cultures was first reported by Myung et al. 2015 [10]. They found that the biosynthesis of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) occurred when methane, supplemented with 3HV precursors (propionate and valerate), was fed to a mixed community dominated by Methlocystis sp., one of the most well-known genera of PHA-producing methanotrophs [37, 39]. The same research group later expanded the study by considering pure cultures of Methylocystis and Methylosinus sp., with a focus on PHA accumulation in the presence and absence of methane [9, 104]. They concluded that the presence of methane is needed to generate the energy required for incorporation of 3HV units into the copolymers [9]. However, previous studies have proposed that PHA functions as a sink of reducing equivalents that can be used drive cometabolic oxidation [140-144]. For example, methanotrophs containing PHB have shown a capacity to transform trichloroethylene (TCE) in the absence of methane [145-147], providing a likelihood that stored PHB may serves as an energy source in the absence of methane. However, to date, there was no evidence support this hypothesis in literature. In this present work, 3HV production in a mixed methanotroph-dominated culture was studied and the capacity of the stored PHA to support 3HV generation, enabling PHBV accumulation in the absence of CH4, was also evaluated.”.

27 4.2 Materials and methods

4.2.1 Semi-continuous enrichment reactor

A semi-continuous reactor (2 L) was established using 30 mL of the cell suspension (S1) described in Section 3.1 plus 210 mL of fresh medium with ammonia (1.6 mL of 28% ammonia solution per 1 L of medium; Merck Millipore, USA) as a nitrogen source. Other components contained in media was provided in Section 3.1.

A large volume of gas in the headspace was maintained to ensure that the culture would have enough methane and oxygen for one cycle run. The headspace was filled with air plus 500 mL of the gas mixture (90% CH4, 10% CO2). The culture was incubated on a magnetic stirrer, with stirring at 200 rpm at 30ᵒC. After an incubation period of 15 days, the culture was subjected to a long-term cycling and wasting regime. This nutrient cycling was established to enable nearly continuous exponential growth (Figure 4.1). Firstly, 200 mL of fresh medium was added to 40 mL of carry-over culture from the previous cycle, then flushed for 10 min with air. 500 mL of the gas mixture (90% CH4, 10% CO2) was then injected into the headspace (Step 1). In Step 2, the culture was incubated at 30ᵒC (200 rpm stirring) over a 72-h period. Finally, 200 mL of cell suspension was removed, completing a cycle (Step 3). The amount of removed culture determined the solids retention time (SRT). The operational SRT was maintained at 3.4±0.3 days SRT over a period of 500 days using a growth repeating cycle (~200 cycles).

Figure 4.1 Overall scheme of 2-L reactor operation and PHA production using methane as a single carbon source. 28 4.2.2 PHA Accumulation

The culture removed from Step 3 was centrifuged at 6300g for 5 min for 5 min (Eppendorf Multipurpose Centrifuge 5804R) and suspended in fresh nitrogen-free medium (Step 4) in 250 mL serum bottles (Wheaton, Mealville, NJ, USA) tighten with rubber stoppers aluminum crimp seal. The final liquid volume was 30 mL, and the headspace volume was 220 mL. Some samples were amended with 100 ppm of propionic acid (Sigma–Aldrich, USA; 101 µL per L medium) and 100 ppm of valeric acid (Sigma-Aldrich, USA; 106 µL per L medium), both with and without methane. These concentrations were chosen according to previous studies done in methanotrophic cultures [9, 10, 28, 38, and 104]. Test conditions were categorized as follows: with methane (M1), without methane (M0), propionic acid with methane (P1M1), propionic acid without methane (P1M0), valeric acid with methane (V1M1), and valeric acid without methane (V1M0). The bottles were flushed with air before addition of gases as follows: 50 mL of the gas mixture (90% CH4, 10% CO2) was added for treatments with methane or 50 mL of nitrogen gas for those without methane. All cultures were grown under non-sterile conditions at 30ᵒC. After 72 h of incubation, cells were harvested by centrifugation (6300g for 5 min) and dried overnight in as oven at 70ᵒC. Preserved samples were assayed for PHA content.

4.3 Results and discussion

4.3.1 Community growth characteristic in the presence of methane and nitrogen

Overall, the culture showed a stable long-term consumption of CH4 at 1800±500 μmol of CH4 day-1. Under these nutrient-sufficient growth conditions, the enrichment showed an exponential growth pattern, doubling every 1.0 days, with the maximum specific growth rate (μmax) being -1 -1 -1 0.7 day [48]. The specific rate of methane oxidation was 19±6 mmol CH4 g TSS h , with -1 -1 - CO2 production at 11±5 mmol CO2 g TSS h . The biomass concentration was 0.5±0.1 gTSS 1 L-1. Under balanced growth conditions, the active biomass contained 4.0±0.3 wt% PHBV (with a 3HB: 3HV ratio of 100:0). The accumulation of PHA under such conditions has been reported previously by Pieja et al. [39].

4.3.2 Community flux in the semi-continuous reactor

The community composition at the genus level for the methanotrophic enrichment was monitored by harvesting cells after the 103rd, 113th and 156th growth repeating cycles (or on Days 261, 288, and 393 respectively), as illustrated in Figure 4.2. Overall, the members of the community affiliated to the OTU1-25 accounted for 86.7-91.9% of the total sequences

29 identified. All OTUs shared a phylogenetic affiliation at the phylum level, with members of the phyla Proteobacteria and Bacteroidetes accounting for 81.3-90.2% and 1.7-5.3%, respectively (Figure 4.2).

Methylosinus was the most abundant genus and accounted for 49.9-60.3% of the total sequences identified, with a non-methane-utilizing methylotroph from genus Methylophilus sp. and Hypomicrobium being next most abundant (Figure 4.2 and Figure 4.3). Hypomicrobium presented at a large proportion initially but was less than 2% relative abundance after the 156th cycle of operation. The population of Methylophilus sp. increased from less than 1% relative abundance to 12.8% over the same time. Methanotrophs typically cohabit with non- methanotrophic microorganisms that survive on methanotrophic by-products [148]. In this case, Methylophilus and Hyphomicrobium are non-methane users that rely on methanol produced as a result of methane oxidation by methanotrophs [149].

100%

90% Methylosinus Methylocystis 80% Methylomonas Methylophilus 70% Hyphomicrobium Brevundimonas 60% Burkholderia Caulobacter 50% Chryseobacterium Methanotrophs

Methanotrophs Comamonas

40% Methanotrophs Ferrovibrio

%Relative abundance Pseudomonas 30% Rhodanobacter Terrimonas 20% Pigmentiphaga Other 10%

0% Cycle 103 Cycle 113 Cycle 156

Figure 4.2 The relative abundance of bacterial 16s rRNA genes at the genus level expressed as % of OTUs in the sample that were present at more than 1%.

The dominant methanotroph associated with the Methylosinus genera shows 97-100% similarity with Methylosinus trichosporium OB3b based on best-matched reference sequences using the Basic Local Alignment Search Tool (BLAST) (Figure 4.3 and Table A1). Methylosinus trichosporium OB3b is a well-known PHA-accumulating methanotroph [150],

30 with poly(3-hydroxybutyrate) (PHB) constituting from 10 to 50% of the dry weight of cells cultured in nitrogen-free media [9, 48, 49, 108, 109, 151, 152]. When its feed is supplemented with valerate, Methylosinus is capable of producing poly(3-hydroxybutyrate-co-3- hydroxyvalerate) (PHBV) under nitrogen-limited conditions [9]. Myung et al (2015) reported the highest 3HV fraction at 22±3 mol% when 100-ppm of valerate was fed with methane [9].

Although methane was the sole carbon source in this methane-driven system, heterotrophs including those capable of accumulating PHA, were observed. For example, Caulobacter (OTU4), Brevundimonas (OTU14), Hyphomicrobium (OTU13, 16, and 25), Pseudomonas (OTU22), Rhodanobacter (OTU6), Comamonas (OTU3), Burkholderia (OTU19), and Terrimonas (OTU21) (Table A1) were all present, and these have capacity to accumulate PHA [153-165]. Further, according to previous studies, Pseudomonas [156, 157], Comamonas [163], Rhodanobacter [154, 164] and Burkholderia [158, 160, 161] can accumulate copolyesters from propionate or valerate under unbalanced-growth conditions. However, the population of these genera presented less than 5% of the total community when the nitrogen starvation experiment was conducted (Table A1). Other OTUs in this study, namely, OTU7, 15, and 24, could not be assigned with confidence to known genera at 97% similarity (Table A1). Thus, a close relative representative sequence was assigned to obtain approximate genus- level taxonomic information. OTU7 was closely related to the genus Methylophilus (with 96% similarity); OTU15 was from the genus Methylosinus (with 94% similarity); OTU24 showed 96% similarity with Methylocystis, a methanotrophic genus known for its capacity to produce PHB and PHBV [38]. Methylocystis shows significant similarities to Methylosinus, including its stoichiometry of methane oxidation and PHAs formation [9, 38, 39].

In short, the community cultivated under methane-driven operation was composed primarily of Proteobacteria with relative abundance of 84-90%. It is well known that most of methanotrophs belong to this phylum. It should be noted that the population of PHA-producing methanotrophs by Cycle 156 accounted for 80% of total sequences identified from PHA-producing microorganisms.

31 Methylosinus trichosporium OB3b (NR 044947) ≥ 50% OTU 12 ≥ 70% ≥ 90% Methylosinus trichosporium OB3b (NR 112024) OTU 1 OTU 10 OTU 5 OTU 15 Methylosinus sporium NCIMB (NR 026512) Methylocystis bryophila H2s (NR 108474) Methylocystis parvus OBBP (NR 044946) Methylocystis heyeri H2 (NR 042531) Methylocystis hirsuta CSC1 (NR 043754) Methylocystis echinoides IMET 10491 (NR 025544) Methylocystis rosea SV97 (NR 042108) OTU 24 OTU 20 OTU 17 OTU 23 Caulobacter segnis ATCC 21756 (NR 074208) Caulobacter vibrioides CB51 (NR 037099) OTU 4 Caulobacter rhizosphaerae 7F14 (NR 156992) Caulobacter henricii ATCC 15253 (NR 025319) OTU 14 Brevundimonas subvibrioides ATCC 15264 (NR 074136) Brevundimonas subvibrioides NBRC 16000 (NR 113834) Brevundimonas vesicularis NBRC 12165 (NR 113586) Brevundimonas nasdae W1-2B (NR 028633) Zavarzinia compransoris Z-1155 (NR 104961) OTU 11 Ferrovibrio xuzhouensis LM-6 (NR 145543) Ferrovibrio denitrificans Sp-1 (NR 117302) Taonella mepensis H1 (NR 132292) Hyphomicrobium denitrificans ATCC 51888 (NR 074189) Hyphomicrobium denitrificans DSM 1869 (NR 114940) OTU 25 Hyphomicrobium chloromethanicum CM2 (NR 025048) Hyphomicrobium methylovorum DSM 5458 (NR 026430) OTU 13 Hyphomicrobium methylovorum NBRC 14180 (AB680579) Hyphomicrobium facile IFAM CO-582 (NR 027610) OTU 16 Pseudomonas mendocina ATCC 25411 (NR 114477) Pseudomonas aeruginosa DSM 50071 (NR 117678) OTU 22 Pseudomonas guguanensis CC-G9A (NR 135725) OTU 18 Methylomonas koyamae Fw12E-Y (NR 113033) Methylophilus glucosoxydans B (NR 122095) Methylophilus methylotrophus NCIMB (NR 041257) Methylophilus rhizosphaerae CBMB127 (NR 116239) Methylophilus flavus Ship (NR 104519) Methylophilus luteus Mim (NR 116847) OTU 2 Methylophilus leisingeri DSM 6813 (NR 041258) OTU 7 Rhodanobacter denitrificans 2APBS1 (NR 102497) Rhodanobacter denitrificans 2APBS1 (NR 108437) Rhodanobacter koreensis THG-DD7 (NR 134797) Rhodanobacter aciditrophus sjH1 (NR 145874) OTU 6 Comamonas terrigena LMG 1253 (NR 114865) Comamonas terrigena DSM 7099 (NR 114856) Comamonas terrigena NBRC 12685 (NR 113597) OTU 3 Pigmentiphaga litoralis JSM 061001 (NR 044530) Pigmentiphaga daeguensis K110 (NR 044082) Pigmentiphaga soli BS12 (NR 132681) OTU 8 Pigmentiphaga kullae K24 (NR 025112) OTU 19 Burkholderia humptydooensis MSMB43 (NR 152631) Burkholderia glebae LMG 29325 (NR 145597) Burkholderia arationis LMG 29324 (NR 145599) Burkholderia calidae LMG 29321 (NR 145602) Burkholderia concitans LMG 29315 (NR 145603) Chryseobacterium zeae JM-1085 (NR 133728) Chryseobacterium hominis NF802 (NR 042517) Chryseobacterium chaponense Sa 1147-06 (NR 117501) OTU 9 Chryseobacterium pallidum 26-3St2b (NR 042504) Terrimonas ferruginea NBRC (NR 113718) OTU 21 Terrimonas terrae T16R-129 (NR 157715) 67 Terrimonas arctica R9-86 (NR 134213) Terrimonas aquatica RIB1-6 (NR 116608) 0.020 Figure 4.3 Maximum composite likelihood phylogenetic tree for 25 OTUs affiliated to Proteobacteria and Bacteroidetes. Bootstrap values ≥50% are represented as open (○) circles, ≥70% as grey (●) circles, and ≥90% as filled (●) circles. 32 4.3.3 PHA accumulation

Six conditions were considered, based on combinations of three variables: methane availability, and propionic acid or valeric acid supplementation. The experiments were labelled as follows: with methane (M1), without methane (M0), with methane and propionic acid (M1P), without methane but with propionic acid (M0P), with methane and valeric acid (M1V), and without methane but with valeric acid (M0V). It should be noted that the biomass grown under nutrient sufficient conditions already contained 4.0±0.3 wt% PHA (with a 3HB: the 3HV ratio of 100:0). Table 4.1 shows methane consumption rate, carbon dioxide production rate, and PHA content and composition after PHA accumulation phase. Figure 4.4 summarises the final PHA content and fractions of 3HB and 3HV on a dry weight basis (wt%) in the biomass.

In the absence of methane (M0), the PHA content was lowest at 1.3 wt% (with a 3HB:3HV ratio of 100:0) (Figure 4.4). The reduction in PHA content relative to the initial content is consistent with PHA degradation in the absence of carbon supply, even under unbalanced growth conditions [141-144]. The positive control with methane (M1) resulted in a PHA content of 8.6 wt% (with a 3HB:3HV ratio of 100:0) (Figure 4.4), which was more than double the initial PHA content and similar to values reported previously [166-168]. The rates of -1 -1 methane consumption and CO2 production were 7.1±1.2 mmol CH4 g TSS h and 2.4±0.3 -1 -1 mmol CO2 g TSS h , respectively (Table 4.1).

In the presence of propionate (M0P and M1P) there was net PHA consumption, with the final PHA content being below the initial 4 wt%. Still, PHA was produced in both trials, evidenced by the generation of 3HV. In the absence of methane (M0P), the final PHA content was 2.8 wt% (with a 3HB:3HV ratio of 80:20), while in the presence of methane (M1P), the final PHA content was 3.5 wt% (with a 3HB:3HV ratio of 77:23). The addition of valerate resulted in higher PHA and 3HV production, with the PHA content in the M0V and M1V experiments more than doubling and tripling relative to the initial, respectively. The highest PHA content overall was obtained in the methane and valeric acid (M1V) experiment: 14.1 wt% (with a 3HB:3HV ratio of 35:65). This result is in accordance with previous study that showed that, while both propionic acid and valeric acid can modify the composition of PHA produced in methane-fed culture, valeric acid usually yields higher PHA and 3HV content [9]. However, in this study, the Methylosinus-dominated enrichment generated high 3HV content (up to 65 mol%) in comparison with previous studies by Myung et al (2015, 2016) who reported 22 mol% 3HV, when the same amount of odd-chain fatty acid was added [9, 10].

33 The production of 3HV was unsurprising given that propionic and valeric acids are precursors for propionyl-CoA and valeryl-CoA respectively, which are intermediates for 3-hydroxyvaleryl-CoA (3HV-CoA) generation [9, 169]. Higher net production in the presence of valeric acid was also unsurprising. Despite 3HV generation from propionic and valeric acid sharing a common pathway in methanotrophic and heterotrophic microorganisms, lower formation of 3HV-CoA from propionic acid compared to valeric acid, which is a more direct precursor to 3HV-CoA, is commonly observed [9, 170, 171].

The significant outcome of this work is the generation of PHA in the absence of methane. This was achieved on both propionic and valeric acids, although net PHA production was only observed when valeric acid was used. Activity in the absence of methane may have been driven by PHA consumption, which could provide reducing power for the assimilation of organic acids. The lower yields for M0P and M0V compared to M1P and M1V are consistent with this hypothesis. Further studies on this topic are required to quantify the relationship between PHA content and composition and energetics of odd-chain fatty acid assimilation and incorporation in methanotrophs.

Table 4.1 PHA content and composition produced by the Methylosinus-dominated culture after 72 h of incubation in the presence and absence of methane with supplementation of odd-chain fatty acids

Odd-chain Presence of PHA Content 3HV fraction Total suspended solid (TSS) Code fatty acids methane (wt%) (mol%) (g TSS L-1) Yes M1 8.6 0.0 0.42±0.01 None No M0 1.3 0.0 0.35±0.01 Propionic Yes M1P 3.5 22.6 0.37±0.01 acid No M0P 2.8 20.5 0.36±0.00 Valeric Yes M1V 14.1 65.0 0.47±0.00 acid No M0V 8.5 56.4 0.45±0.01

CH4 consumed CO2 produced C3 consumed C5 consumed Odd-chain Presence of Code (mmol CH4 (mmol CO2 -1 -1 fatty acids methane (mmol C3 g TSS ) (mmol C5 g TSS ) g TSS-1 day-1) g TSS-1 day-1) Yes M1 7.1±1.2 2.4±0.3 - - None No M0 - N/A - - Propionic Yes M1P 5.2±0.3 1.6±0.3 2.1 - acid No M0P - N/A 1.6 - Valeric Yes M1V 4.3±0.4 0.8±0.2 - 2.2 acid No M0V - N/A - 2.3 N/A: Not applicable because the cultures did not show significant production

34 16

10 3HV 8 3HB

2 PHA, 3HB, and 3HV Content (wt%) Content 3HV and 3HB, PHA, 0 M0 M1 M0P M1P M0V M1V Figure 4.4 PHA content presented as fractions of 3HB (■) and 3HV (■) on a mass basis (wt%) obtained at the end of the PHA accumulation phase in the presence and absence of methane. A bold red dotted line ( - - ) represents the initial PHA content in the culture before being subjected to PHA accumulation.

4.4 Conclusion

It is shown that a semi-continuous reactor can facilitate methanotrophic enrichment and yield a community capable of producing PHA. In this work, the community was dominated by the Proteobacteria with Methylosinus being the most abundant genera making up 60% relative abundance. The population accounted for 80% relative abundance of total sequences identified from PHA-producing microorganisms, playing the main role for PHA production. Addition of propionic acid and valeric acid is enable 3HV producing in PHB-containing culture grown on methane, while only 3HB was produced when fed with methane alone. A higher fraction of 3HV and PHA content was obtained from the valeric acid treatment. Contrary to previous findings, synthesis of 3HV in the absence of methane when PHA-containing cells were incubated with either propionic acid or valeric acid was achieved The 3HV fraction reported in this study far exceeds the previously reported values. The production of PHBV in this chapter is considered as direct synthesis of a PHA copolymer, which odd-carbon substrates are required for 3HV formation; however, evidence of this study could be interfered due to the culture diversity, the presence of non-methanotrophic PHA producers, and the lack of molecular information on metabolic capacity of minor microorganisms that may but unlikely assimilate propionic acid and valeric acid for PHAs production. However, our conclusions are insensitive to the activity of the minor microorganisms undescribed 5% of total community considering that the total PHA content reached 14 wt%; even if the undescribed organisms were all full of PHA there would still have been considerable PHA produced by the identified organisms. 35 Chapter 5 Methanotrophic-heterotrophic symbiosis for 3-hydroxybutyrate and 3-hydroxyvalerate production from methane

This chapter is a modified version of a paper prepared for publication in New Biotechnology. After the establishment of the direct PHBV production from methane by adding fatty acids in Chapter 4, this chapter addresses a novel approach of indirect 3HV generation via microbial symbiosis in consortia solely fed with methane. Together with Chapter 4, this completed the first research objective of this thesis, which is to assess different strategies for PHBV synthesis from methane in mixed microbial cultures (MMC).

The culture used in this chapter was again selected from diverse environmental samples and grown with methane and nitrogen gas as a nitrogen source as discussed in Section 3.1, before a cycling of nutrient deprivation with ammonia was introduced in this current work to promote biomass production. In contrast to the Methylosinus-dominated culture presented in Chapter 4, the culture presented in this current chapter was dominated by Methylomonas sp., which lacks genetic capacity to produce PHA. Dominance of Methylomonas over other non- methanotrophs prevailed over other non-methanotrophic PHA-producing bacteria. The stable production of polyhydoxyalkanoate (PHA) containing both 3-hydroxybutyrate (3HB) and 3-hydroxyvalerate (3HV) monomer units from methane was observed throughout the 400-day operation. The metabolic relationships within the community were investigated using Illumina high- throughput shotgun sequencing, revealing possible interactions benefiting PHA copolymer synthesis within the methane-fed consortium.

The major outcomes of this chapter are: • A methanotrophic-heterotrophic community capable of PHBV production from methane was enriched from environmental samples; • The population was dominated by non-PHA producing methanotrophs from the genus of Methylomonas, which accounted for 35-60% of the community; • The PHA content and composition seems to be sensitive to the community profile, which consequently influences the 3HB:3HV ratio; and • High-throughput shotgun sequencing and Metagenomic Assembled Genomes (MAGs) allows an estimation of potential routes involving microbial interactions for PHBV production.

36 5.1 Introduction

Polyhydroxyalkanoates (PHAs) are biodegradable and biocompatible bioplastics produced by many different bacteria under conditions of unbalanced growth [8, 14]. In order to reduce costs of PHA production, the use of mixed cultures and waste organic carbon as feedstock have been focused in the field of PHA biosynthesis [15, 172-176]. Nevertheless, most waste streams used for PHA production has limitations in term of their abundance and distributed in nature [177]. In contrast, methane is a cheap and abundant carbon source widely available in nature. In addition, the nature of mixed methanotrophic cultures are robust and self-regulating [2, 6, 10, 11, 98], offering the opportunity to operate under non-sterile conditions, thereby reducing operating costs [2].

Methane-driven microbial consortia intermittently subjected to nitrogen limitation have been widely reported to accumulate PHA [10, 39, 60, 100, 166, 167]. In their seminal paper on PHB production by methanotrophs, Asenjo and Suk (1986) presented the overall stoichiometry for biopolymer accumulation in methanotrophs as [60]:

8 CH4 + 12 O2 + FP → C4H6O2 (monomer) + 4 CO2 + 12 ATP + FPH2 where FP = oxidized succinate dehydrogenase and FPH2 = reduced succinate dehydrogenase.

The theoretical yield of biopolymer on methane in methane-raised methanotrophs (neglecting the methane used to regenerate the NADH required for biosynthesis) is therefore 0.67 g PHA g methane-1. In practice, maximum yields are around 0.5 g PHA g methane-1. Wendlandt et al. (2001) obtained yields of 0.55 g PHB g methane-1 in a mixed culture dominated by Methylocystis sp. GB 25 DSMZ 7674, close to the theoretical maximum, with biopolymer content in the biomass being ~50 wt% of total dry weight. However, it is noteworthy that the PHA produced in methane-fed bioreactors is limited to PHB. PHAs are attractive substitutes for petrochemical-derived plastics, but the market for the homopolymer PHB is limited because of its brittle nature and low thermal stability [8, 19].

The physical properties of PHAs are known to vary and are dependent on monomer composition and monomer distribution [178]. For example, by increasing the proportion of 3-hydroxyvalerate (3HV) units in the poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) copolymer the polymer’s flexibility and toughness increase while the melting and glass transition temperatures (Tm and Tg) decrease, making the polymer more readily processable

[8, 179]. It follows that the market for CH4-derived PHA could be substantially expanded by

37 copolymerization of 3-hydroxybutyrate with alternative hydroxyalkanoate monomers (such as 3-hydroxyvalerate (3HV)) [2].

To date, the incorporation of 3HV into PHAs by methanotrophs has been achieved by supplementation with fatty acids with odd carbon number chain lengths, such as propionate or valerate [10] - this was the basis of the previous chapter. These co-substrates are likely metabolized via β-oxidation of fatty acid to form a fatty acid Co-A, such as propionyl-CoA, which is then condensed with acetyl-CoA and subsequently reduced to (R)-3HV-CoA, then copolymerized with (R)-3HB-CoA by PHA synthase (phaC) to form PHBV [9, 104, 180]. However, while this strategy has proven successful, the procurement of these precursors is an additional cost for PHA production.

In this chapter, the generation of PHBV from methane without the requirement for precursor addition is considered. Microorganisms likely responsible for 3HB and 3HV generation in a methane-driven microbial consortia are identified, with the specific objective being to determine possible relationships between organisms characterized to be efficient in the transformation of C1-substrates with organisms capable of forming PHAs, including copolymers containing 3HV. A feature of the work is the long-term observation of the 3HB and 3HV producing methanotrophic-heterotrophic community. Evaluation of population dynamics is undertaken using 16S rRNA gene sequencing and the metabolic capacity of the community identified using metagenomics sequencing.

5.2 Materials and methods

5.2.1 Methane-fed microbial enrichment culture

A semi-continuous reactor (2 L) was established using 30 mL of the cell suspension (S2) grown on nitrogen gas described in Section 3.1 plus 210 mL of fresh medium prepared as described in Section 3.1, amended with 1.6 mL of 28% ammonia solution per 1 L of medium (Merck Millipore, USA) as a nitrogen source. The headspace was filled with air, and 500 mL of the gas feed mixture (90% CH4, 10% CO2) was added. The culture was incubated on a stirrer at 200 rpm at ambient temperature. After 40 days of incubation, a sample was subjected to a 16S rRNA sequence analysis showing that a methanotroph associated with the genus Methylomonas dominated the methane-fed enrichment.

38 Once the methane consumption was detectable in this culture, a cycling of nutrient deprivation with ammonia was introduced to promote biomass production. The system was described as a semi-continuous system with a controlled volume entering or exiting. The operation of the bioreactor was characterized by solid retention time (SRT) as described by Kreuzinger et al (2005) [135]. The culture was subjected to long-term cycling divided into 3 phrases according to different operational SRTs: Phase 1 SRT 5.5 days (from day 0 to 80), Phase 2 SRT 3.0 days (from day 80 to 180), and Phase 3 SRT 3.2-3.6 days (from day 180 to 400). Figure 3.2 illustrates the system operation during Phase 3 in which; Step 1, the bioreactor with 40 mL of leftover culture from the previous cycle received 200 mL of fresh medium to maintain liquid volume at 240 mL. Headspace was flushed for 10 min with air, and then 500 mL of the gas mixture (90% CH4, 10% CO2) was injected into the headspace. In Step 2, the culture was incubated at 30ᵒC (200 rpm) for 72 hr and received another 10 min gas flush with air and CH4:CO2 mixture. In Step 3, 200 mL of liquid was removed, completing a cycle. The volume entering and exiting the system may vary depending on the incubation time within the bioreactor; however, the operational SRT was maintained in the ranges reported above.

5.2.2 PHA Production

The sample removed from the reactor (described above in Step 3) was centrifuged at 6300g for 5 min (Eppendorf Multipurpose Centrifuge 5804R) and suspended in fresh nitrogen-free medium in 250 mL serum bottles (Wheaton, Mealville, NJ, USA) capped with butyl-rubber stoppers and crimp-sealed. The liquid volume was 30 mL and the headspace volume was

220 mL. The headspace was filled with air plus 50 mL of the gas mixture (90% CH4, 10%

CO2). Cultures were maintained in a non-sterile condition at 30ᵒC. After 72 h, cells were harvested by centrifugation (6300g for 5 min) and oven-dried at 70ᵒC. Preserved samples were assayed for PHA content.

5.2.3 Community metagenome sequencing

The community structure and metabolic potential of the parent reactor microbial community was estimated using Illumina high-throughput shotgun sequencing on Day 289 using DNA extracted for 16S rRNA gene sequencing. Metagenomics sequencing generated 4 Gb of paired- end data with the reads assembled using the de novo assembly method using Metaspades [181] and population genomes assembled using Metabat2.0 [182], both with default settings. The quality of contigs, scaffolds, and metagenome-assembled genomes (MAGs) was improved using a combination of FinishM (https://github.com/wwood/finishm) and RefineM 39 (https://github.com/dparks1134/RefineM) programs. The MAGs were taxonomically classified using the Genome Tree Taxonomy Database methods [183]. While the quality of MAGs and the bioreactor community structure were estimated using the CheckM genome classifier [184]. The ability of the microbial community (MAGs and unbinned contigs) from the parent reactor to form PHA was also estimated using the GraftM package graft function [185] using the PHA biosynthesis taxonomic marker gene, phaC. The amino acid sequence of genes detected by GraftM were aligned with database sequences for phaC from organisms known to form PHA and viewed using the MEGA taxonomy tool [186]. The raw metagenome reads, iTag amplicon reads, phaC gene sequences used for trees and MAGs have been deposited in the NCBI Sequence Read Archive under accession number PRJNA486693.

5.3 Results and discussion

5.3.1 Enrichment of a methane-consuming community using a fed-batch process

An enriched methanotrophic-heterotrophic parent community was produced through use of a growth repeating cycle (ammonia sufficiency), with methane as the sole carbon source. Over 400 days of operation, this methanotrophic–heterotrophic community grew under non-sterile conditions under variable solid retention times (SRTs). Initially, the operational SRT was set at 5.5 days. After 80 days, the SRT was reassigned to 3.0 days to reduce the opportunity for non-methanotrophs to proliferate, and then after 180 days of incubation the operational SRT was extended to 3.5 days in order to maintain a stable cell concentration. The pattern of methane consumption, PHA concentration, and mole fraction of 3HB and 3HV in the biomass in the parent reactor during this period of operation is illustrated in Figure 5.1.

The growth characteristics of the methanotrophic-heterotrophic culture from Day 180 onwards (SRT 3.5 days) were examined. The methane-driven mixed culture doubled every 1.1 days. The maximum specific growth rate was 0.03±0.01 h-1 (0.7±0.3 day-1). The specific rate of -1 -1 methane utilization was 0.7 g CH4 g TSS day , with specific CO2 evolution being 1.0 g CO2 g TSS-1 day-1. At the end of batch cycle, biomass concentration was approximately 0.24±0.03 mg TSS mL-1.

PHA was observed in this methanotrophic-heterotrophic community under a microscope when stained with Nile blue A, throughout the entire 400-day study; PHA was always present in the biomass despite nutrient addition to the bioreactor. The average PHA content in the bioreactor was 0.6±0.2 wt% in dry cell, with a varying mole fraction of 3HV. It is noteworthy that the

40 culture grown with methane as the sole carbon supply produced a polymer with a high 3HV fraction: up to 100 mol% HV was achieved in the community on Day 382 without a 3HV precursor being supplied (Figure 5.1). PHA production in the reactor near the end of the study (Day 382 to 385) was examined in detail (Figure 5.2A). The PHA content during the period was 0.8 wt% with 100 mol% of 3HV at 0 h (Day 382) and 0.5 wt% with 86 mol% HV at 72 h (Day 385). Methane consumption, biomass concentration and PHA content are shown in Figure 5.2A. The cycle time for headspace replenishment was 24 h and cell withdrawal occurred every 72 h. The PHA content in the reactor was relatively stable (at around 0.002 C-mmol) over the 72 h of incubation with nutrient sufficiency.

7000 100 SRT = 5.5 day SRT = 3.0 day SRT = 3.5 day 90 6000 ) 1 - 80

5000 70

60 4000 50 3000 40

2000 30 3HB and content3HB 3HV (mol%) 20 Methnae consumption rate (umol day 1000 10

0 0 0 40 80 120 160 200 240 280 320 360 400 Days

Figure 5.1 The pattern of methane consumption and PHA accumulation in methanotrophic– heterotrophic communities grown using different solids retention times (SRTs) over 400 days of a typical growth repeating the operation. Methane consumption rate (μmol day-1) (o), 3HB content on mole basis (mol%) (▲), and 3HV content on mole basis (mol%) (•) in the PHA produced are shown. Dash line (- - -) shows median methane consumption rate for the various SRTs.

5.3.2 Methane-fed PHA production

At the end of the growth repeating cycles, on Days 56, 59, 63, 78, 81, 112, and 385, biomass was withdrawn from the reactor and subjected to nitrogen-deficient conditions to stimulate further PHA production. The cells were washed twice with N-free media to avoid transfer of

41 ammonia from the growth media. The culture was then incubated for 72 h at 30°C in nitrogen- free media. A typical pattern (from biomass harvested on Day 385) of methane utilization and PHA production in the absence of ammonia, on a C-mmol basis, is shown in Figure 5.2B.

A) 20 0.008 Total PHA (C PHA Total 15 0.006

10 0.004 mmol) , and Biomass Biomass , and - 2 - (C mmol)

, CO 5 0.002 4

CH 0 0.000 0 12 24 36 48 60 72 Hour

B) 2.0 0.008 Total PHA (C PHA Total 1.5 0.006

1.0 0.004 mmol) , and Biomass Biomass , and - 2 - mmol) (C 0.5 0.002 , CO 4

CH 0.0 0.000 0 12 24 36 48 60 72 Hour Figure 5.2 Methane consumption and production of carbon dioxide, biomass, and intracellular PHA on a C-mmole basis during a typical 72-h of growth phase in the presence of nitrogen (ammonium) (A) and a 72-h accumulation phase in the absence of nitrogen (B), using cells harvested from the 2-L reactor on Day 382. C-mmole of methane (○), carbon dioxide (x), total biomass (■), and total PHA (¨) are shown.

The removal of ammonia from the methanotrophic-heterotrophic community resulted in an increase in PHA production. On Day 385, the PHA content increased from 0.5 wt% with 86 mol% HV initially to 1.0 wt% with 82 mol% HV within 72 h. However, the average final PHA content in the biomass after accumulation in the absence of nutrients was 0.8±0.2 wt%. When fed with methane alone the generated polymer was composed of 3HB and 3HV repeating units: in the early stages the 3HB content was 58-80 mol%; after over 1 year of operation the polymer composition had switched to nearly all 3HV (82 mol% of 3HV). The relatively high variation in the ratio of 3HB to 3HV may have been a result of the fluctuation in the physiological characteristics of the methanotrophic-heterotrophic parent community across Day 50 to 388.

42 5.3.3 Community dynamics in semi-continuous-batch reactor

The structure of the microbial community was monitored using 16S rRNA gene sequencing (on Day 0 and 45) and Metagenomic Assembled Genomes (MAGs) on Day 289. The community compositions at the genus level for the reactor are summarized in Figure 5.3. Overall, 18 genera with abundance above 1% were identified in the culture. The sequences of each genus, as well as the combined total for the remaining genera (classed as ‘other’), were analyzed based on a %OTU basis. In each genus, the number of sequences was normalized to the total number of sequences obtained for that sample and then converted to a percentage.

The communities at different times were composed mainly of Proteobacteria with an abundance in the range of 59-80%. Methanotrophs are well known to affiliate within this phylum [187]. As methane is the sole carbon source, it is not surprising that Methylomonas, a gammaproteobacterial methanotroph, was the most abundant genus. There was little change in the total relative abundance of the Methylomonas population when the reactor SRT was 5.5 days (from 38% to 35% of the on Day 0 and Day 45, respectively). However, there were other underlying community changes, especially in the observed richness of the microbial communities. Bacteria from the genera Azospirillum (19%), Sporomusa (8.5%), Flavobacterium (5.4%) and Meniscus (4%) were present initially, however these populations were washed out (reduced to ≤1% abundance) from the system within the first 45 days of operation. During this period the abundance of Methylophilus increased from 9% to 22% and the abundance of Flavihumibacter increased from 1% to 8%. In methanotrophic-heterotrophic communities, methanotrophs form the basis of a microbial food web by supplying by-products and metabolites derived from, for example, aerobic methane oxidation and methane-based fermentation under oxygen-limited condition [21, 148]. Methylophilus is a methylotrophic bacterium with an ability to utilize methanol and some other single carbon compounds, but not methane, as the sole carbon and energy source [188]. Flavihumibacter can utilize a diverse range of carbon substrates, including saccharides, organic acids, and amino acids, but not methane [189]. Such carbon compounds would have become available as bacteria decayed [89, 127, 190].

In order to reduce accumulation of secondary metabolites and/or products of cell lysis, the residence time (SRT) was reduced (to 3.0-3.5 days). Most significantly, the proportion of Methylomonas doubled to 61% by Day 289, with the change being primarily associated with decreases in Flavihumibacter and Methylophilus. Interestingly, there were increases in other

43 methanotrophs and heterotrophs, which were initially present in a low portion (<0.5%) in the inoculum, including Burkholderia (9%), Chryseobacterium (1%), Hyphomicrobium (2%), Methylosinus (1%), Pelomonas (3%), and unidentified genera from family Rhodospirillaceae (2%) and Xanthomonadaceae (1%). Most of these species are capable of utilizing a variety of carbon sources including methanol, saccharides, organic acids, or amino acids [191-196]. In addition, they have been found during syntrophic growth with methanotrophs [89, 98, 127, 148, 166, 190]. Significantly, only Methylosinus is recognized as an obligate methanotroph with PHA production capability [9]. In summary, the reduction in SRT both enriched the methanotrophic representation and caused a shift in the community that feeds on secondary metabolites and/or products of decay – though activities related to these substrates are significant even at reduced SRTs.

100

90 Methylomonas Methylosinus* 80 Methylophilus Hyphomicrobium* 70 Burkholderia* Pelomonas* 60 Rhodosprillaceae* Xanthomonadaceae* 50 Azospirillum Magnetospirillum

40 users Methane Caulobacter

Chryseobacterium Methane users Methane Relative abundance (%)

30 users Methane Comamonas Flavihumibacter substrates userssubstrates

substrates userssubstrates

20 C Flavobacterium 1 C substrates userssubstrates

Meniscus 1 C

10 Opitutus

Sporomusa 0 Other Inoculum 45 289 Days Figure 5.3 Bar graph representing relative community compositions of 18 bacterial genera which represented > 1% of total sequence counts, comparing each biomass sampled on Day 0, 45 and 289. Data on Day 0 (inoculum) and Day 45 obtained from 16s rRNA sequencing, while Day 289 retrieved from metagenomic sequencing. The SRT on Day 45 was 5.5 days and on

Day 289 was 3.5 days. Left and right braces highlight total population of C1-substrates and methane utilizers, respectively. (*) represents microorganisms with PHA production capability in literature.

44 5.3.4 Metabolic capacity of the bioreactor microbial community

Despite the shifts in microbial community – particularly in the non-methanotrophic portion of the community – the methanotrophic-heterotrophic consortium was shown to produce a stable low level of PHA during the growth repeating cycles, but with variations in copolymer composition. To further explore the relationship and interactions within the methane-driven community, DNA from the parent reactor was collected on Day 289 for high-throughput sequencing of and subsequent assembly of Metagenomic Assembled Genomes (MAGs), in order to identify the organisms in the system with the potential to form PHA (Table A2).

As already noted: on Day 289, the community was dominated by a methanotrophic population that was most similar to Methylomonas koyamae, a gammaproteobacterial methanotroph, which made up 61% of the community (Figure 5.3; Table A2). This methanotrophic species has been reported to utilize methane and methanol as carbon and energy sources but does not accumulate PHA [197]. This result was confirmed by the metagenomic data, as the genes for PHA biosynthesis (phaABC) were absent in the MAG of the Methylomonas population. Furthermore, phaC genes were absent that matched other gammaproteobacteria phaC genes from both contigs and sequence reads (Figure 5.4), which was consistent with the type strain of this species [197, 198]. However, it should be noted that there were phaC sequances assembled and binned to poor quality MAGs were partially complete with BLAST identity match scores <60%.

Unlike the Methylomonas methanotroph, many of the non-methanotrophic species have the capability to form PHA based on the presence of phaC genes for PHA biosynthesis that are associated with MAGs assembled from the metagenomic data (Figure 5.4; Table A2). Searches of both MAGs and contigs for phaC genes suggest that many species are capable of forming PHA indirectly from methane. A total of twenty phaC sequences from class I, II, III, and IV were identified. Five of these sequences binned to the high-quality MAGs corresponding to Burkholderia, Pelomonas, Hyphomicrobium, Rhodosprillaceae, and Xanthomonadaceae (Table A2), which are PHA-producing heterotrophs known for their capability to utilize a wide variety of carbon sources. For example, Burkholderia and Pelomonas can grow and produce PHA from amino acids, sugars, organic acids, glycerol, and triglycerides [191, 193, 199-201]. This is consistent with our sequencing results, revealing that Burkholderia can utilize formate, lactate, fatty acids, and aromatic compounds. In addition, Hyphomicrobium, a typical obligate methylotroph, was present at a low proportion at the beginning of the experiment (0.5%) before

45 increasing in parallel with the Methylomonas population, and was possibly involved in PHA accumulation as it harbors a complete operon for PHA biosynthesis (phaABC). Furthermore, we have unidentified members from the Rhodosprillaceae and Xanthomonadaceae families, which, according to the MAGs result, can accumulate PHA from sugars, amino acids, and aromatic compounds, which are possibly generated during metabolic activities within the community.

Many other phaC genes were identified that were not associated with genomes (Figure 5.4), suggesting that organisms present at low levels in the bioreactor were also capable of forming PHA. Unexpectedly, there was no crucial gene for PHA synthesis associated with the Methylosinus genus detected from the assembled reads (Figure 5.4). This finding is in contradiction with previous results reported in the literature [24, 101]. There are two possible explanations of this issue: whether the absence of this gene is an authentic signal of its absence from the genome, or this gene was present but not recovered in the MAG. While this methanotroph could possibly contribute to the generation of PHA from methane based on the information in the literature, its activity was excluded in this study because of the lack of the key gene in our result.

In short, the high-throughput metagenomics sequencing allowed the recovery of MAGs that accounted for ~86% of the bacterial community and the identification of phaC genes, revealing that the PHA formed in this representative community was primarily produced by non- methanotrophic species indirectly from methane.

46 Methylocystis rosea (WP_018407079) ≥ 50% ≥ 70% Methylocystis parvus (WP_040579112) Figure 5.4 Phylogenetic ≥ 90% Methylosinus trichosporium (WP_003609868) Methylocapsa acidiphila (WP_026606925) representation of bacterial phaC Methyloferula stellata (WP 040581087) genes from class I, II, III and Microvirga lotononidis (WP 009491520) Methylobacterium extorquens (AAA72330) IV. Bold phaC sequences Methylobacterium radiotolerans (ACB27029) Unbinned contig 78 PhaC sequence corresponding to high-quality CtgCon85 Lutibaculum baratangense (ESR23306) MAGs have been recovered Stappia stellulata (WP 029056879) Labrenzia alba (WP 055676533) from the methanotrophic- Mesorhizobium opportunistum (WP 013895146) heterotrophic community. Sinorhizobium meliloti (AAC61899) Rhizobium gallicum (WP 039844755) Bootstrap values ≥50% are CtgCo104 CtgCo119 represented as open (○) circles, Rhodopseudomonas palustris (WP 027277070) Azorhizobium caulinodans (O66392) ≥70% as grey (●) circles, and Xanthobacter autotrophicus (ABS68752) ≥90% as filled (●) circles. Xanthobacter autotrophicus (WP 049776168) Hyphomicrobium zavarzinii (WP 026868159) Hyphomicrobium denitrificans (AGK57645) Hyphomicrobium MAG PhaC sequence (MAG_5) CtgCon88 CtgCon29 CtgCo122 Caulobacter vibrioides (AAG15582) CtgCo102 CtgCon98 Rhodospillaceae MAG PhaC sequence (MAG_7) CtgCo115 Group I Rhodospirillum rubrum (AAD53179) CtgCon58 Aeromonas caviae (BAA21815) Vibrio cholerae (AAF96588) Rhodobacter capsulatus (CAC85535) Paracoccus denitrificans (BAA07821) CtgCo146 Burkholderia MAG PhaC sequence (MAG_3) Ralstonia eutropha (P23608) Pelomonas MAG PhaC sequence (MAG_4) Azohydromonas lata (AAC83658) Pseudomonas sp. (BAA36198) Rhodococcus ruber (CAA47035) Gordonia rubripertincta (AAB94058) Pseudomonas oleovorans (AAA25934) CtgCo124 CtgCo143 Group II Pseudomonas fuorescens (AAP58480) CtgCon91 CtgCon92 Pseudomonas resinovorans (AAD26365) Myxococcus xanthus (WP 011556797) Bradyrhizobium japoicum (WP 018648127) Roseiflexus castenholzii (WP 011997646) Group IV Chloroflexus aggregans (WP 012615932) Desulfotomaculum gibsoniae (WP 006520325) Syntrophomonas wolfei (WP 011640513) Kurthia massiliensis (WP 044504462) Aneurinibacillus terranovensis (WP 027415498) Bacillus azotoformans (KEF40213) Bacillus megaterium (AJI20472) Bacillus cereus (WP 016133940) Natronococcus jeotgali (WP 008426172) Natronorubrum sulfidifaciens (WP 008159633) Halostagnicola larsenii (AHF99950) Group III Haloferax mediterranei (WP 004056138) Methylocaldum szegediense (WP 036269290) Xanthomonas hyacinthi (WP 046981279) Rhodobacter sp. (WP 008211781) Xanthomonadaceae PhaC sequence (MAG_11) 0.50 Rhodanobacter spathiphylli (WP 007805236)

47 5.3.5 Possible interactions within the methane-driven community for 3HB and 3HV generation

Based on the metagenomics analysis, the suggested interaction between methanotrophs and heterotrophs for 3HB and 3HV generation is illustrated in Figure 5.5. It is suggested that in the methane-driven consortia, when methane and oxygen are available, methanotrophs process methane for biomass growth and, at the same time, produce carbon by-products in the form of methanol and/or organic acids [21, 22, 202, 203]. The analysis of these by-products was undertaken using gas chromatography; however, no secondary by-products such as methanol and ethanol were detected. Only low levels of formic acid and acetic acid (<2 ppm) were observed, indicating that the interactions in the methanotrophic-heterotrophic community have an efficient carbon-cycling system and therefore there are difficulties in the determination of intermediates.

Nevertheless, it was confirmed that Methylomonas does not have the capacity to accumulate PHA, meaning that the biopolymer was generated by either (i) methylotrophs that utilise methanol or, (ii) heterotrophs that feed on byproducts of methanotrophic activity or products of cell decay. According to the literature and the metagenomics result, there are five PHA- producing strains capable of utilising different carbon substrates for growth and PHA biosynthesis; Hyphomicrobium, Burkholderia, Pelomonas, Rhodosprillaceae and Xanthomonadaceae. Hyphomicrobium, for example, are capable of producing PHB from methanol under nutrient-limited conditions [165]. Production of the PHB in the wild type of this organism was accomplished when fed with methanol [165]. The Burkholderia sp., in addition, has been reported to produce PHB from xylose [204], glucose, fructose, and sucrose [201], glycerol [205], myristic acid, lauric acid, palmitic acid, oleic acid, and stearic acid [206]. The potential for 3HV biosynthesis in this microorganism has been demonstrated [207]. However, to the best of our knowledge, there are no reports of 3HV production by these potential PHB producers without the presence of 3HV precursors.

In our system, the routes for 3HV generation likely involve methanotrophic activity and subsequently biomass decay leading to the generation of organic acids, some of which are precursors for 3HV production. The acids could be generated in two ways: (i) by fermentation of cell debris by Opitutus or (ii) by Burkholderia species. In our system, the cells were observed to cluster, potentially resulting in microanaerobic zones, despite overall oxygen-sufficiency. Therefore, such zones would support the Opitutus species, which is capable of carbohydrate

48 fermentation to propionic acid, a well-known precursor for 3HV formation. The reduction in the Opitutus population concurred with the decline in 3HV contetn when the operational SRT was shorten from 5.5 to 3.0 days. However, there is no clrear explanation on the increase of 3HV content at the end of this study. One possible explantion is because there was an increase in the population of the Burkholderia species, which may assimilate branched chain amino acids and release odd-fatty acid compounds as products, precursers for 3HV formation. At this point, we are aware that this study does not provide sufficient evidence to distinhuish the exact mechanism; however, the prime cause of the discrepancy is likely a consequence of the presence of trace genera of facultative aerobes to anaerobes for example Azospirillum, Sporomusa, and Opitutus itself [208-210].

Oxygen is known to be a critical factor relevant not only to 3HV formation in the culture but also for its effects on methane oxidation and associated activities in methanotrophs. A study by Kalyuzhnaya et al. (2013) has shown the potential for methanotrophs to produce organics acids directly from methane in oxygen limited environments via a fermentation process [21]. Under such conditions, the notable change in the Methylomicrobium was the activation of pathways for mixed-acids production, which include formate, acetate, succinate, lactate,

3-hydroxybutyrate and H2. It should be noted that we attempted to determine the concentration of these excreted metabolites during the accumulation phase; however, the concentrations were lower than our detectable range. Despite this, we can still state the possible relationships between the populations based on literature and metagenomics results, with an emphasis on those relationships that would initiate 3HV formation.

49 PHB

) Methanol Hyphomicrobium* 4 PHV or PHBV

Oxygen-limited condition Methylomonas PHB

Burkholderia* ) (PHBV) Lactate and Formate PHV or PHBV*

Methane (CH Organic acids

Lactate PHB Pelomonas* hydroxyvalerate or (PHV), Amino acids and Sugars PHV or PHBV -

Lactate hydroxyvalerate - 3 - co

Amino acids, Sugars, PHB -

Rhodosprillaceae* Products and Aromatics PHV or PHBV

Amino acids, Sugars, PHB and Aromatics Xanthomonadaceae* PHV or PHBV hydroxybutyrate

Decay - products hydroxybutyrate) Poly(3 (PHB), - Carbohydrates Opitutus Propionic acid Poly(3 Poly(3

Figure 5.5 Based on literature, the possible interactions within the methanotrophic-heterotrophic community when methane serves as the sole carbon source and polyhydroxyalkanoates (PHAs) with 3-hydroxybutyrate (3HB) and 3-hydroxyvalerate (3HV) monomer units are being generated. (*) indicates the community members with the phaC gene. The findings of this work are consistent with these interactions. ( ) shows carbon substrates utilization associated with growth and PHB production.( ) show the metabolic formation of 3HV precursor. ( ) shows the co-metabolic transformation of 3HV precursor to PHAs with 3HV composition.

50 5.4 Conclusion

The methanotrophic-heterotrophic community produced in this study has shown its capacity to biosynthesis of multiple repeat units of both 3HB and 3HV from methane without a requirement of co-substrate such as propionic acid and valeric acid. Many non-methanotrophic PHA producers were presented in the culture, along with those who capable of lysed cell fermentation for 3HV precursor formation. According to MAGs sequencing and available information in literature, a number of complex interactions exist between populations, which indirectly lead to biosynthesis of multiple repeat units of both 3HB and 3HV from methane. The most probable interactions leading to the generation of those precursors are those based on the growth and decay of methanotrophs, and subsequent fermentation of decay products. Most of these relationships, some of which may be obligatory and reciprocal for the interacting populations, confer beneficial effects, making the community adaptable for a long-term operation and more efficient in the transformation of C1-substrate to various PHA copolymer compositions than any of the individual populations alone.

51 Chapter 6 Biosynthesis of poly-3-hydroxybutyrate (PHB) in a thermophilic gammaproteobacterial methanotroph

Production of polyhydroxyalkanoates (PHAs) from methane has been limited to mesophilic strains and suffer from the high energy requirements for temperature control. The thermophilic production are instead considered to be energy-efficient because of the removal of a refrigerant needed in the cooling system [3]. The process itself can generate high thermal energy during cultivation, along with heat input generated by the bioreactor ́s stirring system, this contributes to the heating of the process [6]. As a result, both heating and cooling costs can be reduced. The application of thermophilic strains for PHA production is therefore gaining interest as it could reduce the energy usage and pave the way towards cost-efficient PHA production. However, to date, there are no reports on thermophilic or thermotolerant methanotrophs with capability to produce PHA. Hence, the objective of this work was to develop a methane consuming thermophilic culture that formed poly(3-hydroxybutyrate) (PHB).

The culture was enriched from environmental samples collected from a natural hot spring in Australia. Various selective conditions based on nitrogen source, pH, and trace nutrient strength were trialled while methane served as the sole carbon source. The temperature was controlled at 55°C. Nutrient-poor media with nitrate as the nitrogen source and neutral pH selected for a culture that showed evidence of PHA accumulation. The 16S rRNA sequencing revealed dominance of Methylocaldum sp. and other co-existing non-methanotrophic PHA forming bacteria in the methanotrophic enrichment when grown under non-aseptic conditions in a semi-continuous reactor with nitrate as the nitrogen source.

Genome directed metabolic analysis of metagenome-recovered genomes revealed high phylogenetic diversity in the thermophilic community. A total of eleven PHA synthase (phaC) regions from class I, III and IV were recovered; however, only few of these sequences binned to high-quality recovered MAGs (with a completeness level >70%). Investigation of this gene revealed that around 40% of the community carried the PHA synthase gene, with the highest prevalence being in Methylocaldum sp. The MAGs were used to guide the isolation of Methylocaldum sp. from the thermophilic community. An extinction-dilution technique, with addition of antibiotic erythromycin, was applied. When even- and odd-carbon number fatty acids were introduced into the axenic culture of Methylocaldum under nitrogen-limiting conditions, PHB accumulated in all tested conditions either with or without methane supply,

52 suggesting that short chain fatty acids rather than methane were used to form PHA polymers. The subsequent analysis of PHA composition revealed a sole formation of PHB homopolymer with no trace of 3HV units in all tested conditions. Coupling with the presence of β-oxidation- linked PHA polymerase genes (phaCE) and β-oxidation pathway (fadABDG) in the Methylocaldum sp., this suggests an alternative mechanism for PHB formation in Methylocaldum, divergent from the pathways seen in other PHA-producing methanotrophs.

A schematic diagram representing the workflow of this chapter is illustrated in Figure 6.1.

Figure 6.1 Schematic diagram represents the workflow of Chapter 6

Contributions by others

A modified version of this chapter has been prepared for a publication. The community profiling using shotgun metagenomics DNA sequencing, assembly and binning of metagenome-assembled genomes (MAGs), reconstruction of metabolic pathway, and isolation of methanotrophic culture was done in collaboration with Dr. Paul Evans and Samantha MacDonald from the Australian Centre for Ecogenomics (ACE), The University of Queensland. Samantha MacDonald submitted a part of this chapter as her thesis to qualify for her Honors degree in Chemistry and Molecular Biosciences at The University of Queensland submitted in May 2018.

53 6.1 Introduction

The ability of bacteria and archaea to produce polyhydroxyalkanoates (PHAs) is well known and has often been demonstrated under mesophilic conditions using feedstocks such as sugars or other materials that impart a significant cost on PHA production [211]. Methane is a feedstock that is both relatively abundant and cheap, and has been identified as a waste stream that could be used as a feedstock for biosynthesis of PHA in bacteria and archaea [3, 11].

Many studies have shown that methanotrophs have the potential for producing poly(3- hydroxybutyrate) (PHB) or the copolymer poly-(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) in mixed or pure culture [39, 60, 102, 104, 212, 213]. These studies have primarily been conducted with mesophilic Alphaproteobacterial methanotrophs, which are described as Type II species [111, 214]. Conversely, there has been a little study of PHA formation in Gammaproteobacterial methanotrophs that grow under high methane and low oxygen concentrations and are sensitive to the high partial pressure of these substrates [215]. Thermophilic methanotrophs are in this phylum. Considering the scant evidence in the literature, Gammaproteobacterial methanotrophs possibly can form PHA based on the presence of inclusion bodies seen in axenically cultured methanotrophs [112, 216, 217], or their capacity inferred from the genomic evidence [218].

Type II Alphaproteobacterial methanotrophs commonly possess and use the serine cycle for carbon assimilation and encode the phaC gene for PHA synthesis [38, 39]. In contrast, the Gammaproteobacterial methanotrophs assimilate carbon at the oxidation level of formaldehyde into cell carbon via the ribulose monophosphate (RuMP) pathway; they consist of two subdivisions: Type I and Type X methanotrophs [214]. While Type I methanotrophs such as Methylococcus, Methylomonas, Methylothermus, and Methylohalobius solely use the RuMP pathway and test negative for phaC and PHB production [39, 214], the thermophilic and thermotolerant methanotrophic species of Methylocaldum from Type X can use both the RuMP serine cycle pathways, and test positive for genes for PHA biosynthesis (phaC) [106, 120, 219, 220]. However, there is no published quantitative evidence of the capacity of Methylcaldum genus to form PHA.

It should be noted that the current knowledge of Methylocaldum is limited. This is because of its complicated temperature-dependent traits, especially in biochemical and metabolic rearrangements, which usually lead to a loss of culture from culture collections [106, 113]. Methylcaldum sp. shows a wide range of growth temperatures from 37-60°C [106, 113]. By 54 changing temperature, the morphology of this methanotroph changes remarkably and the levels of the key enzymes of RuMP alter along with those of the serine cycle [113]. Although the role of these metabolic rearrangements is unclear, the presence of the phaC gene has suggested the potential for high-temperature PHA production.

The objectives of this chapter are to:

(i) Develop a culture of thermophilic methanotrophs capable of accumulating PHA; (ii) Identify and isolate the thermophilic methanotroph(s) that contribute to PHA accumulation; (iii) Understand the capacity of the isolated methanotroph(s) to accumulate PHA on a range of carbon sources.

6.2 Materials and methods

6.2.1 Sample collection

Environmental samples were collected from Innot hot springs (Ravenshoe, QLD, Australia). Samples were allocated sample code as follows: (H1) soil from the surface; and (H2) soil at 30-cm depths. The sample pH was 8.0±0.3 and 7.2±0.1 for H1 and H2, determined by adding deionized water to the sample and measuring with a combination pH probe (HANNA HI 9125 Portable pH Meter). Inoculation was done within 5 days after sample collection.

6.2.2 Selection experiment

Twelve enrichment series were performed with a combination of three variables: pH (5.0 and 7.0), nitrogen source (ammonia, nitrate, and nitrogen gas), and NMS media concentration (0.5xNMS or 1.0xNMS), as shown in

In some enrichment series, the concentration of the ammonia (AMS) or nitrate (NMS) mineral salts was diluted twofold (1.0x versus 0.5x). The enrichments were performed under two different headspace ratios of Air, CH4:CO2, and Ar:O2 mixture. For enrichment series A-H, the vessels were subjected to vacuum, flushed with O2:Ar mixture (50% O2, 50% Ar) for 2 min to restore atmospheric pressure, and supplemented with 30 mL of CH4:CO2 mixture (90% CH4,

10% CO2). Meanwhile, for enrichment series I-L, the vessels were filled with air and 30 mL of

CH4:CO2 mixture (90% CH4, 10% CO2).

55 Table 6.1. The initial pH of the medium was adjusted with concentrated sodium hydroxide and hydrochloric acid to 5.0 or 7.0 as specified. The mineral salts medium was prepared with either

5 mM NH4Cl or 5 mM KNO3 with other components as listed in Section 3.1.

In some enrichment series, the concentration of the ammonia (AMS) or nitrate (NMS) mineral salts was diluted twofold (1.0x versus 0.5x). The enrichments were performed under two

different headspace ratios of Air, CH4:CO2, and Ar:O2 mixture. For enrichment series A-H, the

vessels were subjected to vacuum, flushed with O2:Ar mixture (50% O2, 50% Ar) for 2 min to

restore atmospheric pressure, and supplemented with 30 mL of CH4:CO2 mixture (90% CH4,

10% CO2). Meanwhile, for enrichment series I-L, the vessels were filled with air and 30 mL of

CH4:CO2 mixture (90% CH4, 10% CO2).

Table 6.1 Selection conditions for thermophilic methanotrophs Enrichment series Nitrogen sources Headspace Nutrient Solution pH

A Ammonia O2/CH4 1.0x NMS (5 mM NH4Cl) 5.0 B Ammonia O2/CH4 1.0x NMS (5 mM NH4Cl) 7.0 C Ammonia O2/CH4 0.5x NMS (2.5 mM NH4Cl) 5.0 D Ammonia O2/CH4 0.5x NMS (2.5 mM NH4Cl) 7.0 E Nitrate O2/CH4 1.0x NMS (5 mM KNO3) 5.0

F Nitrate O2/CH4 1.0x NMS (5 mM KNO3) 7.0 G Nitrate O2/CH4 0.5x NMS (2.5 mM KNO3) 5.0 H Nitrate O2/CH4 0.5x NMS (2.5 mM KNO3) 7.0 I N2 Air/CH4 1.0x NMS (N2 gas in headspace) 5.0 J N2 Air/CH4 1.0x NMS (N2 gas in headspace) 7.0 K N2 Air/CH4 0.5x NMS (N2 gas in headspace) 5.0 L N2 Air/CH4 0.5x NMS (N2 gas in headspace) 7.0

For each series, 8 mg of sample was added to 30 mL of medium in a 160 mL serum bottle sealed with butyl rubber stoppers and aluminium crimp seals. All cultures were incubated at 55°C in an incubator shaker at 200 rpm. When cultures displayed visual turbidity, 5 mL was withdrawn and centrifuged at 6300g for 5 min (Eppendorf Multipurpose Centrifuge 5804R). The pellet was then resuspended in 30 mL medium in a 250 mL serum bottle under the working condition specified previously (

In some enrichment series, the concentration of the ammonia (AMS) or nitrate (NMS) mineral salts was diluted twofold (1.0x versus 0.5x). The enrichments were performed under two

different headspace ratios of Air, CH4:CO2, and Ar:O2 mixture. For enrichment series A-H, the

vessels were subjected to vacuum, flushed with O2:Ar mixture (50% O2, 50% Ar) for 2 min to

56 restore atmospheric pressure, and supplemented with 30 mL of CH4:CO2 mixture (90% CH4,

10% CO2). Meanwhile, for enrichment series I-L, the vessels were filled with air and 30 mL of

CH4:CO2 mixture (90% CH4, 10% CO2).

Table 6.1). The headspace preparation was done as described above (with 60 mL of CH4:CO2 mixture added instead of 30 mL). Sampling was done periodically for turbidity measurement

(OD670) and gas consumption measurement.

The cultures were grown until the OD670 was in the range of 0.3-0.6. 25 mL of each culture was then withdrawn and prepared for a PHA accumulation test (as described in Section 3.1). The working conditions were maintained as shown in

10% CO2). Meanwhile, for enrichment series I-L, the vessels were filled with air and 30 mL of

CH4:CO2 mixture (90% CH4, 10% CO2).

Table 6.1 unless nitrogen was removed from the liquid and gas phase to screen for PHA production. All vessels were vacuumed, flushed with O2:Ar mixture (50% O2, 50% Ar), and supplemented with 60 mL of CH4:CO2 mixture (90% CH4, 10%CO2). An operating volume at this stage was 25 mL in a 250 mL serum bottle. The test was operated for 72 h in an incubator shaker (200 rpm) at 55°C. After 72 h, samples were collected for PHA analysis via fourier- transform infrared spectroscopy (FTIR) as described in Section 3.3.2.

6.2.3 Methanotrophic enrichment in semi-continuous reactors

The culture with the strongest signal of PHA determined by FTIR was used as an inoculum for the next enrichment experiment in a semi-continuous reactor. A seed volume of 5 mL was inoculated in 250 mL serum bottles filled with 25 mL of media containing: 2 mM

MgSO4.7H2O, 2.5 mM KNO3, 1 mM KH2PO4, 0.2 mM Na2HPO4.12H2O, 0.2 mM

CaCl2.6H2O, trace elements 0.36 μM FeSO4.7H2O, 0.0015 μM ZnSO4.7H2O, 0.0075 μM

MnSO4.7H2O, 2.4 μM H3BO3, 0.1 μM CoCl2.6H2O, 2.5 μM CuCl2.2H2O, 0.004 μM

NiCl2.6H2O, 0.005 μM Na2MoO4.2H2O, 0.85 μM EDTA and vitamins 0.04 μM biotin, 0.0225

57 μM folic acid, 0.075 μM thiamine hydrochloride, 0.11 μM D-pantothenic acid, 0.00035 μM vitamin B12, 0.065 μM riboflavin, and 0.4 μM nicotinamide. The pH of the medium was maintained at 6.8-7.0 by phosphate buffer (10 mL of 0.19M KH2PO4 and 0.23M

Na2HPO4.7H2O per L).

When the culture reached a turbidity (OD670) of 0.6, cell liquor suspension was distributed equally, inoculated in triplicates, and subjected to a long-term cyclic feeding and wasting regime, with alternating pulses of methane and nutrients as described in Section 3.1. The operational solids retention time (SRT) was assigned at 10.5 days before being reassigned to 4.0 days on Day 200. It should be noted that the operating volume of bioreactors was doubled from 30 to 60 mL on Day 475. Prior to each feeding regime, the medium was heated to 55°C with a thermostat incubator. The vessels were sealed with butyl rubber stoppers and aluminium crimp seals, vacuumed, flushed with O2:Ar mixture (50% O2, 50% Ar), and supplemented with

60 mL of CH4:CO2 mixture (90% CH4, 10%CO2). All vessels were incubated at 55°C in an incubator shaker at 200 rpm.

6.2.4 Metabolic potential of the thermophilic bioreactor community

The metabolic potential of the bacterial community in the semi-continuous reactor was estimated using Illumina high-throughput shotgun sequencing on Day 169. Metagenomics sequencing generated 4Gb of paired-end data with the reads assembled using the de novo assembly method using Metaspades and population genomes assembled using Metabat2.0 both with default settings. Quality of both contigs, scaffolds, and metagenome-assembled genomes (MAGs) was improved using a combination of GapCloser, FinishM, and RefineM programs. The assembled genomes were taxonomically classified using a combination of CheckM classifier [184] and Genome Tree Taxonomy Database methods [183]. The capacity of the microbial community from parent reactors to form PHA was estimated using the GraftM package graft function [185] to search for the PHA biosynthesis taxonomic marker gene, phaC, from both assembled MAGs and those genes not assembled into genomes. The genes detected by GraftM were aligned with database sequences for phaC from organisms that are known to form PHA, visualized using maximum likelihood taxonomic tree drawing methods, and bootstrapped using 100 iterations.

58 6.2.5 Culture isolation and purity test

Isolation of methanotrophic bacteria was attempted by using repeated sterile serial decimal dilution series and antibiotic treatment of erythromycin, demonstrated by Bodrossy et al (1997) and Danilova et al (2016) [120, 221]. Dilution series took place in 250 mL serum bottles with 30 mL of medium preheated to 45°C with headspace at atmospheric pressure consisted of methane with 1% oxygen, 0.5% carbon dioxide [221]. The growth of the methanotroph in the dilution series was tracked based on the disappearance of methane in the headspace, with the highest dilution showing methane disappearance being subcultured into subsequent dilutions series. After several serial decimal dilution series, erythromycin (10 μg mL-1) was added to inhibit the growth of non-methanotrophic contaminants [121]. When a single organism was presented based on microscopy, DNA extraction was performed using the Fast DNA®SPIN Kit for Soil (MO BIO Laboratories, Inc.) according to the manufacturer’s protocols. Extracted DNA was sent to the Australian Centre for Ecogenomics (ACE) sequencing facility for Illumina iTag sequencing and data processing as described in Section 3.4.

6.2.6 Cultivation condition of the isolate

The methanotrophic isolate was grown under sterile conditions in 30 mL of modified nitrate mineral salts (NMS) that contained the following contents: 0.5 g KNO3, 0.5 g MgSO4∙7H2O,

0.07 g CaCl2∙2H2O, 0.14 g KH2PO4, 0.36 g Na2HPO4.12H2O, 1 mL of trace elements solution -1 and vitamin solution. The trace elements solution contained (g L ) 0.5 g Na2-EDTA, 0.2 g

FeSO4∙7H2O, 0.01 g ZnSO4∙7H2O, 0.0042 g MnSO4∙7H2O, 0.3 g H3BO3, 0.2 g CoCl2∙6H2O,

0.314 g CuSO4∙5H2O, 0.002 g NiCl2∙6H2O, and 0.003 g Na2MoO4∙2H2O. The vitamin solution contained 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 per litre. The pH of the medium was maintained at 6.8-7.0 by phosphate buffer (10 mL of 0.19M KH2PO4 and 0.23M

Na2HPO4.7H2O per 1 L of media). This recipe was modified from Bodrossy et al (1997) and Danilova et al (2016) [120, 221], and differ from the recipe used in Section 6.2.3. All cultures were incubated in 250 mL serum bottles (DWK Life Sciences Wheaton, USA) capped with butyl-rubber stoppers and crimp-sealed at atmospheric pressure that consisted of methane with 1% oxygen, 0.5% carbon dioxide. Prior injection, gas was filtered through a 0.22 µm filter unit (Polyethersulfone Millex-GP Syringe Filter Unit, Merck, US). Bottles filled with media were autoclaved before use and incubated horizontally in a shaker incubator at 200 rpm. The incubation temperature was 45°C [120].

59 30 mL cultures were grown to the final optical density (OD670) of 0.3 and centrifuged at 6300g for 5 min (Eppendorf Multipurpose Centrifuge 5804R). The pellets were suspended in 30 mL of NMS media to create inoculum to replicate 250 mL serum bottles. Each culture received 5 mL inoculum plus 25 mL of fresh media. After growth at 45°C for 48 h, all cultures were subculturing by repeating the described procedure. When the culture showed a stable growth

(OD670), two sets of cultures were prepared: (i) one was simultaneously grown at 45°C, while (ii) subjected to a consequent elevation of temperature risen by 2°C every 48 h until reached 55°C.

6.2.7 Fluorescence in situ hybridization

Microbial cells were visualized using fluorescent in situ hybridization (FISH) staining by applying the method developed by Amann et al. (2001) [222]. A general bacterial probe EUB338 (5’-GCTGCCTCCCGTAGGAGT-3’) and a gammaproteobacterial specific probe GAM42a (5’-GCCTTCCCACATCGTTT-3’) was used targeting the Bacteria domain and Methylocaldum sp., respectively.

6.2.8 PHA accumulation in pure isolate

The isolates were grown to final optical density (OD670) of 0.3 and then harvested by centrifugation (6300g for 5 min by Eppendorf Multipurpose Centrifuge 5804R). Using aseptic technique, the pallets were suspended in 30 mL of fresh media in the absence of nitrogen. The headspace consisted of methane with 1% oxygen, 0.5% carbon dioxide. The rubber septum was placed on top of the vessel during gassing and rapidly sealed with a septum while removing the nozzle. The septa seal at the top of the vessel was tightened with aluminum caps. Some media was amended with 100 ppm mixture of acetic acid (C2), butyrate acid (C4), and hexanoic acid (C6), 100 ppm of propionic acid (C3), or 100 ppm valeric acid (C5) (Sigma-Aldrich, USA) at 45 and 55°C in the presence and absence of methane in order to assess the effects of co- substrate addition on PHA synthesis. The headspace was prepared as mentioned above (section 6.2.6); however, in the absence of methane, the serum bottle was added with argon gas (100% Ar) instead of methane. After 48 h incubation, cells were harvested for PHA analysis by using gas chromatography.

60 6.3 Results and discussion

6.3.1 Selection experiment

Table 6.2 summarizes the specific consumption of methane and total suspended solids (TSS) for all enrichment series measured after a 20-day of incubation. Overall, enrichment showed an increase in turbidity; however, only the B, G, H, and L series showed measurable optical density and methane consumption. Of the three conditions varied, the pH was probably the most important factor in selection for methanotrophs as three out of four successful enrichment series (B, H, and L) were done at pH 7.0.

The highest specific rate of methane consumption was obtained from the 30-cm depth soil (H2) -1 grown under the H series (0.5x NMS at pH 7.0) at 0.2 mg CH4 mg TSS day , followed by 0.2 -1 mg CH4 mg TSS day from the B series (1.0x AMS at pH 7.0) of the same sample. Consistent with methane consumption, most of the enrichment series of 30-cm depth soil (H2) provided higher biomass concentration than surface sample (H1). For example, 0.5 and 0.6 mg TSS mL-1 from B and H series of H2 in comparison with 0.2 and 0.5 mg TSS mL-1 from the same enrichment series of H1 sample. To determine an ability of PHA formation, the B, G, and H series of both H1 and H2 samples grown under balanced-growth conditions were incubated without nitrogen to induce PHA production. The L series was excluded because of low biomass. An FTIR spectrum obtained from selected thermophilic enrichments after the 72-h PHA accumulation phase is shown in Figure 6.2. All tested enrichment series did not show a significant peak of carbonyl at 1728-1738 cm-1, corresponding to the ester carbonyl group of PHA, therefore there was limited evidence of PHA accumulation. However, the strongest evidence of trace levels of PHA was observed in the H series of H2 sample, which was enriched from 30-cm depth soil with nitrate in 0.5x NMS at pH 7.0. For this reason, the H series of H2 sample was selected for enrichment experiment in a semi-continuous reactor and further evaluation of PHA production capability.

61 Table 6.2 Specific methane consumption rate of 24 methanotrophic enrichment series after a 20-day of incubation at 55°C. Enrichment Media Total suspended solids Specific Methane Consumption Samples Nitrogen sources Headspace pH -1 -1 -1 series concentration (mg TSS mL ) (mg CH4 mg TSS day ) H1A 5.0 mM NH Cl O :CH 5.0 n/a n/a 4 2 4 1.0x AMS H1B 5.0 mM NH4Cl O2:CH4 7.0 0.18 0.18 H1C 2.5 mM NH Cl O :CH 5.0 n/a n/a 4 2 4 0.5x AMS H1D 2.5 mM NH4Cl O2:CH4 7.0 n/a n/a H1E 5.0 mM KNO O :CH 5.0 n/a n/a 3 2 4 1.0x NMS Surface soil H1F 2.5 mM KNO3 O2:CH4 7.0 n/a n/a sample (H1) H1G 5.0 mM KNO O :CH 5.0 0.16 0.10 3 2 4 0.5x NMS H1H 2.5 mM KNO3 O2:CH4 7.0 0.49 0.20 H1I N Air:CH 5.0 n/a n/a 2 4 1.0x MS H1J N2 Air:CH4 7.0 n/a n/a H1K N Air:CH 5.0 n/a n/a 2 4 0.5x MS H1L N2 Air:CH4 7.0 0.17 0.05 H2A 5.0 mM NH Cl O :CH 5.0 n/a n/a 4 2 4 1.0x AMS H2B 5.0 mM NH4Cl O2:CH4 7.0 0.52 0.07 H2C 2.5 mM NH Cl O :CH 5.0 n/a n/a 4 2 4 0.5x AMS H2D 2.5 mM NH4Cl O2:CH4 7.0 n/a n/a H2E 5.0 mM KNO O :CH 5.0 n/a n/a 3 2 4 1.0x NMS 30-cm depth H2F 2.5 mM KNO3 O2:CH4 7.0 n/a n/a sample (H2) H2G 5.0 mM KNO O :CH 5.0 0.46 0.11 3 2 4 0.5x NMS H2H 2.5 mM KNO3 O2:CH4 7.0 0.62 0.12 H2I N Air:CH 5.0 n/a n/a 2 4 1.0x MS H2J N2 Air:CH4 7.0 n/a n/a H2K N Air:CH 5.0 n/a n/a 2 4 0.5x MS H2L N2 Air:CH4 7.0 0.17 0.06 n/a – not available; the condition highlighted in red was the condition selected for enrichment experiment.

62 0.04 A). 0.02

0.00 Absorbance -0.02 1900 1850 1800 1750 1700 1650 1600 1550 1500 Wavelength (cm-1)

0.04 B). 0.02

0.00 Absorbance -0.02 1900 1850 1800 1750 1700 1650 1600 1550 1500 Wavelength (cm-1)

0.04 C). 0.02

0.00 Absorbance -0.02 1900 1850 1800 1750 1700 1650 1600 1550 1500 Wavelength (cm-1)

0.04 D). 0.02

0.00 Absorbance -0.02 1900 1850 1800 1750 1700 1650 1600 1550 1500 Wavelength (cm-1)

Figure 6.2 FTIR Scans for selected thermophilic enrichment series after the 72-h PHA accumulation phase under nitrogen-deficient condition. Grey area indicates a peak area of the carbonyl group of PHA. (A) H1 sample in enrichment H series. (B, C, and D) are H2 sample in enrichment series of G, B, and H, respectively. See

63 In some enrichment series, the concentration of the ammonia (AMS) or nitrate (NMS) mineral salts was diluted twofold (1.0x versus 0.5x). The enrichments were performed under two different headspace ratios of Air, CH4:CO2, and Ar:O2 mixture. For enrichment series A-H, the vessels were subjected to vacuum, flushed with O2:Ar mixture (50% O2, 50% Ar) for 2 min to restore atmospheric pressure, and supplemented with 30 mL of CH4:CO2 mixture (90% CH4,

10% CO2). Meanwhile, for enrichment series I-L, the vessels were filled with air and 30 mL of

CH4:CO2 mixture (90% CH4, 10% CO2).

Table 6.1 for specific details of each enrichment series.

6.3.2 Methanotrophic enrichment in semi-continuous reactors

Figure 6.3 illustrates total methane consumption and specific substrate utilization of thermophilic enrichment in semi-continuous reactors over 750 days of operation, which can be divided into three phases according to operational solids retention time (SRT) and working volume: Phase 1 (Day 0-220), a 10.5-day SRT; Phase 2 (Day 220-475), a 4-day SRT with 30 mL operating volume; and Phase 3 (Day 475-750), a 4-day SRT with 60 mL operating volume.

-1 Overall, there were fluctuations and variations in total methane consumption (μmol CH4 day ) between three periods of the operation. A specific consumption rate of methane was therefore determined, measured as total methane consumed divided by total suspended solids (TSS) -1 -1 concentration on a mass basis (mg CH4 mg TSS day ). At the initial stage of the operation (Phase 1), there was an increase in average specific methane consumption from 0.1±0.0 mg -1 -1 -1 -1 CH4 mg TSS day on Day 90 to 0.4±0.1 mg CH4 mg TSS day on Day 203, before a period -1 -1 of stability at 0.5±0.1 mg CH4 mg TSS day throughout Phase 2. In Phase 3, although it showed a higher total consumption of methane, an average of the specific consumption rates showed no significant difference between Phase 2 and 3. The specific consumption rate of this -1 -1 -1 phase was 0.4±0.13 mg CH4 mg TSS day with 0.4±0.1 mg TSS mL . When the amount of substrate consumption and the concentration of total suspended solids (TSS) (as indicated by OD), at the end of each feeding cycle were constant, kinetic parameters were measured. It was found that the exponentially growing methanotrophic enrichment doubled every 2.8 days with specific growth rate 0.25 day-1.

64 1600 1.6

1400 1.4 ) 1 - day 1 1200 1.2 - ) 1 - mg TSS

1000 1.0 4

800 0.8

600 0.6 Methane Consumption (umol day 400 0.4 Specific Methane Consumption (mg CH 200 0.2

0 0.0 0 50 100 150 200 250 300 350 400 450 500 550 600 650 700 750 Days

Figure 6.3 Total methane consumption rate and specific consumption rate of thermophilic enrichment in semi-continuous reactors. (ᴏ), (∆), (□) -1 -1 -1 represents total methane consumption (μmol CH4 day ), and (●), (▲), (■) represents specific consumption of methane (mg CH4 mg TSS day ) of Phase 1 (Day 0-220), Phase 2 (Day 220 -475, and Phase 3 (Day 475-750). The trendlines represent a direction of specific consumption rates. Error bar represents the standard error of three replicates. 65 6.3.3 PHA production in the thermophilic community

Over the 750 days of operation, biomass collected at the end of the growth cycle was periodically used for PHA production. Under the nitrogen-deficient condition, an average of 3.3±0.3 wt% PHB was obtained during Phase 2 (Day 220-475), while 0.2±0.1 wt% PHB was obtained during Phase 3. This demonstrated that the thermophilic culture was able to produce the homopolymer PHB from methane at 55°C. Figure 6.4 shows images of PHA-containing cells stained with Nile Blue A. It is striking that PHA was detected in multiple cell morphologies in the parent reactor. To further investigate the number of organisms in the parent bioreactor with the potential to form PHA, DNA from the parent reactor was subjected to 16S rRNA sequencing for bacterial identification (Section 6.3.4), and, in addition, a high- throughput sequencing and subsequent assembly of Metagenomics Assembled Genomes (MAGs) was performed for a sample taken on Day 169 (Section 6.3.5).

Figure 6.4 Bright-field photomicrograph of PHA-accumulating thermophilic biomass overlaid with Nile Blue A stain for PHA viewed under fluorescence.

6.3.4 Monitoring of the microbial community structure in semi-continuous reactors

Microbial profiling in semi-continuous reactors was monitored using a 16s rRNA sequencing (Figure 6.5). Microorganism names were assigned based on various operational taxonomic units (OTUs). All communities collected at different time points were classified as members of phyla Proteobacteria, Bacteroidetes, Chloroflexi, Acidobacteria, Armatimonadetes, Deinococcus, and Planctomycetes.

66 Proteobacteria was found to be the major member of the community at the phylum level (47.2±5.5% relative abundance). It is well known that almost all methanotrophs belong to this phylum. A methanotroph within the Gammaproteobacteria class was the most abundant genera with a relative abundance of 32.1±4.0% of total DNA sequences identified. This methanotroph showed 100% similarity with Methylocaldum szegediens, a phaC-possessing bacterium. Methylocaldum has an ability to metabolize methane as a carbon and energy source by possessing a simultaneous operation of three C1 assimilation pathways: the RuMP cycle, the serine pathway, and the Calvin-Benson-Basham (CBB) cycle [106, 113, 120, 219, 220, 223]. Of most interest is the serine pathway, which is known for carbon assimilation for PHA production in methanotrophs [38]. Additionally, there are unidentified methylotrophic and methanotrophic members from the order Rhizobiales accounting for 11.2% of the population on Day 114. Some genera of methylotrophs and methanotrophs such as Methylobacterium, Methyloceanibactes, and Methylococcus phylogenetically belong to these orders.

The phylum Bacteroidetes is the second most dominant organism (21.6±7.0%) in the culture. It is a group of uncultivated microorganisms broadly distributed in thermal environments [224]. Sometimes this phylum is grouped with Chlorobi in phylogenetic trees, indicating a close relationship between two phyla [225]. They are known to grow with lysed biomass as the sole carbon source [226-228]. Although their metabolic capacity is limited and not fully understood, their relationships with other microorganisms during biomass deconstruction in thermophilic consortia have been reported [229, 230]. Throughout this experiment, sequences related to the orders of OPB56 and Ignavibacteriales were presented simultaneously.

Also present were Planctomycetes, Acidobacteria, Armatimonadetes, Chloroflexi, and Deinococcus. Together these made up for 26.9±4.0% relative abundance. Most of these phyla are commonly found in diverse environmental habitats excluding Deinococcus, a phylum of extremophilic bacterium found in hazardous or extreme conditions. Planctomycetes are chemolithoautotrophs which have been reported having a close relationship with methylotrophs and methanotrophs [231]. They possess two unique metabolic features: (i) they possess genes encoding enzymes for formaldehyde detoxification, which had previously been found in only methane-generating archaea and a methane-oxidizing group of Proteobacteria, and (ii) these microorganisms oxidize ammonium to dinitrogen using nitrite as an electron acceptor, with accompanying reduction of carbon dioxide [232]. However, Planctomycetes are not strict chemolithoautotrophs [233-235]. Instead, they are capable of using a variety of

67 organic compounds including carboxylic acids, for example, formate, acetate, propionate, and methylamines [236].

The presence of various microorganisms in the methane-driven consortia shows that methanotrophs are able to provide secondary carbon substrates for non-methane utilizers to survive. Nevertheless, these bacteria possibly contribute to PHA accumulation in the culture since members of the phyla including Alpha-, Beta-, Gamma-, Delta-proteobacteria, Bacteroidetes and Chloroflexi have been shown to accumulate PHA [237].

100%

90% g__Methylocaldum p__Bacteroidetes 80% o__Rhizobiales 70% p__Deinococcus p__Actinobacteria 60% p__Armatimonadetes p__Chloroflexi 50% p__Planctomycetes c__Betaproteobacteria 40% f__Comamonadaceae g__Thermomonas 30% o__Burkholderiales %Relative abundance o__Methylophilales 20% o__Rhodospirillales 10% Other

0% 55 90 114 321 482 524 599 630 700 Phase 1 Phase 2 Phase 3 Operating Day

Figure 6.5 The relative abundances of bacterial 16S rRNA genes expressed as % of OTUs in methanotrophic enrichment grown in semi-continuous reactors at 55°C (Table A3).

6.3.5 Metabolic potential of the thermophilic bioreactor community

The microbial community and metabolic potential for PHA formation in the thermophilic parent reactor were analyzed using metagenomically assembled genomes (MAGs) produced from the reactor on Day 169. A total of 39 MAGs were generated with 22 being of high quality (>70% CheckM quality metric) and accounted for approximately 83% of the community (Table A4). A further 17% of the sequencing data remained unmapped with the remainder of the sequence data making up the low abundance or poor quality MAGs (Table A4).

By concatenated alignments of these MAGs across 122 bacterial marker genes, a diverse thermophilic community from range of classes including Alpha-, Beta-, Gamma-, and Delta-

68 proteobacteria, Acidobacterriaceae, Ignavibacteria, Chlorobi, Plantomycetales, Actinobacteria, Deinococci, Chloroflexus, Caldilineae, and Armatimonadetes was discovered (Figure 6.6). The three predominant community MAGs from the genera Chlorobi (27.1%, MAG_32), Methyloceanibacter (16.0%, MAG_30) and Methylocaldum (16.2%, MAG_10) made up ~59% of the community (Figure 6.6 and Table A4). The result corresponds with the 16s rRNA gene taxonomic results (Figure A2); however, they were reported as Bacteroidetes (28.1%), Rhizobiales (11.2%), and Methylocaldum (28.4%) on Day 114.

The diversity of thermophilic methanotrophs in the bioreactor was confirmed by the presence of a particulate methane monooxygenase gene operon (pmoA) (Figure A1). A total of three methanotroph pmoA genes were identified from assembled contigs using a custom pmoA GraftM package. Two of these sequences binned to MAGs from the class Gammaproteobacteria identified as Methylocaldum szegediense (MAG_10) and Methylothermus subterraneus (MAG_22). The third sequence belonged to the Methylococcus capsulatus (<1% community). However, no soluble (smo) or alternate particulate methane monoxygenases (xmo) were identified.

Eleven PHA synthase (phaC) gene sequences from class I, III, and IV were identified as assembled reads using a custom phaC GraftM package (Figure 6.7). All of these PHA gene- harboring organisms belong to the proteobacterial lineages. Four of these sequences binned to recovered MAGs spanning a range of bacteria with corresponding 16S rRNA gene sequences for the Rhodosprillum and Methyloceanibacter (group I), Chloroflexus (group IV) and Methylocaldum species (group III). Remaining phaC sequences assembled and binned to poor quality MAGs (Table A4) and were partially complete with BLAST identity (Figure 6.6). Interestingly, the Methylocaldum MAG phaC gene occurs in an operon with a phaE gene which is indicative of group III phaC genes and is distinct from the group I phaC genes from Type II methanotrophs (Figure 6.7).

Analysis of the metabolic capabilities of the PHA-forming Methylocaldum, Methyloceanibacter, and Methylococcus identified genes corresponding to methanotrophic carbon and energy metabolism pathways. A reconstruction of identified metabolic pathways of methanotrophs is illustrated in Figure 6.8. The presence of serine, rubisco monophosphate (RuMP), and Calvin-Benson-Basham (CBB) carbon assimilation pathways in the Methylocaldum methanotroph MAGs (Figure 6.8), along with pmo (Figure A1) and 16S rRNA gene (Figure A2) taxonomies confirm this organism is Type X methanotroph. 69 A complete pathway for methane oxidation to carbon dioxide via the C1-group carrier tetrahydromethanopterin (H4MPT) was also observed in the methanotroph MAGs (Figure 6.8). The two methanotrophs (Methylocaldum and Methylococcus) MAGs also possess the carbon dioxide fixation pathway (Figure 6.8). Only the Methyloceanibacter MAG encodes the ethylmalonyl-CoA (EMC) pathway; it does not encode genes for PHA biosynthesis. As mentioned, the Methylocaldum genome does encode PHA biosynthesis genes including taxonomic marker gene phaC which fell adjacent to a phaC sequence from a Methylocaldum isolate, Methylocaldum szegediense OR2 (Figure 6.7). However, this MAG does not encode the EMC pathway, which is typically associated with PHB biosynthesis in type II methanotrophs [238]. Instead, it has a β-oxidation pathway and partial glyoxylate pathway that is typical for this methanotroph type [219, 239], suggesting this organism is able to assimilate fatty acids as an alternative carbon source.

MAGs from all three methanotrophs contained complete NADH-quinone oxidoreductase, succinate dehydrogenase, cytochrome-bd oxidase, cytochrome-c oxidase and F-type ATPase genes required for oxidative phosphorylation in aerobic organisms (Figure 6.8). The presence of the high-affinity cytochrome-bd ubiquinol oxidase indicates that these organisms can grow under microaerophilic conditions. In addition, the presence of assimilatory nitrate reductase (nasDE) genes in these methanotrophs suggest they utilize nitrate as nitrogen source (Figure 6.8), which is consistent with the NMS media these organisms were cultivated on. The nitrogenase complex (nifDHKB), in addition, indicates their capability for nitrogen fixation. Nitrate, potassium, sulfonate, and ABC membrane transport systems were evident across the genomes (Figure 6.8).

Figure 6.8 Metabolic reconstruction of methanotroph and Methyloceanibacter MAGs from the thermophilic community Abbreviations: pMMO, particulate methane monooxygenase; sMMO, soluble methane monooxygenase; pXMO, particulate methane monooxygenase; XoxF, MDH, methanol dehydrogenase; MxaF, MDH, methanol dehydrogenase; NiFe, Nickel-Iron containing hydrogenases; I, Complex I NADH dehydrogenase; II, Complex II succinate dehydrogenase; III, Complex III cytochrome bc; IV cbb3, Complex IV cytochrome cbb3; IV cyd, Complex IV high affinity cytochrome C; Rnf, Rhodococcus nitrogen factor; Nif, Nitrogen fixation; LPS, lipopolysaccharide; H4F, tetrahydrofolate; H4MPT, tetrahydromethanopterin; CBB, Calvin-Benson-Basham cycle; EMC, Ethylmalonyl-CoA pathway; TCA, Tricarboxycyclic acid pathway; RuMP, Rubisco Monophosphate pathway; PHB, poly-(3-hydroxybutyrate biosynthesis pathway; FDH, formate dehydrogenase. 73 6.3.6 Culture isolation and purity check

The Methylocaldum phaC-positive methanotroph present in the PHA accumulation enrichment was targeted for isolation by using a combination of isolation strategies [120, 221, 242]. The combination of the extinction dilution and erythromycin treatment led to the isolation of the Methylocaldum strain; however, concentrations of methane and oxygen, as well as incubation temperature, are marked as important factors that influenced the isolation.

Many methanotrophs have been isolated from various habitats and shown to live preferentially under high methane and high oxygen levels [215, 243]. However, our attempt to isolate the Methylocaldum under such condition (methane: oxygen ratio of 1:1) failed. The presence of the high-affinity cytochrome-bd ubiquinol oxidase (Section 6.3.5) and previous findings [120, 215, 243, 244] are clues that this gammaproteobacterial Methylocaldum strain likely prefers microaerophilic conditions. This, therefore, led to the development of an axenic culture of Methylocaldum under low oxygen level. The culture was successfully developed under this condition.

Besides, temperature seems to be additional factor that influenced the isolation of Methylocaldum. The mixed culture grown at 55°C was used as an inoculum for the isolation experiment; however, the attempt to isolate the strain at 55oC failed. When the temperature was reduced to 45°C, the culture was successfully isolated. The isolated culture was later shown to maintain growth at both 45 and 55°C. Although further studies on the effect of temperature and oxygen availability are required, the results in this thesis are consistent with previous results. Bodrossy et al (1997) isolated two methanotrophic bacteria at 45°C, despite optimum growth temperatures above 50°C [120]. Similarly, Methylobacter vinelandii, a moderate thermophilic methanotroph grown in enrichments at 55°C, was isolated at 45°C [134, 245].

As a result, at both 45°C and 55°C, a single OTU obtained from amplicon sequencing of 16s rRNA confirmed a 100% similarity of the isolate Methylocaldum organism with the Methylocaldum szegediense OR2 isolated from a thermal spring (Figure 6.9) [120]. In addition, the concatenated marker proteins showed 100% identity between the two organisms (Tree 15 in Figure A2), but only 98% similarity across the whole genomes based on nucleotide sequence. A fluorescence microscopy image of the isolate Methylocaldum organism stained with a control universal Cy3-labeled EUB338 bacterial probe and a gammaproteobacterial specific probe labeled with FITC (GAM42a) is illustrated in Figure 6.10.

Figure 6.10 Images of the isolate Methylocaldum organism after serial dilutions and erythromycin treatment (left) and a Fluorescent in situ hybridization (FISH) image of the isolate stained with the Cy3-labelled control general EUB338 bacterial probe and FITC- labelled specific gammaproteobacterial GAM42a probe (right). The image was taken by Samantha McDonald (2018) [241]. Scale bars represent 5µm.

6.3.7 PHA production in the Methylocaldum isolate

The presence of phaC gene and β-oxidation pathway in the isolate Methylocaldum genome suggests that this methanotroph is able to form PHA from methane and fatty acids Section

75 6.3.5). Therefore, cells of the isolate Methylocaldum were harvested and fed with methane, acetate, propionate, butyrate, valerate, and hexanoate. These substrates were added to the nitrogen-free media at a concentration of 100-ppm and incubated at 45 and 55°C.

At 55°C, the Methylocaldum isolate showed no evidence of PHA formation in all tested conditions. But, at 45°C, when acetate, propionate, butyrate, valerate, and hexanoate were introduced, PHA was detected. The PHA content of the cells after 48 h in nitrogen starvation phase was found to be in the range of 3.2-4.6 wt%. No PHA was detected when fed with methane alone. No HV units were detected; the PHA was all PHB. Table 6.3 summarizes PHA content and composition obtained from gas chromatography analysis and PHA testing conditions done in this study.

The biosynthesis of PHA from fatty acids but not from methane suggests that the PHB metabolism in the Methylocaldum isolate is diverged from the conventional PHA-producing methanotrophs (type II) that have been shown to form the PHAs when fed methane or methane with either even- or odd- fatty acids [9, 10, 104, 246]. In Type II methanotrophs, PHA can be formed directly from methane through assimilation of acetyl-CoA by PHA biosynthesis genes via the shared ethylmalonyl-CoA (EMC) pathway [49-51]. The co-feeding substrates alternatively assimilate via unrevealed mechanisms of fatty acid oxidation and form CoA intermediates before incorporation into PHAs [40, 52].

A complete EMC pathway in the Methylocaldum isolate is absent. Its genome instead harbors both serine and CBB pathways as well as other pathways such as β-oxidation for carbon assimilation, suggesting non-conventional PHA-forming mechanisms in this organism (Figure 6.8). The Methylocaldum isolate has a phaC gene in an operon with a phaE gene (Figure 6.7), which is often associated with PHA formation via β-oxidation in organisms harboring this enzyme complex [247-249]. Therefore, in the Methylocaldum isolate, the presence of an (R)- specific enoyl-coenzyme A (CoA) hydratase identified formed β-oxidation would likely facilitate the conversion of CoA intermediates into precursors for the phaCE complex polymerase to form PHA.

76 Table 6.3 PHB production by the Methylocaldum isolate after 48 h incubation at 45°C in the presence and absence of methane with or without co-substrate addition. Co-substrate Methane Nitrogen PHA Content (wt%) 3HB Content (mol%) None Yes - - - Even-fatty acidsa Yes - 4.4 100.0 100-ppm - - - - Propionic acid Yes - 4.1 100.0 100-ppm - - 4.0 100.0 Valeric acid Yes - 3.2 100.0 100-ppm - - 4.6 100.0 a A mixture of acetic acid, butyrate acid, and hexanoic acid. The total concentration of acids was 100 ppm.

77 6.4 Conclusion

This chapter investigated PHA formation in a thermophilic methane-fed enrichment. The selection and enrichment criteria were established by testing combinations of nitrogen sources, pH, and nutrient concentration. The capability of PHA production from a C1-substrate was demonstrated in the mixed community. Genomic analyses of recovered metagenome- assembled genomes (MAGs) identified a PHA forming methanotroph based on the presence of PHA biosynthase genes. The methanotroph, which showed 100% similarity with the Methylocaldum szegediense OR2, was successfully isolated by using a combination of isolation strategies. Divergent from other methanotrophs, this methanotroph produced only PHB, and produced PHB from even- and odd-carbon number fatty acids but not from methane. The PHA mechanism of this methanotroph therefore seems to be closely related to fatty acid degradation rather than methane oxidation. In short, this study has shown – for the first time - that the Methylocaldum, a gammaproteobacterial methanotroph, can accumulate PHA and its metabolism is alternate from the traditional pathways seen in Type II methanotrophs.

78 Chapter 7 Thermophilic biotechnology for production of PHA with high ratio of 3-hydroxyvalerate (3HV) to 3-hydroxybutyrate (3HB)

A thermophilic enrichment capable of PHA production was successfully selected and enriched in Chapter 6, and its microbial composition was stable over long-term operation. In this chapter, cells harvested from the parent reactors on Day 630 were incubated under nitrogen deﬁcient conditions with various combinations of methane, propionic acid, and valeric acid, with the view to expand the range of PHAs synthesized. When the methane-fed enrichment was fed with methane - but deprived of nitrogen for 48 h - it only produced a low amount of 3- hydroxybutyrate (3HB). When fed with propionic acid or valeric acid – and deprived of nitrogen for 48 h - 3-hydroxyvalerate (3HV) was additionally produced. This was true regardless of whether methane was supplied or not. The 3HV fractions in the PHA ranged from 15-99 mol%. The rate of 3HV formation depended on the types of co-substrate added. The addition of acetic acid, a precursor for hydroxyalkanoate (HA) units, increased the synthesis of 3HB units, but not 3HV. Degradation of PHA polymer was noticed following the exhaustion of carbon feeds, especially methane. The experiment was conducted at 55 and 58°C; however, the temperature did not have a significant effect on the PHA production.

7.1 Introduction

The copolymer of 3-hydroxybutyrate (3HB) and 3-hydroxyvalerate (3HV), called poly(3- hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), has attracted industrial attention as a practical candidate to replace conventional plastics (i.e. polyethylene (PE) and polypropylene (PP)) [251]. This is because PHBV provides better mechanical properties than poly(3- hydroxybutyrate) (PHB) homopolymer yet retains biodegradability [2, 8, 19].

Compared to PHB, the copolymer has better impact strength, flexural modulus, and melting temperature [3, 5]. With higher 3HV content, the copolymer exhibits isodimorphism, is found to be tougher and has a lower melting point [6]. A high 3HV fraction copolymer has been produced by many microorganisms [5, 7-9]. The most common strategy for maximizing 3HV is feeding propionic acid and valeric acid [5]. Alcaligenes eutrophus (now called Ralstonia eutropha), for example, produces copolymers with 3HV fraction varying from 0 to 47 mol% 3HV from glucose and propionic acid [10, 11]. By integrating knowledge of the biosynthetic pathways of A. eutrophus, Doi et al (1988) maximized the proportion of 3HV up to 90 mol% 3HV, thus providing wider opportunities for commercialization [9].

79 Methylocystis parvus OBBP and Methylosinus trichosporium OB3b, on the other hand, are methane-utilizing microorganisms known for their PHB synthesis capabilities [9, 10, 48, 49, 108, 109, 152, 252]. Myung et al. (2017) have demonstrated their capacity to produce PHBV copolymer with 3HV contents in a range from 0 to 22 mol% from methane, propionic acid, and valeric acid [9]. However, the low content of 3HV in the biomass makes the production processes and downstream processing expensive, consequently restricting the application of the polymer on an industrial scale [253]. As a production challenge, the enhancement of the 3HV content of PHBV and a rethinking of the process for thermophilic PHA production are being considered [2, 4, 6, 254].

The previous chapter (Chapter 6) considered the production of PHB by a thermophilic methanotroph. In this chapter, an expansion of the range of PHAs synthesized by the thermophilic culture from which the methanotroph was isolated is studied. The effects of different 3HV producing substrates and of culture conditions for 3HV biosynthesis are investigated. In addition, the supplementation of acetic acid to maximize the pool of acetyl- CoA, a limiting intermediate for both 3HB and 3HV formation, is trialled. Finally, the performance of PHA production is evaluated at higher temperatures (55 and 58°C), so as to further minimise cooling water costs for heat removal.

7.2 Materials and methods

7.2.1 Culture condition

All experiments were conducted using the thermophilic mixed culture from Chapter 6 harvested on Day 630. Forty-eight replicates of the culture were established in 250-mL serum bottles (Wheaton Science Products, Millville, NJ) as illustrated in Figure 7.1. Each bottles contained 60 mL of media as described in Section 6.2.4. The combined culture volume of the replicates was three litres. The gas feeding and composition and incubation condition of these replicates is described in the Section 6.2.4.

7.2.2 PHB and PHBV production

All replicates were grown in the presence of methane and nitrogen to exponential phase (Figure 7.1; Step 5) before being collected by centrifugation at 6300 g for 5 min (Eppendorf Multipurpose Centrifuge 5804R, Germany), and being washed twice with nitrogen-free media, and resuspended in 60 mL nitrogen-free to induce PHA production. A summary of sixteen conditions tested in this study was shown in Table 7.1. All of these conditions were divided

80 into three groups for three comparative assessments as shown in Section 7.2.2.1, 7.2.2.2, and 7.2.2.3. The results of these assessments were later discussed in Section 7.3.2, 7.3.3, and 7.3.4, respectively, and all information and figures related to each assessment were given in its own section. No cross comparison between sections is required.

7.2.2.1 Effect of temperature and odd-fatty acids on PHA production in the presence of methane To assess PHB and PHBV production and determine the effect of temperature on the production, eight groups of serum bottles (four at 55°C and four at 58°C) were established as following: methane alone (M1C), methane with 100-ppm propionic acid in the medium (M1P, 101 µL per L medium, Sigma–Aldrich, USA), methane with 100-ppm valeric acid in the medium (M1LV, 106 µL per L medium, Sigma–Aldrich, USA), and methane with 200-ppm valeric acid in the medium (M1HV, 212 µL per L medium, Sigma–Aldrich, USA). These bottles were vacuum evacuated, flushed with O2:Ar gas mixture (50% O2, 50% Ar), and injected with 60 mL CH4:CO2 (90% CH4, 10% CO2) before being incubated at 55°C and 58°C in shaking incubators at 200 rpm for 48 h. The pH of the medium was controlled at 6.8-7.0 by phosphate buffer (10 mL of 0.19M KH2PO4 and 0.23M Na2HPO4.7H2O per L).

7.2.2.2 PHA production in the absence of methane To evaluate the effect of methane on PHA production, the following conditions were additionally prepared: no methane and no fatty acid supplementation (M0C), no methane with 81 100-ppm propionic acid in the medium (M0P), no methane with 100-ppm valeric acid in the medium (M0LV), and no methane with 200-ppm valeric acid in the medium (M0HV). These bottles were vacuum evacuated, flushed with O2:Ar gas mixture (50% O2, 50% Ar), and, instead of methane injection, 60 mL of argon gas (100% Ar) was added. Argon was used in this chapter, instead of nitrogen gas used in Chapter 4 and Chapter 5, to minimise the influence of nitrogen gas on PHA accumulation and to maintain partial pressure of gases in headspace. Addition of argon gas provides a constant gas pressure in all bottles and These bottles were incubated at 55°C in shaking incubators at 200 rpm for 48 h.

7.2.2.3 Effect of acetic acid on PHA production The remaining bottles were divided into 4 groups: 100-ppm acetic acid only (M0A, 95.2 µL per L medium, 100% glacial, Merck, Germany), 100-ppm acetic acid with methane (M1A), 100-ppm acetic acid with 100-ppm propionic acid (M0AP, 95.2 µL 100% glacial acetic acid, Merck, Germany and 101 propionic acid, Sigma–Aldrich, USA per L medium), and 100-ppm acetic acid with 100-ppm propionic acid and methane (M1AP), to examine how adding acetyl- CoA precursor influences PHA synthesis at 55°C. These bottles were vacuum evacuated and flushed with O2:Ar gas mixture (50% O2, 50% Ar). The treatment with methane presence was injected with 60 mL CH4:CO2 (90% CH2, 10% CO2). The treatment with methane absence was injected with 60 mL of argon gas (100% Ar). These bottles were incubated at 55°C in shaking incubators at 200 rpm for 48 h.

All testing conditions in this study were run in triplicate. 5-10 mL samples were withdrawn every 24 h for optical density (OD670) measurement, analysis of volatile fatty acid (VFAs) concentrations, and PHA content and composition analysis. The sample was immediately centrifuged at 10,000g for 5 min (Sigma 1-14 Microcentrifuge, Germany). Pellets were oven dried overnight at 70°C for PHA analysis and the supernatant was stored separately at 4°C for VFAs analysis.

7.2.1 Statistical analysis All testing conditions and chemical analyses were run in triplicate. A two-way factorial analysis was performed using Minitab (Version 18, Minitab Inc.) to test for significant differences and quantify main effects at α=0.05 (95% confidence level). The statistics used in the comparison of means are given in Appendix (Table A5 - Table A7). The significance of the differences in temperature, methane availability, and types of co-substrate and concentrations are discussed in the text.

82 Table 7.1 A summary of PHA accumulation conditions at 55°C and 58°C with and without methane availability and different supplementation of organic acids. Carbon available during PHA accumulation Operating Liquid Headspace Headspace composition No. Code* Methane Propionic acid Valeric acid Acetic acid temperature (°C) volume (mL) volume (mL) (% by volume) 1 55M1C Yes - - - 55°C 60 190 2 55M1P Yes 100-ppm - - 55°C 60 190 3 55M1LV Yes - 100-ppm - 55°C 60 190

4 55M1HV Yes - 200-ppm - 55°C 60 190 21.6% CH4, 2.4% CO2,

5 58M1C Yes - - - 58°C 60 190 38% O2, 38% Ar 6 58M1P Yes 100-ppm - - 58°C 60 190 7 58M1LV Yes - 100-ppm - 58°C 60 190 8 58M1HV Yes - 200-ppm - 58°C 60 190 9 55M0C No - - - 55°C 60 190 10 55M0P No 100-ppm - - 55°C 60 190 38% O and 62% Ar 11 55M0LV No - 100-ppm - 55°C 60 190 2 12 55M0HV No - 200-ppm - 55°C 60 190

13 55M1A Yes - - 100-ppm 55°C 60 190 21.6% CH4, 2.4% CO2,

14 55M1AP Yes 100-ppm - 100-ppm 55°C 60 190 38% O2, 38% Ar 15 55M0A No - - 100-ppm 55°C 60 190 38% O and 62% Ar 16 55M0AP No 100-ppm - 100-ppm 55°C 60 190 2 *The abbreviations represent each testing conditions were used throughout this chapter.

83 7.3 Results

7.3.1 Microbial community in thermophilic enrichment

Microbial community analysis of the thermophilic mixed culture on Day 630 was performed to reveal community composition prior to the PHA accumulation test (Figure 6.5; Table A3). In short, Methylocaldum dominated the thermophilic culture at 29±5% relative abundance. Other major bacterial members presented in the culture were Bacteroidetes (21±7%) and Meiothermus from Deinococcus phylum (15±1%), with minor microorganisms accounting for the remaining bacteria (Table A3). The community of the thermophilic enrichment remain stable over PHA production period (data not shown). According to the genome directed metabolic analysis of metagenome-recovered genomes in Chapter 6, three microorganisms from Elioraea, Chloroflexus, and Methylocaldum genera identified with phaC gene were presented in the thermophilic culture (Figure 6.6 and Figure 6.7).

7.3.2 Effect of temperature and odd-fatty acids on PHA production in the presence of methane

Figure 7.2 illustrates total PHA, 3HB, and 3HV content on dry weight (wt%) and mole (mol%) basis when the thermophilic enrichment was grown under balanced-growth conditions at 55°C and 58°C and incubated without nitrogen to induce PHA accumulation in the presence and absence of odd-fatty acids. Eight different conditions were evaluated (four at 55oC and four at 58oC): methane alone (M1C), methane with 100-ppm propionic acid (M1P), methane with 100- ppm valeric acid (M1LV), and methane with 200-ppm valeric acid (M1HV) at 24 and 48 h.

When cultures were incubated with methane, only 3HB was produced. And although an increase in temperature appeared to result in a slightly increased PHA yield, from 0.2±0.1 wt% at 55°C (55M1C) to 0.3±0.2 wt% at 58°C (58M1C), there was no significant difference in the production of PHB when the temperature was increase.

When odd number fatty acid was fed along with methane, PHA content increased significantly and 3HV was synthesized. The highest final PHA content was 10±3 wt% obtained from the treatment with 200-ppm valeric acid at 55°C (55M1HV). The highest final 3HV content was 85±14 mol% obtained from the treatment with 100-ppm propionic acid at 58°C (58M1P). However, it should be noted that under the same conditions, 99±1 mol% 3HV was obtained within the first 24 h of the accumulation. Again, the increase in temperature did not result in a statistically significant difference in the rates of biosynthesis of PHA or of relative 3HV

84 formation; however, a slight decline in PHA contents (wt%) and an increase in final 3HV mole fraction (mol%) was observed. The final PHA contents reported from the treatments incubated at 55°C were in the range of 2.6±0.2 to 10±3 wt% with the highest 3HV mole fraction of 70±3 mol%. Meanwhile, among those treatments at 58°C, the highest PHA content of 8±1 wt% and the highest 3HV content of 85±14 mol% was obtained.

20 100 3HB and 3HV fraction (mol%) 3HB3HVand fraction 15 75

10 50

5 25 PHA, 3HB, and 3HV Content (wt%)

0 0 24h 48h 24h 48h 24h 48h 24h 48h 24h 48h 24h 48h 24h 48h 24h 48h

55M1C 58M1C 55M1P 58M1P 55M1LV 58M1LV 55M1HV 58M1HV Figure 7.2 PHA (♦), 3HB (●), and 3HV (▲) content presented on a mass basis (wt%) and 3HB (■) and 3HV (■) fraction on a mol basis (mol%) obtained during PHA accumulation phase in the presence of methane at 55 and 58°C. Error bars are standard deviations (n=3). Statistic letters denote significant differences within each analyte for 3HV and 3HB content with p<0.05 were derived in Table A5.

To further understand the effect of odd carbon number fatty acids and temperature on PHA metabolism, specific substrate consumption (methane, propionic acid, and valeric acid) and production (carbon dioxide, PHA, 3HB, 3HV, and biomass) rates at 24-h and 48-h were calculated on a carbon mole basis (Section 3.6) and are illustrated in Figure 7.3. The calculations reveal a small carbon flux distributed from consumed substrates to PHA formation in all conditions. The highest carbon flux to 3HB was observed in the systems fed only methane; meanwhile, a distribution for 3HV synthesis was noticed when odd-number fatty acids were fed along with methane. Although different patterns in carbon flux to 3HB and 3HV formation were observed, depending on types and concentrations of co-substrates, there was a no statistically significant difference as a function of temperature.

85 In the presence of a propionic acid-methane mixture (M1P), the formation rates of 3HV units dominated in the early stage of the PHA production (0-24 h), with 52±4 C-mmol 3HV C-mol X-1 at 55°C and 26±7 C-mmol 3HV C-mol X-1 at 58°C and with nearly zero 3HB production (Figure 7.3B). These rates dropped sharply to -12±1 C-mmol 3HV C-mol X-1 at 55°C and -6±2 C-mmol 3HV C-mol X-1 at 58°C treatments when no propionic acid was available in the later stage of the accumulation (24-48 h). 3HV degradation was likely, evidenced by a reduction in 3HV content on the mass basis, from 2.6±0.2 wt% to 1.8±0.3 wt% at 55°C and from 1.8±0.5 to 1.2±0.3 wt% at 58°C (Figure 7.3B). When propionic acid was completely consumed and 3HV monomers started to degrade, and an increase in the 3HB production rates was observed with no difference between temperatures. As a result, there was an increase in 3HB content on both mass basis and mole fraction by the end of the accumulation phase, with mole fraction reaching up to 30±3 mol% when incubated at 55°C.

A different pattern of 3HB and 3HV formation was observed when propionic acid was replaced by valeric acid in the presence of methane (M1LV and M1HV). The synthesis of 3HB and 3HV was co-occurring and started at the initiation of the accumulation phase (Figure 7.3C and Figure 7.3D). The highest 3HV rate (95±5 C-mmol 3HV C-mol X-1 and 29±2 C-mmol 3HB C-mol X-1) was obtained from the treatment with 200-ppm valeric acid at 55°C (55M1HV), followed in order by 100-ppm at 55°C (55M1LV), 200-ppm at 58°C (58M1HV), and 100-ppm at 58°C (58M1LV). Under all conditions, the formation rate of 3HV was about three times higher than for 3HB and all were higher than for the treatments with propionic acid. As a result, higher 3HV contents on a mass basis were obtained in treatments with valeric acid, yet lower mole fractions were observed because of the co-synthesis of 3HB and 3HV.

Overall, addition of valeric acid did significantly increase the PHA content on a dry mass basis (wt%) when compared to PHA accumulation on methane and propionic acid (Figure 7.2). However, an increase in the concentration of valeric acid did not significantly enhance the overall production rate, as there was no significant increase in the production rates of 3HB and 3HV. Nevertheless, it did apparently restrict the degradation of 3HV during the 24-48 h period of the accumulation phase, suggesting that the absence of odd-carbon-number fatty acids stimulates the consumption of 3HV. For example, the treatment with 100-ppm valeric acid provided an initial formation rate of 3HV at 93±5 C-mmol 3HV C-mol X-1, which later dropped sharply to -11±4 C-mmol 3HV C-mol X-1 at 55°C (Figure 7.3C). In contrast, when the culture was supplemented with 200-ppm valeric acid, the rate increased slightly to 95±5 C-mmol 3HV C-mol X-1 before sharply declining to 19±8 C-mmol 3HV C-mol X-1 at 55°C (Figure 7.3D).

86 Consequently, the final 3HV content of 6±2 wt% of 3HV was obtained from 200-ppm valeric acid addition when only 3.6±0.5 wt% of 3HV was obtained from the treatment with 100-ppm valeric acid (Figure 7.2).

A) 48 hr.

24 hr. 58M1C

48 hr.

24 hr. 55M1C

-500.0 -400.0 -300.0 -200.0 -100.0 0.0 100.0 200.0

B) 48 hr.

24 hr. 58M1P

48 hr.

24 hr. 55M1P

-500.0 -400.0 -300.0 -200.0 -100.0 0.0 100.0 200.0

C) 48 hr.

24 hr. 58M1LV

48 hr.

24 hr. 55M1LV

-500.0 -400.0 -300.0 -200.0 -100.0 0.0 100.0 200.0

48 hr. D)

24 hr. 58M1HV

48 hr.

24 hr. 55M1HV

-500.0 -400.0 -300.0 -200.0 -100.0 0.0 100.0 200.0 Specific consumption and production (C-mmol C-mol X-1)

87 7.3.3 PHA production in the absence of methane To investigate the role of methane in PHA production in the thermophilic community, a set of PHA accumulations with and without co-substrate supplementation was performed in the absence of methane. All testing conditions were prepared in triplicate to assess each of the following conditions: no methane and no fatty acid supplementation (M0C), no methane with 100-ppm propionic acid (M0P), no methane with 100-ppm valeric acid (M0LV), and no methane with 200-ppm valeric acid (M0HV). These conditions were evaluated in comparison with identical conditions in the presence of methane at 55°C.

Figure 7.4 illustrates total PHA, 3HB, and 3HV content on dry weight (wt%) and mole (mol%) basis obtained at 24 and 48 h of PHA accumulation in the presence and absence of methane at 55°C. Concentrations of propionic acid and valeric acid available in cultures are reported in parts per million (ppm) in Figure 7.5. The specific formation of PHA, 3HB, and 3HV and consumption rates of propionic acid and valeric acid are illustrated in Figure 7.6.

20 100 3HB and 3HV fraction (mol%) 3HB3HVand fraction 15 75

10 50

5 25 PHA, 3HB, and 3HV Content (wt%)

0 0 24h 48h 24h 48h 24h 48h 24h 48h 24h 48h 24h 48h 24h 48h 24h 48h

55M1C 55M0C 55M1P 55M0P 55M1LV 55M0LV 55M1HV 55M0HV

88 240 240 A) M1P B) M1LV M0P M0LV 200 200 M1HV M0HV 160 160

120 120

80 80 Valeric acid concentration (ppm)

Propionic acid concentration (ppm) 40 40

0 0 0 12 24 36 48 0 12 24 36 48 Time (Hr) Time (Hr)

Figure 7.5 The concentration of (A) propionic acid and (B) valeric acid expressed as percentage concentration (ppm) presented in cell suspension during 48-h PHA accumulation phase in the presence and absence of methane at 55°C. Error bars are standard deviations (n=3).

Overall, in the absence of methane, PHA content in the range of 0.5±0.0 to 3.8±0.7 wt% was observed with HV mole fractions being 63±1 to 79±3 mol% (Figure 7.4). Under the base accumulation process, when methane was removed and no co-substrates were available, 0.5±0.0 wt% PHB was observed with no significant difference in the PHB content when compared with the methane-fed condition.

When propionic acid was added in the absence of methane (M0P), PHA content increased to 3.6±0.1 wt% within 24 h, before reducing to 2.2±0.6 wt% at the end of the accumulation phase. In this condition, there was no significant difference in total PHA concentration in either the presence or absence of methane (Figure 7.4). However, there was a major difference in the pattern of 3HB and 3HV formation (Figure 7.6). The absence of methane enabled 3HB formation along with 3HV in the initial stage of PHA accumulation: 24±1 C-mmol 3HB C-mol X-1 and 21±1 C-mmol 3HV C-mol X-1 with a total production rate of PHA at 45±1 C-mmol PHA C-mol X-1 (Figure 7.6B). As a result, 55±1 mol% 3HB and 45±1 mol% 3HV was produced (Figure 7.4). The consumption rate of propionic acid fell significantly in the absence of methane (Figure 7.5A). In addition, the degradation rate of 3HB dramatically increased (Figure 7.6B), which leads to a decline in 3HB content from 2.0±0.1 to 0.8±0.2 wt% of 3HB within the last 24 h of the accumulation phase.

89 A) 48 hr.

24 hr. 55M0C

48 hr.

24 hr. 55M1C

-500.0 -400.0 -300.0 -200.0 -100.0 0.0 100.0 200.0

B) 48 hr.

24 hr. 55M0P

48 hr.

24 hr. 55M1P

-500.0 -400.0 -300.0 -200.0 -100.0 0.0 100.0 200.0

C) 48 hr.

24 hr. 55M0LV

48 hr.

24 hr. 55M1LV

-500.0 -400.0 -300.0 -200.0 -100.0 0.0 100.0 200.0

D) 48 hr.

24 hr. 55M0HV

48 hr.

24 hr. 55M1HV

-500.0 -400.0 -300.0 -200.0 -100.0 0.0 100.0 200.0 Specific consumption and production (C-mmol C-mol X-1)

90 When the culture was incubated with valeric acid with no methane (M0LV and M0HV), there was a significant decrease in PHA content, with 4.8±0.5 and 4.1±0.1 wt% PHA being obtained at 24 h the accumulation phase when fed with 100 and 200-ppm of only valeric acid, which is significantly lower than for the treatments fed with methane (Figure 7.4). At the end of the accumulation phase, the final PHA content was 3.7±0.6 and 3.8±0.7 wt%, respectively, when treated with 100-ppm and 200-ppm of valeric acid, respectively, in the absence of methane (Figure 7.4). The decline in PHA content was a result of (i) a significant drop in the formation rate of 3HV during the 0-24 h period and (ii) an increase in the degradation rate of 3HB during the 24-48 h period of the accumulation, which is possibly associated with the presence of methane. In the end, only 2.9±0.5 and 3.0±0.7 wt% 3HV was produced. The 3HB content reduced sharply from 2.3±0.2 to 0.9±0.1 wt% when treated with 100-ppm valeric acid and from 2.1±0.1 to 0.8±0.1 wt% when treated with 200-ppm valeric acid (Figure 7.4), giving no significant difference in the effect of co-substrate concentration on PHA production when methane was absent.

7.3.4 Effect of acetic acid on PHA production

Figure 7.7 illustrates the effects of addition of 100-ppm acetic acid on PHA production in the thermophilic enrichment fed with and without methane and propionic acid. Concentrations of acetic acid and propionic acid available in cultures are reported in parts per million (ppm) in Figure 7.8. The specific substrate consumption and production of PHA, 3HB, and 3HV on a carbon mole basis are illustrated in Figure 7.9. Four additional testing conditions were prepared as follows: 100-ppm acetic acid with and without methane (M1A and M0A), and 100-ppm acetic acid and 100-ppm propionic acid with and without methane (M1AP and M0AP), all at 55°C. These conditions were evaluated in comparison with the normal PHA accumulation process (M1C and M0C) and the addition of 100-ppm propionic acid (P) in the presence (M1) and absence (M0) of methane.

91 20 100 3HB and 3HV fraction (mol%) 3HB3HVand fraction 15 75

10 50

5 25 PHA, 3HB, and 3HV Content (wt%) 0 0 24h 48h 24h 48h 24h 48h 24h 48h 24h 48h 24h 48h 24h 48h 24h 48h

55M1C 55M0C 55M1P 55M0P 55M1A 55M0A 55M1AP 55M0AP

The production of PHB in a culture with acetic acid (M1A) was increased compared to that of a culture without acetic acid (M0A), either in the presence or absence of methane, at 2.4±0.2 and 1.0±0.1 wt% of total biomass, respectively. In the absence of methane (M0A), acetic acid was completely consumed within 24 h (Figure 7.8A), with associated PHB production, before the degradation of PHB took place after the exhaustion of exogenous carbon supplies. In the presence of methane (M1A), a comparable production rate of 3HB was observed, with a delay in PHB consumption. The culture under M1A conditions consumed acetic acid concurrently with methane at a lower rate compared with M0A and M1C condition. These results suggest that acetic acid and methane possibly provide a similar function as precursors for acetyl-CoA starting material for PHB production. The production rate of 3HB can be enhanced by the addition of acetic acid; however, the presence of methane can delay PHB degradation.

In the presence of methane and propionic acid, there was a positive trend in 3HB formation due to the presence of acetic acid: when 100-ppm acetic acid was added, there was a significant

92 increase in the 3HB production rate, with 33±8 C-mmol 3HB C-mol X-1 with methane (M1AP) and 49±8 C-mmol 3HB C-mol X-1 without methane (M0AP) (Figure 7.9D). In a culture amended with acetic acid and propionic acid, there was a significant decrease in the 3HV formation rate in either the presence or absence of methane, with the rate decreasing to 23±3 C-mmol 3HV C-mol X-1 in the absence (M0AP) and zero rate in the presence of methane (M1AP) at 24 h. This indicates that, in the presence of methane and propionic acid (M1P), 3HV formation was not limited by the pool of acetyl-CoA, as acetic acid addition did not increase either the total PHA or the 3HV content when compared with the M1AP run. However, acetic acid had a positive effect on the 3HB formation, enabling higher 3HB content in M1AP and M0AP conditions when compared with M1P and M0P.

180 180 A) M1A B) M1P M0A M0P 150 150 M1AP M1AP M0AP M0AP 120 120

90 90

60 60 Acetic acid concentration (ppm)

30 Propionic acid concentration (ppm) 30

0 0 0 12 24 36 48 0 12 24 36 48 Time (Hr) Time (Hr)

93 A) 48 hr.

24 hr. 55M1C

48 hr.

24 hr. 55M0C

-500.0 -400.0 -300.0 -200.0 -100.0 0.0 100.0 200.0

B) 48 hr.

24 hr. 55M1A

48 hr.

24 hr. 55M0A

-500.0 -400.0 -300.0 -200.0 -100.0 0.0 100.0 200.0

C) 48 hr.

24 hr. 55M1P

48 hr.

24 hr. 55M0P

-500.0 -400.0 -300.0 -200.0 -100.0 0.0 100.0 200.0

48 hr. D)

24 hr. 55M1A1P

48 hr.

24 hr. 55M0A1P

-500.0 -400.0 -300.0 -200.0 -100.0 0.0 100.0 200.0 Specific consumption and production (C-mmol C-mol X-1)

94 7.4 Discussion Thermophilic PHB and PHBV biosynthesis in a thermophilic culture – enriched on methane – was investigated and strategies for the production of PHBV with different 3-hydroxyvalerate (3HV) mole fractions were demonstrated. The production of PHB and PHBV was carried out using the thermophilic methane-utilizing mixed culture (dominant species Methylocaldum sp., 29±5% of total relative abundance) grown at 55°C in semi-continuous reactors, which were described in Chapter 6. The high-throughput metagenomics sequencing revealed three organisms harboring the phaC gene for PHA synthesis (Section 6.3.5; Figure 6.7). Corresponding to 16S rRNA gene sequences, these phaC sequences belong to Methylocaldum, Elioraea, and Chloroflexus sp (Section 6.3.5; Figure 6.7). Methylocaldum, Elioraea, and Chloroflexus sp. are known as moderate thermophiles growing in a range of temperatures between 41°C and 67°C [261-264] with an optimal growth temperature between 50°C and 55°C [106, 263-265]. In this work, net PHA accumulation at 55oC and 58oC was very similar.

The types of 3HV-inducing substrates used have an important role in determining total PHA content and 3HV content. While both propionic acid and valeric acid can be added to modify 3HV content, valerate addition commonly yields PHBV with a higher fraction of 3HV (mol%) and total PHA content (wt%) [9, 10, 213]. In this study, there was a significant increase in total PHA content obtained from valeric acid treatments, compared to propionic acid treatment, which is in accordance with previous studies [9, 10, 213]. However, in the presence of methane, the mole fraction of 3HV was nearly 100 mol% (after 24h accumulation) when propionic acid was fed. When valeric acid was fed, the 3HV mole fraction was only 73±2 mol%.

The production of PHV homopolymer from odd-number-carbon fatty acids has been demonstrated previously in literature [266-269], however, there is no clear explanation for the synthesis in the culture used in this study. In many bacteria, 3HV formation from propionic acid is restricted by a limited pool of acetyl-CoA, a crucial intermediary metabolite for the biosynthesis of PHA [124, 270-273]. However, in the present study, when thermophilic culture was exposed to both propionic acid and acetic acid in the presence of methane, PHA content did not increase. Instead, acetic acid suppressed 3HV formation and consumption of propionic acid, while 3HB productivity increased. As a result, only 19±1 mol% of 3HV was observed. This indicates that 3HV accumulation from propionic acid was not prevented by a deficiency in acetyl-CoA in this mixed culture.

95 While the genomic evidence revealed that there were three main organisms harboring the high- quality phaC gene for PHA biosynthesis in the thermophilic culture, the results in Chapter 6 show that the Methylocaldum was unable to synthesize 3HV units from propionate and valerate fatty acids under nitrogen-limited conditions (See section 6.3.7). In the axenic culture of Methylocaldum, only 3HB was formed, without a trace of 3HV units when propionic acid or valeric acid was fed either with or without methane. This indicates that, in the thermophilic mixed culture, (i) PHB may be indirectly formed by the Elioraea and Chloroflexus when methane served as the sole carbon source, and (ii), when propionic acid or valeric acid was present in the medium, Methylocaldum assimilates these co-substrates for 3HB formation while Elioraea and Chloroflexus may undertake both 3HB and 3HV biosynthesis, resulting in an increase in PHA productivity and content.

Although Methylocaldum did not play a direct role in 3HV production, its activity in the presence of methane apparently did affect the productivity of both the 3HB and 3HV monomers and the degradation of the polymer. When the thermophilic culture was exposed to propionic acid in the absence of methane, the 3HV fraction significantly decreased from 96±7 to 45±1 mol% after 24 h of accumulation. Similarly, the fraction of 3HV obtained from valeric acid treatment declined from 73±12 to 53±2 mol%, which was in accordance with a significant reduction in 3HV productivity. A likely explanation is that there is a key symbiotic relationship between Methylocaldum and the other two organisms present in these co-cultures where, in the presence of methane, each provides secondary metabolites or supplements required for enhancing 3HV formation.

Nevertheless, the removal of methane did increase the degradation rate of the 3HB monomer. In many bacteria, the utilization of stored PHB occurs normally in the absence of exogenous carbon when nutrients are available [39, 274, 275]. In this study, when the thermophilic culture was incubated with 100-ppm of either propionic acid or valeric acid and no methane was available, degradation of 3HB took place after the exhaustion of co-substrates. Therefore, it is initially assumed that these co-substrates served as a carbon supply for both 3HB and 3HV production. However, in the 200-ppm valeric acid treatment, the degradation of 3HB still occurred even though there was availability of valeric acid in the medium, suggesting that the exhaustion of odd carbon number fatty acids did not contribute to a degradation of PHB. Instead, it is likely affected by other methane-related activities, as no degradation of either 3HB

96 or 3HV was observed when an excess of valeric acid (200-ppm) was available in the presence of methane.

Overall, symbiotic relationships between Methylocaldum and Elioraea and Chloroflexus seem to be key to the production of PHB and PHBV in the thermophilic culture. Further study is needed for a better understanding of microbial interactions between these organisms in order to increase PHA productivity. However, this study highlights the potential for thermophilic production of PHB and PHBV – in a culture enriched on methane. And significantly, a PHV near-homopolymer could be produced by the culture by simply feeding propionic acid with methane; the resulting PHA was shown to be nearly 100 mol% 3HV. Alternatively, PHBV having a 3HV fraction of 39±1% could be produced when fed propionic acid with acetic acid.

97 Chapter 8 Conclusion and future perspective

The core idea of this thesis is to develop new strategies for PHB and PHBV production from methane to offer an opportunity for production cost reduction and provide commercially relevant mechanical properties of bio-derived and biodegradable PHA. The significant outcomes of this thesis relative to the stated thesis research objectives are summarised in this chapter. The specific research objectives of this study were as follows:

1. To assess different strategies for PHBV synthesis from methane in mixed microbial cultures (MMC); 2. To enrich a thermophilic methanotrophic mixed culture capable of PHA production; 3. To investigate the feasibility of PHB and PHBV production from methane in the thermophilic mixed culture; 4. To isolate and characterize thermophilic methanotrophs with PHA producing capability.

Regarding the first objective, there are two strategies considered for poly(3-hydroxybutyrate- co-3-hydroxyvalerate) (PHBV) synthesis: (i) a conventional approach for PHBV synthesis by the addition of odd-carbon-number fatty acids demonstrated in Chapter 4 and (ii) a novel approach of indirect 3HV generation from methane in Chapter 5. Both strategies resulted in PHBV production from methane, therefore benefiting the value of PHA materials. It is noted that the PHA contents (wt%), corresponding to the 3HV contents (wt%), obtained from the mixed microbial cultures were low compared to previous studies, hence an optimization and improvement of PHA production must be considered in the future in order to achieve a higher PHA content for industrial production. However, this study is significant as it demonstrates the generation of 3HV units from methane without any supplementation of fatty acids.

In order to develop an insightful understanding of how 3HV was accumulated in the culture fed solely with methane, the whole community metagenome sequencing was implemented (Chapter 5). The metagenomics analysis revealed that one methanotrophic microorganism from the genus of Methylomonas known for lacking in the genetic capacity for PHA generation dominated the community in company with multiple non-methanotrophic PHA producers. This led to the assumption that the dominant methanotroph converts methane into secondary metabolites, which are utilized by other non-methanotrophic organisms to accumulate PHBV

98 (Figure 5.5). This is in accordance with the literature, which reports that methanotrophs can form the base of a microbial food chain and play important role in microbial ecology.

The methanotrophic mixed cultures used to assess the two strategies for PHBV production in Chapter 4 and Chapter 5 were originally collected and enriched from the same environmental samples. While the community used for the novel approach of indirect 3HV generation from methane was dominated by Methylomonas sp., which has no capability for PHA formation, the culture involving the conventional approach of odd-carbon-number fatty acid supplementation was dominated by Methylosinus sp., a well-known PHA-producing methanotroph. This indicates that apparently variation in the nitrogen source led to the deviation in methanotrophic populations.

Most significantly, this thesis presents, for the first time, a comprehensive enrichment of a thermophilic PHA-producing community solely fed with methane. The remaining research objectives 2, 3, and 4 are all related to this thermophilic biotechnology.

The successful selection and long-term operation of the thermophilic PHA-producing mixed microbial culture grown on methane are described in Chapter 6. PHA was routinely observed in the culture under nitrogen-deficient condition. The metagenomics sequencing was again conducted to reveal the metabolic capacity of the community and to understand how PHB was generated. The investigation of the PHA synthase (phaC) gene showed multiple phaC high- quality sequences and one of those sequences was belonging to a thermophilic methanotroph from Methylocaldum genus, which was the most prevalent organism accounted for 40% of phaC-harbouring microorganisms presented in the community. The genome directed metabolic analysis revealed the presence of β-oxidation-linked PHA polymerase genes and β-oxidation pathway in Methylocaldum sp., which is distinct from other methanotrophic PHA producers. The organism was later isolated for a better understanding of its behaviors and potential for thermophilic PHA production from methane. Isolating the organism was rather challenging but eventually, an isolate was obtained, and microbial analysis confirmed it was Methylocaldum sp. The isolate was confirmed to accumulate PHA – this was the first quantitative evidence of PHA accumulation in a thermophilic methanotroph – but interestingly: (i) the organism did NOT accumulate PHA when only methane was supplied; PHA accumulation was only observed when fatty acids were available, and (ii) the organism only accumulated 3HB units; 3HV was never accumulated, even when odd-fatty acids were available and consumed.

99 The parent thermophilic culture from which the Methylocaldum sp. was isolated, on the other hand, did accumulate 3HV as shown in Chapter 7. When the community was supplemented with odd-carbon-number fatty acids, the PHA content was found to be up to 10±3 wt%. The PHA content at 10 wt% may be considered low for industrial production, but the community was able to generate PHA with up to 100 mol% of 3HV, providing higher HV fraction than that reported in other methanotrophic studies. However, according to the knowledge developed on the isolate Chapter 6, it can be concluded that the 3HV was NOT generated by the thermophilic methanotroph, but instead, Elioraea and/or Chloroflexus converted the odd-fatty acids to 3HV units. Nevertheless, it is significant that the highest 3HV mole fraction was observed when methane was available, indicating that the methanotroph in some way indirectly contributed to the very high 3HV fraction.

Overall, this study has highlighted the potential of mixed microbial cultures for the production of PHAs from methane and, for the first time, identified a novel thermophilic methanotroph capable of PHA production. However, more research remains to be done to achieve higher rates and productivities. It is suspected that maximizing gas-liquid mass transfer – for methane and oxygen (although, in some cases, microaerophilic conditions might be advantageous) would be a significant help.

Tailoring polymer composition also remains an important consideration. In this thesis, 3HV units are generated in cultures enriched on methane in both mesophilic and thermophilic conditions – in the presence and absence of methane. But without further efforts on understanding and development of microbial community structure beneficial for an indirect conversion of methane into PHAs, it seems that supplementation with odd-fatty acids would be necessary. Nevertheless, this research has made progress towards the development of a cost- efficient (mixed culture, thermophilic biotechnology using methane as the feedstock) high- value biomaterial (PBHV) production.

Overall, this study has highlighted the potential of mixed microbial cultures for the production of PHAs from methane and, for the first time, we identified a novel thermophilic methanotroph capable of PHA production. The application of mixed microbial culture and fundamental of thermophilic fermentation has been highlighted the feasibility of a low-cost and sustainable manufacturing of PHAs. However, besides investigations done in this work, more research remains to be done on maximizing gas-liquid mass transfer and productivities and strategies being applied to manipulate microbial diversity or microbial metabolism for enhanced

100 production of co-polymer production. The balance between co-substrates cost and value of altered polymer composition also remains an important consideration, unless efforts should be made on understanding and development of microbial community structure beneficial for an indirect conversion of carbon substrate into PHAs. Nevertheless, this research has paved the way towards the development of a cost-efficient PHA production.

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129 Appendix Table A1 Phylogenetic classification of the 16S rRNA gene sequences (relative abundance >1%) in the mesophilic methanotrophic–heterotrophic communities. BLASTs Phylogenetic classification of the 16S rRNA gene sequences %Relative abundance Cycle Top match %Similarity Phylum Class Order Family Genus Cycle 103 Cycle 113 Cycle 156 OTU_1 Methylosinus 100 Proteobacteria Alphaproteobacteria Rhizobiales Methylocystaceae Methylosinus 8.3 4.6 51.5 OTU_10 Methylosinus 97 Proteobacteria Alphaproteobacteria Rhizobiales Methylocystaceae Methylosinus 0.0 0.0 1.6 OTU_11 Ferrovibrio 98 Proteobacteria Alphaproteobacteria Rhodospirillales Rhodospirillaceae Phaeospirillum 0.0 1.9 1.4 OTU_12 Methylosinus 98 Proteobacteria Alphaproteobacteria Rhizobiales Methylocystaceae Methylosinus 1.2 0.0 1.3 OTU_13 Hyphomicrobium 99 Proteobacteria Alphaproteobacteria Rhizobiales Hyphomicrobiaceae Hyphomicrobium 6.4 2.8 1.3 OTU_14 Brevundimonas 100 Proteobacteria Alphaproteobacteria Caulobacterales Caulobacteraceae - 0.0 0.0 1.2 OTU_15 Methylosinus 94 Proteobacteria Alphaproteobacteria Rhizobiales Methylocystaceae Methylosinus 0.0 0.0 1.1 OTU_16 Hyphomicrobium 99 Proteobacteria Alphaproteobacteria Rhizobiales Hyphomicrobiaceae Hyphomicrobium 11.5 5.1 0.0 OTU_17 Hyphomicrobium 99 Proteobacteria Alphaproteobacteria Rhizobiales Hyphomicrobiaceae Hyphomicrobium 1.1 0.0 0.0 OTU_18 Methylomonas 100 Proteobacteria Gammaproteobacteria Methylococcales Methylococcaceae Methylomonas 0.0 1.8 0.0 OTU_19 Burkholderia 100 Proteobacteria Betaproteobacteria Burkholderiales Burkholderiaceae Burkholderia 2.9 0.0 0.0 OTU_2 Methylophilus 100 Proteobacteria Betaproteobacteria Methylophilales Methylophilaceae Methylophilus 0.0 0.0 10.6 OTU_20 Methylocystis 99 Proteobacteria Alphaproteobacteria Rhizobiales Methylocystaceae Methylosinus 1.2 0.0 0.0 OTU_21 Terrimonas 100 Bacteroidetes [Saprospirae] [Saprospirales] Chitinophagaceae - 1.0 1.9 0.0 OTU_22 Pseudomonas 100 Proteobacteria Gammaproteobacteria Pseudomonadales Pseudomonadaceae Pseudomonas 0.0 1.4 0.0 OTU_23 Hyphomicrobium 99 Proteobacteria Alphaproteobacteria Rhizobiales Hyphomicrobiaceae Hyphomicrobium 1.9 3.4 0.0 OTU_24 Methylocystis 96 Proteobacteria Alphaproteobacteria Rhizobiales Methylocystaceae Methylosinus 1.2 0.0 0.0 OTU_25 Hyphomicrobium 99 Proteobacteria Alphaproteobacteria Rhizobiales Hyphomicrobiaceae Hyphomicrobium 2.9 1.3 0.0 OTU_3 Comamonas 100 Proteobacteria Betaproteobacteria Burkholderiales Comamonadaceae - 0.0 0.0 6.6 OTU_4 Caulobacter 98 Proteobacteria Alphaproteobacteria Caulobacterales Caulobacteraceae - 0.0 0.0 3.4 OTU_5 Methylosinus 100 Proteobacteria Alphaproteobacteria Rhizobiales Methylocystaceae Methylosinus 40.4 55.7 3.3 OTU_6 Rhodanobacter 100 Proteobacteria Gammaproteobacteria Xanthomonadales Xanthomonadaceae Rhodanobacter 5.5 3.4 2.5 OTU_7 Methylophilus 96 Proteobacteria Betaproteobacteria Methylophilales Methylophilaceae Methylophilus 0.0 0.0 2.3 OTU_8 Pigmentiphaga 100 Proteobacteria Betaproteobacteria Burkholderiales Alcaligenaceae Pigmentiphaga 0.0 0.0 2.2 OTU_9 Chryseobacterium 99 Bacteroidetes Flavobacteriia Flavobacteriales - - 1.2 3.4 1.7 Other 14.6 16.8 9.8

130 Table A2 The mesophilic methanotrophic-heterotrophic culture Metagenomic Assembled Genomes (MAGs).

Bin % % % Mapped % Binned % MAG ID size Mapped Quality* GTDB Taxonomy Completeness Contamination reads Population Community (Mbp) reads 2 4.9 98.7 0.4 23322784 43.9 65.8 60.6 97.9 d__Bacteria;p__Proteobacteria;c__Gammaproteobacteria;o__Methylococcales;f__Methylococcaceae;g__Methylomonas 3 8.4 99.1 0.1 12472702 23.5 10.0 9.2 99.0 d__Bacteria;p__Proteobacteria;c__Betaproteobacteria;o__Burkholderiales;f__Burkholderiaceae;g__Burkholderia;s__cepacia 4 4.8 97.1 0.5 2126474 4.0 3.0 2.7 96.1 d__Bacteria;p__Proteobacteria;c__Betaproteobacteria;o__Burkholderiales;f__Comamonadaceae;g__Pelomonas 5 4.3 98.4 1.6 1705603 3.2 2.7 2.5 95.3 d__Bacteria;p__Proteobacteria;c__Alphaproteobacteria;o__Rhizobiales;f__Hyphomicrobiaceae;g__Hyphomicrobium 6 5.5 100.0 0.0 1529576 2.9 1.9 1.7 100.0 d__Bacteria;p__Bacteroidetes;c__Saprospirae;o_Chitinophagles;f__Chitinophagaceae;g__Flavihumibacter 7 7.5 97.0 8.1 1831899 3.5 1.7 1.5 80.8 d__Bacteria;p__Proteobacteria;c__Alphaproteobacteria;o__Rhodosprillaceae 8 2.9 99.0 0.0 696866 1.3 1.6 1.5 99.0 d__Bacteria;p__Bacteroidetes;c__Flavobacteriia;f__Weeksellaceae;g__Chryseobacterium; s__Epilithonimonas 9 4.8 96.4 1.1 1078230 2.0 1.5 1.4 94.1 d__Bacteria;p__Proteobacteria;c__Alphaproteobacteria;o__Rhizobiales;f__Methylocystaceae;g__Methylosinus 11 4.0 92.8 1.8 679785 1.3 1.1 1.1 89.2 d__Bacteria;p__Proteobacteria;c__Gammaproteobacteria;o__Xanthomonadales;f__Xanthomonadaceae 12 2.7 76.7 0.0 434487 0.8 1.1 1.0 76.7 d__Bacteria;p__Proteobacteria;c__Betaproteobacteria;o__Methylophilales;f__Methylophilaceae;g__Methylotonera 14 4.4 95.3 0.4 597791 1.1 0.9 0.9 94.5 d__Bacteria;p__Proteobacteria;c__Gammaproteobacteria;o__Enterobacteriales;f__Enterobacteriaceae;g__Enterobacter 15 4.3 98.8 3.0 468726 0.9 0.7 0.7 92.8 d__Bacteria;p__Firmicutes;c__Negativicutes;o__Selenomonadales;f__Sporomusaceae 16 3.6 98.1 2.3 305624 0.6 0.6 0.5 93.5 d__Bacteria;p__Proteobacteria;c__Gammaproteobacteria;o__Legionellales;f__Coxiellaceae 23 4.3 73.3 0.5 228376 0.4 0.4 0.3 72.2 d__Bacteria;p__Bacteroidetes;c__Bacteroidia;o__Meniscales;f__WCHB1-32 Unbinned 78.3 N/A N/A 5649001 10.64 N/A 14.34 N/A N/A *Quality = %Completeness - (2 X %Contamination), <70% Quality Score was used as a cutoff Value [184]. MAGs highlighted in red indicate the presence of phaC genes

131 Table A3 Physiological groups present in the thermophilic reactors over long-term cultivation at 55°C. Operating day

55 90 114 321 482 524 630 599 700 p__Deinococcus 3.7 3.2 10.5 10.2 7.2 11.0 15.1 11.5 11.6 p__Acidobacteria 1.5 2.1 3.6 4.6 1.6 2.3 4.0 3.1 2.6 p__Armatimonadetes 0.3 0.4 2.5 0.0 14.5 9.2 0.0 7.6 5.6 p__Bacteroidetes 12.8 14.0 28.1 32.2 13.1 23.4 20.7 25.0 25.2 p__Chloroflexi 9.0 10.6 2.2 4.0 5.2 5.9 0.5 5.8 4.7 p__Planctomycetes 10.1 15.0 3.1 9.7 1.2 1.6 0.0 1.7 2.0 c__Betaproteobacteria 4.4 2.8 5.7 2.2 12.1 7.1 6.6 6.2 7.3 f__Comamonadaceae 0.0 0.0 0.0 0.6 0.0 0.1 0.0 0.0 0.2 g__Methylocaldum 34.9 39.4 28.4 33.8 36.0 28.6 29.0 29.3 29.6 g__Thermomonas 0.0 0.0 0.0 0.0 0.0 0.0 12.2 0.0 0.0 o__Burkholderiales 0.3 0.2 1.5 0.0 7.0 9.3 0.0 8.7 9.5 o__Methylophilales 2.8 1.4 0.0 0.0 0.0 0.0 0.0 0.0 0.0 o__Rhizobiales 0.0 0.0 11.2 0.2 1.0 0.3 0.0 0.4 0.3 o__Rhodospirillales 2.2 2.3 2.1 0.9 0.5 0.0 6.1 0.0 0.0 Other 18.0 8.6 1.1 1.6 0.6 1.2 5.8 0.7 1.4

132 Table A4 Genome completeness, contamination, and quality of 33 MAGs recovered from the thermophilic community. The remaining 6 low- quality MAGs and unbinned reads are given in the ‘other’ category. Bin ID Lineage Completeness Contamination Bin size (Mbp) % Mapped Reads % Binned Populations % Abundance 32 p__Bacteroidetes 93.6 1.1 2.2 19.3 30.2 27.1 10 c__Gammaproteobacteria 98.4 5.9 4.6 24.6 18.1 16.2 30 c__Alphaproteobacteria 98.4 0.6 2.8 14.9 17.8 16.0 22 p__Deinococcus 85.0 1.5 2.9 4.1 4.7 4.2 11 p__Acidobacteria 94.0 3.2 2.6 2.5 3.3 2.9 12 p__Bacteroidetes 91.5 2.3 2.7 2.5 3.1 2.8 23 o__Betaproteobacteriales 98.7 2.1 3.3 2.9 3.0 2.7 16 c__Deltaproteobacteria 96.1 6.3 7.3 4.2 2.0 1.8 9 c__Alphaproteobacteria 98.8 0.0 3.7 2.1 1.9 1.7 2 o__Betaproteobacteriales 99.1 2.1 4.1 1.9 1.6 1.4 37 p__Armatimonadetes 88.6 0.9 2.3 0.9 1.3 1.1 36 c__Deltaproteobacteria 91.3 4.2 3.6 0.9 0.8 0.7 5 p__Chloroflexi 91.2 3.7 3.0 0.7 0.7 0.7 25 c__Gammaproteobacteria 92.4 2.8 2.8 0.6 0.7 0.6 18 p__Armatimonadetes 92.5 0.2 3.9 0.8 0.7 0.6 8 p__Plantomycetaceae 95.4 0.0 4.9 0.7 0.5 0.5 39 p__Chloroflexi 99.7 0.0 4.8 0.6 0.4 0.4 27 o__Betaproteobacteriales 89.7 1.1 3.7 0.4 0.4 0.4 21 p__Acidobacteria 95.7 3.6 5.4 0.6 0.4 0.3 31 p__Chloroflexi 99.1 5.5 5.0 0.6 0.4 0.3 7 p__Actinobacteria 85.3 0.4 3.2 0.3 0.3 0.2 4 c__Deltaproteobacteria 72.7 0.9 1.5 0.1 0.2 0.2 Other 13.8 7.7 17.2

133 76 Methylocaldum marinum (BAO51827) 68 Methylocaldum sp. 14B (WP_086132856) 97 Methylocaldum sp. SAD2 (WP_077732836) 96 Methylocaldum MAG_10 89 Methylocaldum szegediense (WP_026609851) 86 Methylomagnum ishizawai (WP_085214205) Methylogaea oryzae (WP_054774741) 95 Methylococcus capsulatus (AAB49821) 95 Methylococcus capsulatus (WP_010961050) 97 Methylococcus Contig_11073 100 Methyloterricola oryzae (WP_045226820) 88 Methylothermus thermalis (AAX37294) 66 Methylothermus subterraneus (BAI52935) Methylothermus MAG_22 55 Methylothermus sp. HB (AAD02578) 99 Methylohalobius crimeensis (WP_022947315) Methylomarinovum caldicuralii (BAF62078) 91 Methylomonas koyamae (WP_054761029) 54 Methylomonas methanica (WP_013817026) 100 Methylomonas lenta (WP_066986567) Methylomarinum vadi (WP_031433836) 99 Methyloglobulus morosus (WP_023494957) 91 Methylosarcina lacus (WP_024298805) 98 Methylobacter whittenburyi (WP_036297036) 93 Methylobacter luteus (WP_027159170) Methylobacter tundripaludum (WP_031437564) 98 Methylovulum psychrotolerans (WP_088620576) 100 Methylovulum miyakonense (WP_019865090) 77 Methylomicrobium alcaliphilum (WP_014147021) 88 Methylomicrobium japanense (BAE86885) 0.10 Methylomicrobium buryatense (WP_017841993)

Figure A1 Thermophilic bioreactor methanotroph PmoA sequence tree. A taxonomic tree of thermophilic bioreactor pmoA sequences identified with GraftM and representative database sequences generated using Maximum-Likelihood treeing methods with bootstrap values from 100 iterations. Numbers assigned to bioreactor PmoA sequences refer to the MAGs from which these sequences were derived in Table A4.

134 Tree 1: Aratimonadetes p__Armatimonadetes;c__CG2-30-59-28;o__CG2-30-59-28;f__CG2-30-59-28;g__CG2-30-59-28;s__, GB_GCA_001872605.1 p__Armatimonadetes;c__CG2-30-66-41;o__CG2-30-66-41;f__CG2-30-66-41;g__CG2-30-66-41;s__, GB_GCA_001871675.1 p__Armatimonadetes;c__UBA5829;o__UBA5829;f__UBA5829;g__RBG-16-58-9;s__, GB_GCA_001768085.1 H2H_Bioreactor_r2_18, None, U_94229 p__Armatimonadetes;c__Chthonomonadetes;o__Chthonomonadales;f__Chthonomonadaceae;g__Chthonomonas;s__Chthonomonas calidirosea, RS_GCF_000427095.1 p__Armatimonadetes;c__Chthonomonadetes;o__Chthonomonadales;f__Chthonomonadaceae;g__Chthonomonas;s__Chthonomonas calidirosea, RS_GCF_001457975.1 p__Armatimonadetes;c__Fimbriimonadia;o__OS-L;f__GBS-DC;g__GBS-DC;s__GBS-DC sp1, GB_GCA_001442985.1 p__Armatimonadetes;c__Fimbriimonadia;o__OS-L;f__GBS-DC;g__GBS-DC;s__GBS-DC sp1, GB_GCA_001442875.1 H2H_Bioreactor_r2_37, None, U_94240 p__Armatimonadetes;c__Fimbriimonadia;o__Fimbriimonadales;f__Fimrbiimonadaceae;g__OLB18;s__, GB_GCA_001567425.1 p__Armatimonadetes;c__Fimbriimonadia;o__Fimbriimonadales;f__Fimrbiimonadaceae;g__55-13;s__, GB_GCA_001898035.1 p__Armatimonadetes;c__Fimbriimonadia;o__Fimbriimonadales;f__Fimrbiimonadaceae;g__Fimbriimonas;s__Fimbriimonas ginsengisoli, RS_GCF_000724625.1

Tree 2: Chloroflexi p__Chloroflexi;c__Anaerolineae;o__Anaerolineales;f__RBG-16-64-43;g__RBG-16-64-43;s__, GB_GCA_001796965.1 p__Chloroflexi;c__Anaerolineae;o__Anaerolineales;f__UBA6663;g__RBG-16-63-12;s__, GB_GCA_001796945.1 p__Chloroflexi;c__Anaerolineae;o__Caldilineales;f__CG2-30-64-16;g__CG2-30-64-16;s__, GB_GCA_001871755.1 H2H_Bioreactor_r2_31, None, U_94237 p__Chloroflexi;c__Anaerolineae;o__Caldilineales;f__Caldilineaceae;g__Caldilinea;s__Caldilinea aerophila, RS_GCF_000281175.1 p__Chloroflexi;c__Chloroflexia;o__54-19;f__54-19;g__54-19;s__, GB_GCA_001898225.1 p__Chloroflexi;c__Chloroflexia;o__Chloroflexales;f__Chloroflexaceae;g__Oscillochloris;s__Oscillochloris trichoides, RS_GCF_000152145.1 H2H_Bioreactor_r2_39, p__Chloroflexi;c__Chloroflexia;o__Chloroflexales;f__Chloroflexaceae;g__Chloroflexus;s__Chloroflexus aurantiacus, RS_GCF_000022185.1 p__Chloroflexi;c__Chloroflexia;o__Chloroflexales;f__Chloroflexaceae;g__Chloroflexus;s__Chloroflexus aurantiacus, RS_GCF_000018865.1 p__Chloroflexi;c__Chloroflexia;o__Chloroflexales;f__Chloroflexaceae;g__Chloroflexus;s__Chloroflexus sp1, RS_GCF_000735195.1 p__Chloroflexi;c__Chloroflexia;o__Chloroflexales;f__Chloroflexaceae;g__Chloroflexus;s__Chloroflexus sp1, RS_GCF_000516515.1 p__Chloroflexi;c__Chloroflexia;o__Chloroflexales;f__Chloroflexaceae;g__Chloroflexus;s__Chloroflexus aggregans, RS_GCF_000021945.1 p__Chloroflexi;c__Chloroflexia;o__Chloroflexales;f__Chloroflexaceae;g__Chloroflexus;s__, RS_GCF_001650695.1

135 Tree 3: Actinobacteria p__Actinobacteria;c__Acidimicrobiia;o__Microtrichales;f__TK06;g__MedAcidi-G3;s__MedAcidi-G3 sp4, GB_GCA_000817105.1

H2H_Bioreactor_r2_7, None, U_94244

p__Actinobacteria;c__Acidimicrobiia;o__Microtrichales;f__MedAcidi-G1;g__MedAcidi-G2A;s__, GB_GCA_000817095.1

p__Actinobacteria;c__Acidimicrobiia;o__Microtrichales;f__MedAcidi-G1;g__MedAcidi-G1;s__MedAcidi-G1 sp1, GB_GCA_000817085.1

p__Actinobacteria;c__Acidimicrobiia;o__Microtrichales;f__MedAcidi-G1;g__MedAcidi-G2B;s__, GB_GCA_000817115.1

p__Actinobacteria;c__Acidimicrobiia;o__Microtrichales;f__MedAcidi-G1;g__S20-B6;s__S20-B6 sp1, GB_GCA_001626165.1

p__Actinobacteria;c__Acidimicrobiia;o__Microtrichales;f__MedAcidi-G1;g__S20-B6;s__S20-B6 sp1, GB_GCA_001628575.1

p__Actinobacteria;c__Acidimicrobiia;o__Microtrichales;f__MedAcidi-G1;g__S20-B6;s__S20-B6 sp1, GB_GCA_001628605.1

p__Actinobacteria;c__Acidimicrobiia;o__Microtrichales;f__MedAcidi-G1;g__S20-B6;s__S20-B6 sp1, GB_GCA_001626125.1

p__Actinobacteria;c__Acidimicrobiia;o__Microtrichales;f__MedAcidi-G1;g__S20-B6;s__S20-B6 sp1, GB_GCA_001628595.1

Tree 4: Chloroflexi H2H_Bioreactor_r2_5, None, U_94243

p__Chloroflexi;c__Ellin6529;o__CSP1-4;f__CSP1-4;g__RBG-16-72-14;s__, GB_GCA_001797035.1

p__Chloroflexi;c__Ellin6529;o__CSP1-4;f__CSP1-4;g__RBG-16-69-14;s__, GB_GCA_001797015.1

p__Chloroflexi;c__Ellin6529;o__CSP1-4;f__CSP1-4;g__UBA5189;s__, GB_GCA_001795245.1

p__Chloroflexi;c__Ellin6529;o__CSP1-4;f__CSP1-4;g__GWC2-73-18;s__, GB_GCA_001794945.1 p__Chloroflexi;c__Ellin6529;o__CSP1-4;f__CSP1-4;g__CSP1-4;s__, GB_GCA_001443375.1

136 Tree 5: Deinococccaeota p__Deinococcaeota;c__Deinococci;o__Deinococcales;f__Thermaceae;g__Meiothermus;s__Meiothermus rufus, RS_GCF_000423425.1

p__Deinococcaeota;c__Deinococci;o__Deinococcales;f__Thermaceae;g__Meiothermus;s__Meiothermus cerbereus, RS_GCF_000620065.1

p__Deinococcaeota;c__Deinococci;o__Deinococcales;f__Thermaceae;g__Meiothermus;s__Meiothermus ruber, RS_GCF_000024425.1

p__Deinococcaeota;c__Deinococci;o__Deinococcales;f__Thermaceae;g__Meiothermus;s__Meiothermus ruber, RS_GCF_000376665.1

p__Deinococcaeota;c__Deinococci;o__Deinococcales;f__Thermaceae;g__Meiothermus;s__Meiothermus taiwanensis, RS_GCF_000482765.1

p__Deinococcaeota;c__Deinococci;o__Deinococcales;f__Thermaceae;g__Meiothermus;s__Meiothermus taiwanensis, RS_GCF_000836395.1

p__Deinococcaeota;c__Deinococci;o__Deinococcales;f__Thermaceae;g__Meiothermus_A;s__Meiothermus_A chliarophilus, RS_GCF_000430045.1

p__Deinococcaeota;c__Deinococci;o__Deinococcales;f__Thermaceae;g__Meiothermus_A;s__Meiothermus_A timidus, RS_GCF_000373205.1

H2H_Bioreactor_r2_22, None, U_94232 Tree 6: Placnctomycetes p__Planctomycetes;c__Planctomycetia;o__Pirellulales;f__Pirellulaceae;g__Rhodopirellula_A;s__Rhodopirellula_A maiorica, RS_GCF_000346295.1

H2H_Bioreactor_r2_8, None, U_94245

p__Planctomycetes;c__Planctomycetia;o__Pirellulales;f__Pirellulaceae;g__Pirellula_A;s__Pirellula_A staleyi, RS_GCF_000025185.1

p__Planctomycetes;c__Planctomycetia;o__Pirellulales;f__Pirellulaceae;g__Blastopirellula;s__Blastopirellula marina, RS_GCF_000153105.1

p__Planctomycetes;c__Planctomycetia;o__Pirellulales;f__Ga0077529;g__Ga0077529;s__, GB_GCA_001464525.1 Tree 7: Bacteroidetes 1 p__Bacteroidetes;c__Ignavibacteria;o__OLB7;f__OLB7;g__OLB7;s__, GB_GCA_001567195.1 p__Bacteroidetes;c__Ignavibacteria;o__OPB56;f__UBA2268;g__UBA2268;s__, GB_GCA_001804565.1 p__Bacteroidetes;c__Ignavibacteria;o__OPB56;f__UBA2268;g__RIFOXYC2-FULL-35-21;s__, GB_GCA_001804705.1 p__Bacteroidetes;c__Ignavibacteria;o__OPB56;f__NICIL-2;g__NICIL-2;s__, GB_GCA_001302225.1 H2H_Bioreactor_r2_32, None, U_94238 p__Bacteroidetes;c__Ignavibacteria;o__OPB56;f__OLB6;g__OLB6;s__, GB_GCA_001567175.1 p__Bacteroidetes;c__Ignavibacteria;o__OPB56;f__OLB6;g__Kapabacteria;s__, GB_GCA_001899175.1

137 Tree 8: Bacteroidetes 2 p__Bacteroidetes;c__Ignavibacteria;o__UBA10030;f__UBA10030;g__RIFCSPLOWO2-02-FULL-55-14;s__, GB_GCA_001802915.1

H2H_Bioreactor_r2_12, None, U_94227

p__Bacteroidetes;c__Ignavibacteria;o__Ignavibacteriales;f__RBG-13-36-8;g__RBG-13-36-8;s__, GB_GCA_001804605.1

p__Bacteroidetes;c__Ignavibacteria;o__Ignavibacteriales;f__Melioribacteraceae;g__BRH-c32;s__, GB_GCA_001516025.1

p__Bacteroidetes;c__Ignavibacteria;o__Ignavibacteriales;f__Melioribacteraceae;g__Melioribacter_A;s__Melioribacter_A roseus, RS_GCF_000279145.1

p__Bacteroidetes;c__Ignavibacteria;o__Ignavibacteriales;f__Melioribacteraceae;g__RIFOXYB12-FULL-38-5;s__, GB_GCA_001803365.1

p__Bacteroidetes;c__Ignavibacteria;o__Ignavibacteriales;f__Melioribacteraceae;g__Melioribacter;s__, GB_GCA_001803345.1 Tree 9: Acidobacteria 1 p__Acidobacteria;c__Blastocatellia;o__Chloracidobacteriales;f__Chloracidobacteriaceae;g__Chloracidobacterium;s__Chloracidobacterium thermophilum, RS_GCF_001482755.1

p__Acidobacteria;c__Blastocatellia;o__Chloracidobacteriales;f__Chloracidobacteriaceae;g__Chloracidobacterium;s__Chloracidobacterium thermophilum, RS_GCF_000226295.1

H2H_Bioreactor_r2_11, None, U_94226 p__Acidobacteria;c__Blastocatellia;o__Pyrinomonadales;f__OLB17;g__OLB17;s__, GB_GCA_001464665.1

p__Acidobacteria;c__Blastocatellia;o__Pyrinomonadales;f__OLB17;g__OLB17;s__, GB_GCA_001464455.1 p__Acidobacteria;c__Blastocatellia;o__Pyrinomonadales;f__OLB17;g__OLB17;s__, GB_GCA_001567505.1

p__Acidobacteria;c__Blastocatellia;o__Pyrinomonadales;f__RB41;g__13-1-20CM-3-53-8;s__, GB_GCA_001920415.1

p__Acidobacteria;c__Blastocatellia;o__Pyrinomonadales;f__Pyrinomonadaceae;g__Pyrinomonas;s__Pyrinomonas methylaliphatogenes, RS_GCF_000820845.2

Tree 10: Acidobacteria 2 p__Acidobacteria;c__Acidobacteriia;o__LOWO2-12-FULL-54-10;f__LOWO2-12-FULL-54-10;g__RIFCSPLOWO2-02-FULL-61-28;s__, GB_GCA_001767035.1

p__Acidobacteria;c__Acidobacteriia;o__LOWO2-12-FULL-54-10;f__LOWO2-12-FULL-54-10;g__RIFCSPLOWO2-12-FULL-54-10;s__, GB_GCA_001767075.1

p__Acidobacteria;c__Acidobacteriia;o__Solibacterales;f__Solibacteraceae;g__Solibacter;s__Solibacter usitatus, RS_GCF_000014905.1

H2H_Bioreactor_r2_21, None, U_94231

p__Acidobacteria;c__Acidobacteriia;o__Solibacterales;f__Solibacteraceae;g__KBS-96;s__, RS_GCF_000381625.1

p__Acidobacteria;c__Acidobacteriia;o__Solibacterales;f__Solibacteraceae;g__Ga0077553;s__, GB_GCA_001464575.1 p__Acidobacteria;c__Acidobacteriia;o__Solibacterales;f__Solibacteraceae;g__Bryobacter;s__Bryobacter aggregatus, RS_GCF_000702445.1

138 Tree 11: Deltaproteobacteria p__Deltabacteraeota;c__Polyangia;o__Polyangiales;f__Polyangiaceae;g__Pajaroellobacter;s__Pajaroellobacter abortibovis, RS_GCF_001931505.1

p__Deltabacteraeota;c__Polyangia;o__Polyangiales;f__Polyangiaceae;g__Labilithrix;s__Labilithrix luteola, GB_GCA_001263205.1

p__Deltabacteraeota;c__Polyangia;o__Polyangiales;f__Polyangiaceae;g__Labilithrix;s__, GB_GCA_001899635.1

H2H_Bioreactor_r2_16, None, U_94228 Tree 12: Alphaproteobacteria 1 p__Proteobacteria;c__Alphaproteobacteria;o__Rhizobiales;f__Methyloligellaceae;g__Methyloligella;s__Methyloligella halotolerans, RS_GCF_001708935.1

H2H_Bioreactor_r2_30, None, U_94236

p__Proteobacteria;c__Alphaproteobacteria;o__Rhizobiales;f__Methyloligellaceae;g__Methyloceanibacter;s__Methyloceanibacter superfactus, RS_GCF_001723305.1

p__Proteobacteria;c__Alphaproteobacteria;o__Rhizobiales;f__Methyloligellaceae;g__Methyloceanibacter;s__Methyloceanibacter marginalis, RS_GCF_001723295.1

p__Proteobacteria;c__Alphaproteobacteria;o__Rhizobiales;f__Methyloligellaceae;g__Methyloceanibacter;s__Methyloceanibacter methanicus, RS_GCF_001723285.1

p__Proteobacteria;c__Alphaproteobacteria;o__Rhizobiales;f__Methyloligellaceae;g__Methyloceanibacter;s__Methyloceanibacter caenitepidi, RS_GCF_000828475.1

p__Proteobacteria;c__Alphaproteobacteria;o__Rhizobiales;f__Methyloligellaceae;g__Methyloceanibacter;s__Methyloceanibacter stevinii, RS_GCF_001723355.1

Tree 13: Alphaproteobacteria 2 p__Proteobacteria;c__Alphaproteobacteria;o__Acetobacterales;f__Acetobacteraceae;g__Acidocella;s__Acidocella aminolytica, RS_GCF_000964385.1

p__Proteobacteria;c__Alphaproteobacteria;o__Acetobacterales;f__Acetobacteraceae;g__Acidocella;s__Acidocella facilis, RS_GCF_000687875.1

p__Proteobacteria;c__Alphaproteobacteria;o__Acetobacterales;f__Acetobacteraceae;g__Acidocella;s__Acidocella facilis, RS_GCF_000306035.1

p__Proteobacteria;c__Alphaproteobacteria;o__Acetobacterales;f__Acetobacteraceae;g__Acidiphilium;s__Acidiphilium angustum, RS_GCF_000701585.1

p__Proteobacteria;c__Alphaproteobacteria;o__Acetobacterales;f__Acetobacteraceae;g__Acidiphilium;s__Acidiphilium multivorum, RS_GCF_000202835.1

p__Proteobacteria;c__Alphaproteobacteria;o__Acetobacterales;f__Acetobacteraceae;g__Acidiphilium;s__Acidiphilium cryptum, RS_GCF_000724705.2

p__Proteobacteria;c__Alphaproteobacteria;o__Acetobacterales;f__Acetobacteraceae;g__Acidiphilium;s__Acidiphilium cryptum, RS_GCF_000016725.1

H2H_Bioreactor_r2_9, None, U_94246 p__Proteobacteria;c__Alphaproteobacteria;o__Acetobacterales;f__Acetobacteraceae;g__Elioraea;s__Elioraea tepidiphila, RS_GCF_000378465.1

139 Tree 14: Gammaproteobacteria 1 H2H_Bioreactor_r2_23, None, U_94233 p__Proteobacteria;c__Gammaproteobacteria;o__Betaproteobacteriales;f__SG8-41;g__Ga0077527;s__, GB_GCA_001464815.1

p__Proteobacteria;c__Gammaproteobacteria;o__Betaproteobacteriales;f__SG8-41;g__SG8-41;s__, GB_GCA_001303585.1

p__Proteobacteria;c__Gammaproteobacteria;o__Betaproteobacteriales;f__SG8-41;g__SG8-41;s__, GB_GCA_001771935.1

p__Proteobacteria;c__Gammaproteobacteria;o__Betaproteobacteriales;f__SG8-41;g__RIFCSPLOWO2-02-64-14;s__, GB_GCA_001770325.1

p__Proteobacteria;c__Gammaproteobacteria;o__Betaproteobacteriales;f__SG8-41;g__LOWO2-12-FULL-62-58;s__, GB_GCA_001772005.1 p__Proteobacteria;c__Gammaproteobacteria;o__Betaproteobacteriales;f__SG8-41;g__LOWO2-12-FULL-62-58;s__, GB_GCA_001771985.1 Tree 15: Gammaproteobacteria 2 p__Proteobacteria;c__Gammaproteobacteria;o__Methylococcales;f__Methylococcaceae;g__Methylogaea;s__Methylogaea oryzae, RS_GCF_001312345.1 p__Proteobacteria;c__Gammaproteobacteria;o__Methylococcales;f__Methylococcaceae;g__73a;s__, RS_GCF_000934725.1

H2H_Bioreactor_r2_25, None, U_94234 p__Proteobacteria;c__Gammaproteobacteria;o__Methylococcales;f__Methylococcaceae;g__Methylococcus;s__Methylococcus capsulatus, RS_GCF_000424685.1 p__Proteobacteria;c__Gammaproteobacteria;o__Methylococcales;f__Methylococcaceae;g__Methylococcus;s__Methylococcus capsulatus, RS_GCF_000008325.1 p__Proteobacteria;c__Gammaproteobacteria;o__Methylococcales;f__Methylococcaceae;g__Methylocaldum;s__Methylocaldum szegediense, RS_GCF_000427385.1 H2H_Bioreactor_r2_10 Tree 16: Gammaproteobacteria 3 p__Proteobacteria;c__Gammaproteobacteria;o__Betaproteobacteriales;f__Burkholderiaceae;g__GJ-E10;s__, RS_GCF_000828975.1 H2H_Bioreactor_r2_27, None, U_94235 H2H_Bioreactor_r2_2, None, U_94230 p__Proteobacteria;c__Gammaproteobacteria;o__Betaproteobacteriales;f__Burkholderiaceae;g__Parasutterella;s__, RS_GCF_001688905.1 p__Proteobacteria;c__Gammaproteobacteria;o__Betaproteobacteriales;f__Burkholderiaceae;g__Parasutterella;s__Parasutterella excrementihominis, GB_GCA_000980495.1 p__Proteobacteria;c__Gammaproteobacteria;o__Betaproteobacteriales;f__Burkholderiaceae;g__Parasutterella;s__Parasutterella excrementihominis, RS_GCF_000144975.1 p__Proteobacteria;c__Gammaproteobacteria;o__Betaproteobacteriales;f__Burkholderiaceae;g__Parasutterella;s__Parasutterella excrementihominis, RS_GCF_000205025.1

Figure A2 Thermophilic reactor derived MAGs 122 bacterial concatenated single marker genes. Numbers assigned to bioreactor sequences refer to the MAGs from which these sequences were derived in Supplementary Table 6.2.

140 Table A5 PHA content on a mass basis (wt%) and 3HB and 3HV content on a mol basis (%mol) obtained during PHA accumulation phase in the presence of methane at 55 and 58°C.

Time (h) Time (h) Condition Content Condition Content 0 24 48 0 24 48 PHA Content %wt* 0.0±0.0h 0.2±0.1h PHA Content %wt* 0.0±0.0h 0.3±0.2h %wt* 0.0±0.0f 0.2±0.1ef %wt* 0.0±0.0f 0.3±0.2ef 3HB Content 3HB Content 55M1C %mol** 0.0±0.0 0.0±0.0g 100±0.0a 58M1C %mol** 0.0±0.0 0.0±0.0g 100±0.0a %wt* 0.0±0.0h 0.0±0.0h %wt* 0.0±0.0h 0.0±0.0h 3HV Content 3HV Content %mol** 0.0±0.0g 0.0±0.0g %mol** 0.0±0.0g 0.0±0.0g PHA Content %wt* 2.7±0.2efgh 2.6±0.2efgh PHA Content %wt* 1.8±0.5fgh 1.4±0.5gh %wt* 0.1±0.2f 0.8±0.0cdf %wt* 0.0±0.0f 0.3±0.3ef 3HB Content 3HB Content 55M1P %mol** 0.0±0.0 3.9±6.8fg*** 30.4±3.4bcd 58M1P %mol** 0.0±0.0 0.5±0.9g 15.3±13.6ef %wt* 2.6±0.2defg 1.8±0.3fg %wt* 1.8±0.5fg 1.2±0.3gh 3HV Content 3HV Content %mol** 96.1±6.8ab*** 69.6±3.4def %mol** 99.5±0.9a 84.7±13.6bc PHA Content %wt* 6.2±0.3bc 5.5±0.9cd PHA Content %wt* 4.2±0.5cdef 3.1±0.4defg %wt* 1.7±0.2cd 1.9±0.3bc %wt* 0.9±0.2cdef 1.0±0.2cdef 3HB Content 3HB Content 55M1LV %mol** 0.0±0.0 26.8±1.9bcde 34.5±1.2bc 58M1LV %mol** 0.0±0.0 21.7±1.9de 31.6±1.8bcd %wt* 4.6±0.2abc 3.6±0.5cde %wt* 3.3±0.3cdef 2.1±0.3efg 3HV Content 3HV Content %mol** 73.2±1.9cdef 65.5±1.2ef %mol** 78.3±1.9cde 68.4±1.8def PHA Content %wt* 6.2±0.3bc 9.6±3.0a PHA Content %wt* 5.3±0.7cde 8.4±1.2ab %wt* 1.5±0.1cd 3.5±1.2a %wt* 1.3±0.3cde 3.0±0.5ab 3HB Content 3HB Content 55M1HV %mol** 0.0±0.0 24.7±1.2bcde 36.4±2.2ef 58M1HV %mol** 0.0±0.0 23.8±2.7cde 35.2±1.0bc %wt* 4.6±0.2abc 6.1±1.8a %wt* 4.1±0.5bcd 5.4±0.7ab 3HV Content 3HV Content %mol** 75.3±1.2cdef 63.6±2.2f %mol** 76.2±2.7cde 64.8±1.0ef * %wt means weight percentage based on total dry biomass; ** %mol means mole percentage based on total mole of PHA produced; *** Data obtained from the condition were 0, 0, and 11.7 mol% 3HB and 100, 100, and 88.3 mol% 3HV; ± represents standard deviations (n=3) Note: - M1 and M0 stand for the presence and absence of methane, 55 and 58 stands for treatments at 55°C and 58°C, P stands for treatments with 100 ppm of propionic acid, LV and HV stands for treatments with 100 ppm and 200 ppm of valeric acid, respectively. - Statistic letters denote significant differences within each analyte for PHA content (wt%), 3HB, and 3HV content on weight (wt%) and mole (mol%) basis with p<0.05 were derived. The letters denote interactions between t = 24 and t = 48 h. There is no interaction between the data presented in this table and in the others

141 Table A6 PHA content on a mass basis (wt%) and 3HB and 3HV content on a mol basis (%mol) obtained during PHA accumulation phase in the presence and absence of methane at 55°C. Time (h) Time (h) Condition Content Condition Content 0 24 48 0 24 48 PHA Content %wt* 0.0±0.0g 0.2±0.1fg PHA Content %wt* 0.0±0.0g 0.5±0.0fg %wt* 0.0±0.0d 0.2±0.1d %wt* 0.0±0.0d 0.5±0.0d 3HB Content 3HB Content 55M1C %mol** 0.0±0.0 0.0±0.0g 100±0.0a 55M0C %mol** 0.0±0.0 0.0±0.0g 100±0.0a %wt* 0.0±0.0e 0.0±0.0e %wt* 0.0±0.0e 0.0±0.0e 3HV Content 3HV Content %mol** 0.0±0.0g 0.0±0.0g %mol** 0.0±0.0g 0.0±0.0g PHA Content %wt* 2.7±0.2def 2.6±0.2def PHA Content %wt* 3.6±0.1cde 2.2±0.6efg %wt* 0.1±0.2d 0.8±0.0cd %wt* 2.0±0.1b 0.8±0.2cd 3HB Content 3HB Content 55M1P %mol** 0.0±0.0 3.9±6.8g*** 30.4±3.4de 55M0P %mol** 0.0±0.0 54.8±1.4b 36.8±1.0d %wt* 2.6±0.2cd 1.8±0.3d %wt* 1.6±0.1de 1.4±0.4de 3HV Content 3HV Content %mol** 96.1±6.8a*** 69.6±3.4cd %mol** 45.2±1.4f 63.2±1.0d PHA Content %wt* 6.2±0.3bc 5.5±0.9bc PHA Content %wt* 4.8±0.5bcde 3.7±0.6bcde %wt* 1.7±0.2bc 1.9±0.3b %wt* 2.3±0.2b 0.9±0.1cd 3HB Content 3HB Content 55M1LV %mol** 0.0±0.0 26.8±1.9ef 34.5±1.2d 55M0LV %mol** 0.0±0.0 47.4±1.9c 23.9±1.2ef %wt* 4.6±0.2ab 3.6±0.5bc %wt* 2.5±0.3cd 2.9±0.5cd 3HV Content 3HV Content %mol** 73.2±1.9bc 65.5±1.2d %mol** 52.6±1.9ef 76.1±1.2bc PHA Content %wt* 6.2±0.3bc 9.6±3.0a PHA Content %wt* 4.1±0.1bcde 3.8±0.7bcde %wt* 1.5±0.1bc 3.5±1.2a %wt* 2.1±0.1b 0.8±0.1cd 3HB Content 3HB Content 55M1HV %mol** 0.0±0.0 24.7±1.2ef 36.4±2.2d 55M0HV %mol** 0.0±0.0 50.3±2.0bc 21.3±3.0f %wt* 4.6±0.2ab 6.1±1.8a %wt* 2.0±0.1cd 3.0±0.7bcd 3HV Content 3HV Content %mol** 75.3±1.2bc 63.6±2.2d %mol** 49.7±2.0ef 78.7±3.0b * %wt means weight percentage based on total dry biomass; ** %mol means mole percentage based on total mole of PHA produced; *** Data obtained from the condition were 0, 0, and 11.7 mol% 3HB and 100, 100, and 88.3 mol% 3HV; ± represents standard deviations (n=3) Note: - M1 and M0 stand for the presence and absence of methane, 55 and 58 stands for treatments at 55°C and 58°C, P stands for treatments with 100 ppm of propionic acid, LV and HV stands for treatments with 100 ppm and 200 ppm of valeric acid, respectively. - Statistic letters denote significant differences within each analyte for PHA content (wt%), 3HB, and 3HV content on weight (wt%) and mole (mol%) basis with p<0.05 were derived. The letters denote interactions between t = 24 and t = 48 h. There is no interaction between the data presented in this table and in the others

142 Table A7 PHA content on a mass basis (wt%) and 3HB and 3HV content on a mol basis (%mol) obtained during PHA accumulation phase in the presence and absence of methane at 55°C. Time (h) Condition Content Time (h) Condition Content 0 24 48 0 24 48 PHA Content %wt* 0.0±0.0f 0.2±0.1f PHA Content %wt* 2.8±0.3bcd 2.4±0.2cd %wt* 0.0±0.0f 0.2±0.1f %wt* 2.8±0.3bcd 2.4±0.2cd 3HB Content 3HB Content 55M1C %mol** 0.0±0.0 0.0±0.0g 100±0.0a 55M1A %mol** 0.0±0.0 100±0.0a 100±0.0a %wt* 0.0±0.0d 0.0±0.0d %wt* 0.0±0.0d 0.0±0.0d 3HV Content 3HV Content %mol** 0.0±0.0g 0.0±0.0g %mol** 0.0±0.0g 0.0±0.0g PHA Content %wt* 0.0±0.0f 0.5±0.0f PHA Content %wt* 3.7±0.6b 1.0±0.1ef %wt* 0.0±0.0f 0.5±0.0f %wt* 3.7±0.6ab 1.0±0.1e 3HB Content 3HB Content 55M0C %mol** 0.0±0.0 0.0±0.0g 100±0.0a 55M0A %mol** 0.0±0.0 100±0.0a 100±0.0a %wt* 0.0±0.0d 0.0±0.0d %wt* 0.0±0.0d 0.0±0.0d 3HV Content 3HV Content %mol** 0.0±0.0g 0.0±0.0g %mol** 0.0±0.0g 0.0±0.0g PHA Content %wt* 2.7±0.2bcd 2.6±0.2bcd PHA Content %wt* 2.9±0.8bcd 1.9±0.1de %wt* 0.1±0.2f 0.8±0.0ef %wt* 2.9±0.8abc 1.6±0.1de 3HB Content 3HB Content 55M1P %mol** 0.0±0.0 3.9±6.8f*** 30.4±3.4g 55M1AP %mol** 0.0±0.0 100±0.0a 81.6±1.1b %wt* 2.6±0.2a 1.8±0.3b %wt* 0.0±0.0d 0.4±0.0c 3HV Content 3HV Content %mol** 96.1±6.8a*** 69.6±3.4b %mol** 0.0±0.0g 18.4±1.1f PHA Content %wt* 3.6±0.1bc 2.2±0.6de PHA Content %wt* 5.7±1.0a 6.8±0.2a %wt* 2.0±0.1d 0.8±0.2e %wt* 3.9±0.7ab 4.1±0.1a 3HB Content 3HB Content 55M0P %mol** 0.0±0.0 54.8±1.4d 36.8±1.0e 55M0AP %mol** 0.0±0.0 69.5±0.5c 60.7±0.7d %wt* 1.6±0.1b 1.4±0.4b %wt* 1.7±0.3b 2.7±0.1a 3HV Content 3HV Content %mol** 45.2±1.4d 63.2±1.0c %mol** 30.5±0.5e 39.3±0.7d * %wt means weight percentage based on total dry biomass; ** %mol means mole percentage based on total mole of PHA produced; *** Data obtained from the condition were 0, 0, and 11.7 mol% 3HB and 100, 100, and 88.3 mol% 3HV; ± represents standard deviations (n=3) Note: - M1 and M0 stand for the presence and absence of methane, 55 and 58 stands for treatments at 55°C and 58°C, P stands for treatments with 100 ppm of propionic acid, LV and HV stands for treatments with 100 ppm and 200 ppm of valeric acid, respectively. - Statistic letters denote significant differences within each analyte for PHA content (wt%), 3HB, and 3HV content on weight (wt%) and mole (mol%) basis with p<0.05 were derived. The letters denote interactions between t = 24 and t = 48 h. There is no interaction between the data presented in this table and in the others

143 Table A8 Specific consumption and production of methane, carbon dioxide, propionic acid, valeric acid, PHA, 3HB, and 3HV on carbon mole basis during PHA accumulation phase in the presence of methane at 55 and 58°C. Specific rate (x100 C-mol C-mol X-1) Conditions Time Methane Carbon dioxide Acetic acid Propionic acid Valeric acid PHA 3-Hydroxybutyrate 3-Hydroxyvalerate (qCH4) (qCO2) (qAc) (qPr) (qVal) (qPHA) (q3HB) (q3HV) 0-24 hr -109.6±23.5 48.0±8.3 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0 55M1C 24-48 hr -6.5±3.5 21.4±7.7 0.0±0.0 0.0±0.0 0.0±0.0 0.2±0.1 0.2±0.1 0.0±0.0 0-24 hr -103.8±12.4 73.7±12.0 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0 58M1C 24-48 hr -42.4±5.2 31.3±1.6 0.0±0.0 0.0±0.0 0.0±0.0 0.4±0.3 0.4±0.3 0.0±0.0 0-24 hr -96.5±20.4 58.8±4.6 0.0±0.0 -28.6±0.2 0.0±0.0 5.4±0.4 0.2±0.4 5.2±0.4 55M1P 24-48 hr -17.2±10.1 24.8±1.7 0.0±0.0 -4.7±1.6 0.0±0.0 -0.4±0.2 0.7±0.4 -1.2±0.1 0-24 hr -77.2±7.9 74.3±6.8 0.0±0.0 -33.6±1.7 0.0±0.0 2.6±0.7 0.0±0.0 2.6±0.7 58M1P 24-48 hr -30.4±2.7 20.9±4.2 0.0±0.0 0.0±0.0 0.0±0.0 -0.2±0.3 0.4±0.3 -0.6±0.2 0-24 hr -85.3±5.7 62.6±6.4 0.0±0.0 0.0±0.0 -36.8±6.2 12.5±0.7 3.2±0.3 9.3±0.5 55M1LV 24-48 hr -27.5±1.3 24.3±0.4 0.0±0.0 0.0±0.0 -4.9±2.6 -0.7±0.9 0.4±0.5 -1.1±0.4 0-24 hr -64.2±0.4 60.8±2.3 0.0±0.0 0.0±0.0 -42.7±1.7 6.4±1.1 1.3±0.4 5.1±0.8 58M1LV 24-48 hr -25.2±0.0 20.1±6.2 0.0±0.0 0.0±0.0 0.0±0.0 -1.7±0.3 0.0±0.1 -1.6±0.2 0-24 hr -81.3±2.9 61.2±1.1 0.0±0.0 0.0±0.0 -42.0±2.5 12.4±0.6 2.9±0.2 9.5±0.5 55M1HV 24-48 hr -27.0±0.6 27.8±2.8 0.0±0.0 0.0±0.0 -22.6±0.8 4.4±4.3 2.5±1.5 1.9±2.8 0-24 hr -73.4±1.6 74.5±4.3 0.0±0.0 0.0±0.0 -44.0±3.6 8.7±1.6 1.8±0.3 6.8±1.3 58M1HV 24-48 hr -27.6±2.4 28.7±3.6 0.0±0.0 0.0±0.0 -24.9±1.8 4.8±1.0 2.5±0.6 2.3±0.4 Note: M1 and M0 stands for the presence and absence of methane, 55 and 58 stands for treatments at 55°C and 58°C, P stands for treatments with 100 ppm of propionic acid, LV and HV stands for treatments with 100 ppm and 200 ppm of valeric acid, respectively

144 Table A9 Specific consumption and production of methane, carbon dioxide, propionic acid, valeric acid, PHA, 3HB, and 3HV on carbon mole basis during PHA accumulation phase in the presence and absence of methane at 55°C.

Specific rate (x100 C-mol C-mol X-1) Conditions Time Methane Carbon dioxide Acetic acid Propionic acid Valeric acid PHA 3-Hydroxybutyrate 3-Hydroxyvalerate (qCH4) (qCO2) (qAc) (qPr) (qVal) (qPHA) (q3HB) (q3HV) 0-24 hr 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0 55M0C 24-48 hr 0.0±0.0 4.3±0.5 0.0±0.0 0.0±0.0 0.0±0.0 0.5±0.1 0.5±0.1 0.0±0.0 0-24 hr 0.0±0.0 4.3±1.8 0.0±0.0 -11.8±1.8 0.0±0.0 4.5±0.4 2.4±0.1 2.1±0.1 55M0P 24-48 hr 0.0±0.0 9.8±0.2 0.0±0.0 -17.2±1.4 0.0±0.0 -1.9±0.8 -1.5±0.2 -0.4±0.6 0-24 hr 0.0±0.0 4.5±0.6 0.0±0.0 0.0±0.0 -12.4±1.7 5.9±0.6 2.7±0.2 3.2±0.4 55M0LV 24-48 hr 0.0±0.0 6.6±0.6 0.0±0.0 0.0±0.0 -11.8±1.4 -1.3±1.5 -1.7±0.3 0.4±1.2 0-24 hr 0.0±0.0 6.0±0.5 0.0±0.0 0.0±0.0 -14.4±0.1 5.2±0.2 2.5±0.1 2.7±0.1 55M0HV 24-48 hr 0.0±0.0 9.4±0.2 0.0±0.0 0.0±0.0 -17.4±0.6 -0.6±.6 -1.6±0.2 1.0±0.8 0-24 hr -24.9±3.1 30.2±4.3 -5.5±0.2 0.0±0.0 0.0±0.0 3.6±0.0 3.6±0.3 0.0±0.0 55M1A 24-48 hr -31.3±1.1 52.0±3.9 -6.3±1.1 0.0±1.1 0.0±0.0 -0.5±0.2 -0.5±0.2 0.0±0.0 0-24 hr 0.0±0.0 6.7±0.3 -15.3±1.7 0.0±0.0 0.0±0.0 4.6±0.8 4.6±0.8 0.0±0.0 55M0A 24-48 hr 0.0±0.0 3.6±0.2 0.0±0.0 0.0±0.0 0.0±0.0 -3.5±0.7 -3.5±0.7 0.0±0.0 0-24 hr -9.0±3.1 15.2±1.8 -3.0±0.1 -3.5±0.2 0.0±0.0 3.3±0.8 3.3±0.8 0.0±0.0 55M1AP 24-48 hr -4.1±3.9 25.0±4.5 -5.5±0.3 -6.2±0.4 0.0±0.0 -1.1±0.9 -1.6±1.0 0.5±0.0 0-24 hr 0.0±0.0 6.9±0.3 -10.5±1.3 -4.5±0.9 0.0±0.0 7.2±1.2 4.9±0.8 2.3±0.3 55M0AP 24-48 hr 0.0±0.0 8.8±1.1 -5.0±1.0 -16.4±1.5 0.0±0.0 1.2±1.3 0.1±0.9 1.2±0.4 Note: M1 and M0 stands for the presence and absence of methane, 55 stands for treatment at 55°C, A and P stands for treatment with 100 ppm of acetic acid or propionic acid, AP stands for treatment with 100-ppm of acetic acid with 100-ppm propionic acid

145