Methanol Production from Biogas with a Thermotolerant

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Methanol production from biogas with a thermotolerant methanotrophic consortium isolated from an anaerobic digestion system Zhongliang Su, Xumeng Ge, Wenxian Zhang, Lingling Wang, Zhongtang Yu, and Yebo Li Energy Fuels, Just Accepted Manuscript • DOI: 10.1021/acs.energyfuels.6b03471 • Publication Date (Web): 17 Feb 2017 Downloaded from http://pubs.acs.org on February 18, 2017

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Methanol production from biogas with a thermotolerant methanotrophic consortium

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isolated from an anaerobic digestion system

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Zhongliang Su a,b,1, Xumeng Ge a,c,1,*, Wenxian Zhang a,d, Lingling Wang e, Zhongtang Yu e,

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Yebo Li a,*

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a. Department of Food, Agricultural and Biological Engineering, The Ohio State University/Ohio

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Agricultural Research and Development Center, 1680 Madison Ave., Wooster, OH, 44691-4096,

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USA

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b. Department of Biotechnology, Qingdao University of Science and Technology, Box 47, 53

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Zhengzhou Road, Qingdao, 266042, China

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c. quasar energy group, 2705 Selby Rd., Wooster, OH 44691

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d. Engineering Research Center of Industrial Microbiology, Ministry of Education, College of

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Life Sciences, Fujian Normal University, Fujian 350108, China

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e. Department of Animal Sciences, The Ohio State University, 2029 Fyffe Court, Columbus, OH

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43210, USA

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* Corresponding authors. Tel.: +1 330 202 3561; fax: +1 330 263 3670.

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E-mail: [email protected] (X. Ge); [email protected] (Y. Li);

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1

Authors contributed equally to this work.

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1 ACS Paragon Plus Environment

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Abstract

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Thermotolerant methanotrophic consortia are desirable for their robustness under stressful

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environments during industrial applications. A thermotolerant methanotrophic consortium, MC-

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AD3, was enriched from digestate in an anaerobic digestion (AD) system, and evaluated for cell

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growth and methanol production with biogas. MC-AD3 obtained cell yields of 0.22-0.40 g cells/

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g methane at temperatures from 30°C to 55°C and pH from 5.5 to 7.5, and achieved the highest

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cell yield of 0.4 g cells/ g methane at 47°C at a biogas to air ratio of 1:4 (v/v) and pH of 6.8. MC-

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AD3 produced 0.33 g/L of methanol at 47°C, with a methanol conversion ratio of 0.47 mol

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methanol/ mol methane. A biogas to air ratio of 1:1 (v/v) was found to be optimal for methanol

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production with MC-AD3. The cell growth and methanol production performance of MC-AD3 at

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47°C fell within the range of those obtained by other methanotrophic strains/consortia at lower

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temperatures.

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Keywords: biogas upgrading; thermotolerant methanotrophic consortia; anaerobic digestate;

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methane; methanol

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Highlights

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A thermotolerant methanotrophic consortium was isolated from anaerobic digestate.

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The consortium grew stably at 30-55°C and pH of 5.5-7.5 on biogas.

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Cell yield up to 0.4 g cells/ g methane consumed was obtained.

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The thermotolerant consortium produced 0.33 g/L of methanol from biogas at 47°C.

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A conversion ratio of 0.47 mol methanol/ mol methane consumed was achieved at 47°C.

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1. Introduction Biogas (mainly methane and carbon dioxide) is produced from anaerobic digestion (AD) of

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organic wastes, such as crop residues, municipal solid waste, food waste, sewage sludge, and

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animal manure 1,2. As a renewable source of energy, biogas has been used to produce electricity

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using combined heat and power (CHP) systems 3. Nevertheless, biogas is in a gaseous form at

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ambient temperatures, and is difficult and costly to store, transport, and distribute 4. This issue

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can be addressed by conversion of methane to methanol, which is an important building block

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for transportation fuels and chemicals 5. Methane-to-methanol conversion is commonly

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conducted via thermochemical conversion, which involves high pressure and/or temperature and

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expensive chemical catalysts 6. Compared to thermochemical conversion methods, biological

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conversion of biogas to methanol is more attractive for biogas upgrading due to efficient

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conversion reactions under mild conditions 7–9.

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Methanotrophs are a group of bacteria that can use methane as only carbon source for

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growth 4. Currently, all enriched/isolated methanotrophs are aerobic. Although anaerobic

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methanotrophs (ANME) have also been identified, no ANME have been isolated either in pure

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culture or in a consortium up to date 4. Methanotrophs generally convert methane to methanol

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with methane monooxygenase (MMO), consuming two reducing equivalents per molecule of

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methane oxidized 1,10. Under normal conditions, methanotrophs further oxidize methanol to CO2

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with formaldehyde and formate as intermediates, which are catalyzed by methanol

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dehydrogenase (MDH), formaldehyde dehydrogenase (FalDH), and formate dehydrogenase

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(FDH), in a sequential manner 1,10. Methanol accumulation is generally conducted with addition

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of MDH inhibitors, including phosphate, EDTA, MgCl2, high salinity, and cyclopropanol 4,11,12.

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When MDH is inhibited, external electron donors, mainly formate, are needed to maintain cell


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vitality of methanotrophs, which can only utilize mono-carbon compounds 4. Production of

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formic acid from CO2 and H2O has been achieved via electrochemical methods, which offer a

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cheap supply of formate as an electron donor 13,14. However, studies on biological conversion of

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biogas to methanol are still limited and there are challenges for industrial scale production 4.

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One of these challenges is the lack of robust methanotrophic strains with high tolerance to

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environmental stresses that may occur during industrial applications 4,15. Pure methanotrophic

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strains, mainly Methylosinus trichosporium, are routinely used for methanol production from

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methane at a lab scale 4,16. However, pure methanotrophs are generally vulnerable to

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contamination by other microorganisms, posing a high risk in industrial scale applications. It is

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believed that diversity in bacterial communities can promote stability of ecosystems 17. For

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example, researchers in microalgae-based biofuel production have proposed to use wild consortia

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of microalgae rather than monospecific microalgal cultures to reduce the risk of contamination

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by “weed” microalgae or predators 18. Similar strategies might be effective for methanol

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production with methanotrophs. However, only one study has reported methanol production

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from biogas with a methanotrophic consortium isolated from landfill cover soil 19.

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Another strategy to minimize contamination by other microorganisms is to use

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thermophilic or thermotolerant strains that can grow at relatively high temperatures 20. Besides,

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thermotolerant strains are also desirable for reduced cooling requirement which could be

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challenging for large scale fermentation process during which heat is generally removed by using

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a cooling device 21. Several thermophilic and thermotolerant methanotrophs have been identified

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with optimum growth at 37-57ºC, although they have not been used for methanol production 22.

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Isolation of robust methanotrophic consortia is a promising option for methanol production from


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biogas. To date, there have been no reports on the isolation of thermophilic/thermotolerant

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methanotrophic consortia for methanol production from biogas.

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The objectives of this study were to: 1) isolate thermophilic/thermotolerant methanotrophic

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consortia from digestate in AD systems; and 2) evaluate environmental conditions on cell growth

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and methanol production with selected consortia. Digestate from different AD systems were used

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as sources for isolation of methanotrophic consortia. One thermotolerant methanotrophic

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consortium that accumulated higher methanol than others was selected. The effect of the biogas

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to air ratio, temperature, and/or pH on cell growth and methanol production with this consortium

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were investigated.

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2. Materials and methods 2.1. Enrichment of methanotrophic consortia from digestate Enrichment of methantrophic consortia were conducted on the digestate from AD

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experiments that used different feedstocks (expired dog food, corn stover, and giant reed) with

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different total solids (TS) contents and feedstock to inoculum (F/I) ratios. These AD experiments

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were conducted for previous studies and specific conditions for each are shown in Table 1. AD

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effluent collected from a mesophilic liquid anaerobic digester (KB BioEnergy, Akron, OH, USA)

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was activated at 37ºC or 55ºC for one week, and used as an inoculum for AD. The activated AD

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effluent, and digestate collected after AD experiments were used as sources prior to use for

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enrichment.

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The enrichment was conducted according to protocols reported by Bowman 23, Dedysh and

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Dunfield 24, and Sheets et al. 25. Briefly, 5 g of digestate sample was mixed with 20 ml of nitrate

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mineral salts (NMS) medium in a 125-mL flask (in duplicate). The NMS medium contained


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MgSO4·7H2O (1.0 g L-1), KNO3 (1.0 g L-1), KH2PO4 (0.272 g L-1), Na2HPO4 (0.284 g L-1),

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CaCl2·2H2O (0.134 g L-1), chelated Fe solution (0.2% (v/v)), and a trace element solution (0.05%

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(v/v)). The chelated Fe solution was prepared by dissolving ferric (III) ammonium citrate (1.0 g

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L-1), EDTA (2.0 g L-1), and concentrated HCl (0.3% (v/v)) in deionized (DI) water. The trace

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element solution was prepared by dissolving EDTA (500 mg L-1), FeSO4·7H2O (200 mg L-1),

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ZnSO4·7H2O (10 mg L-1), MnCl2·4H2O (3.0 mg L-1), H3BO3 (30 mg L-1), CoCl2_6H2O (20 mg

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L-1), CaCl2·2H2O (1.0 mg L-1), NiCl2·6H2O (2.0 mg L-1), and Na2MoO4·2H2O (3.0 mg L-1) in DI

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water. Five milliliters of each suspension was inoculated into 50 mL of NMS medium in a 250-

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mL flask. The flask was filled with 20% (v/v) CH4 (99% purity purchased from Praxair®,

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Danbury, CT, USA) and 80% (v/v) air in its headspace, and sealed with a rubber stopper. The

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flasks were incubated at 37°C or 55°C, and stirred at 150 rpm for enrichment of methanotrophs

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(Table 1). Every 5 days, 5 mL of the culture was successively transferred to 50 mL of fresh NMS

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medium in another 250-mL flask, which was then filled with the gas mixture (CH4/air = 1:4, v/v)

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and incubated under the same conditions. The enrichment process was repeated 3 times.

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Consortia were cultured in 2 mL of NMS medium supplemented with 2 µM of CuCl2 and

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50 mM of sodium formate in 15-mL sealed culture tubes. The tubes were filled with CH4 and air

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(1:4, v/v) in their headspace, and incubated at 37°C and stirred at 150 rpm for 48 h. Cell

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suspensions were filtered (0.2 µm) and the filtrates were subjected to methanol analysis using gas

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chromatography (GC). The methanotrophic consortium from AD3 (MC-AD3) which obtained

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the highest methanol concentration was selected for further evaluation.

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Microbial community in MC-AD3 was analyzed with 16S rRNA gene sequencing. It was revealed that MC-AD3 contained methanotrophic bacterium, Methylocaldum, (87.21%), and


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other bacteria including Agrobacterium (7.02%), Alcaligenaceae (4.66%), Stenotrophomonas

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(0.42%), Limnohabitans (0.11%), and Paenibacillus (0.05%).

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2.2. Cell growth on biogas under different conditions Biogas samples (A: 72.04% CH4, 26.02% CO2, 1.66% N2, and 0.28% O2; or B: 64.09%

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CH4, 34.39% CO2, 0.97% N2, and 0.55% O2) were collected from a commercial scale digester

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(quasar energy group, Wooster, OH, USA) which was fed with food waste 26. MC-AD3 was

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inoculated into 30 mL of NMS medium in 250-mL flasks to reach an initial cell density of about

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0.4 g/L. Each flask was connected to a 500-mL Tedlar gas bag, and the headspace of the flask

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and Tedlar gas bag was filled with a specified biogas/air mixture with a total gas volume of 500

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mL. The flasks were inoculated at the designated temperature and shaken at 150 rpm for 192 h.

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Gas composition (CH4, O2, N2, and CO2) was monitored every day. Cell density was measured

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after the cultivation for determination of cell yield from methane (g dry biomass produced/ g

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methane consumed).

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The effect of biogas to air ratio, temperature, and pH on cell growth of MC-AD3 was

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evaluated in a sequential manner. Briefly, the optimal biogas to air ratio was first determined

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with cell cultivation at 37°C and a pH of 6.8 with different biogas to air ratios (1:2, 1:4, and 1:6,

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v/v) according to a previous study 25. The second run of cell cultivation was then conducted at

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the optimal biogas to air ratio and a pH of 6.8, but at different temperatures (30°C, 37°C, 42°C,

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47°C, 50°C, and 55°C), in order to determine optimal temperature. Optimal pH was determined

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with the third run of cell cultivation at the optimal biogas to air ratio and temperature with

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different pH levels (5.5, 6.0, 6.8, and 7.5). Three replicates were used for each condition. Biogas

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sample A was collected first and used for the experiment of the effect of biogas to air ratio on


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cell growth. In order to allow more repeated trails for further experiments, biogas sample B was

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collected with a larger volume. After the experiment on optimization of temperature for cell

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growth, the gas bag of sample B was accidentally broken. As a result, biogas sample A was used

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for all the rest of experiments.

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2.3. Methanol production from biogas under different conditions MC-AD3 was grown in 30 mL of NMS medium in 250-mL flasks at 37°C and a pH of 6.8

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with a biogas/air (1:4, v/v) mixture in the headspace. Four flasks were setup for each condition.

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After 8 days, MC-AD3 cultures in the four flasks were combined, and the cells were harvested

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by centrifugation at 8,000 g for 10 min. The harvested cells were re-suspended in 120 mL of

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fresh NMS medium (pH 6.8) with 5 µM of CuCl2 and 100 mM of sodium formate. The cell

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suspension was mixed well and transferred into four 250-mL flasks (30 mL in each flask). One

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of the flasks was used to determine initial cell density (about 0.23 g/L, dry weight). The other

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three were used for methanol production. Each of the three flasks was connected to a 500-mL

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Tedlar gas bag. The headspace of the flask and Tedlar gas bag was filled with a biogas/air (1:4,

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v/v) mixture to reach a total volume of 700 mL, and the flask was inoculated at a designated

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temperature with shaking at 150 rpm for 168 h. Gas composition (CH4, O2, N2, and CO2) and

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methanol concentration were monitored every 12-24 h. Methanol yield from methane (mol

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methanol produced/ mol methane consumed) was calculated to determine optimal conditions.

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Methanol production with MC-AD3 was conducted at a biogas to air ratio of 1:1 (v/v) and

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different temperatures (37°C, 47°C, and 55°C), in order to evaluate effect of temperature on

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methanol production. Effects of the biogas to air ratio on methanol production was further


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evaluated for methanol production at 47°C with different biogas to air ratios (2:1, 1:1, 1:2, 1:4,

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and 1:6, v/v). Three replicates were used for each condition.

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2.4. Analytical methods

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Cell density was determined using a method originated by Zhu and Lee 27 and modified by

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Sheets et al. 25. Briefly, 30 mL of cell suspension was centrifuged at 10,000 rpm for 15 min, and

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the supernatant was discarded. In order to remove residual salts, the cell pellet was re-suspended

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in 25 mL of 0.5 M NH4HCO3, and centrifuged again at 10,000 rpm for 15 min. After the

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supernatant was discarded, the cell pellet was transferred to a pre-ignited (550°C) porcelain

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crucible with 3 mL of 0.5 M NH4HCO3 and dried in a Thelco Model 18 oven (Precision

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Scientific, Chennai, India) at 105°C for 12 h to determine the dry weight of total biomass. The

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dry biomass sample was heated in an Isotemp muffle furnace (Fisher Scientific, IA, USA) at

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550°C for 4 h to determine the ash weight. The ash-free dry weight was calculated as the

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difference between the dry weight of total biomass and the weight of the residual ash.

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Gas composition (CH4, CO2, N2, and O2) was analyzed using a gas chromatograph (Agilent,

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HP 6890, Wilmington, DE, USA), which was equipped with a 30 m × 0.53 mm × 10 µm Rt®-

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Alumina Bond/KCl deactivation column and a thermal conductivity detector. Helium gas was

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used as the carrier gas with a flow rate of 5.2 mL/min. Temperatures of the injector and detector

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were set at 150°C, and 200°C, respectively. The temperature of the column oven was initially set

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at 40°C for 4 min and later increased to 60°C at 20°C/minute and held at 60°C for 5 min.

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Methanol concentration in the filtrate samples was analyzed using a gas chromatograph

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(Shimadzu, 2010PLUS, Columbia, MD, USA), which was equipped with a Stabilwax polar

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phase column (30 m × 0.32 mm × 0.5 µm) and flame ionization detector. The temperatures of


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both the injector and the detector were set at 250°C, while the temperature of the column oven

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was initially set at 50°C and gradually increased to 80°C at a rate of 5.0°C/min. Helium was used

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as the carrier gas with a total flow rate of 24.8 mL/min and a split ratio of 15.

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2.5. Statistical analysis Analysis of variance (one-way ANOVA, α = 0.05, i.e. confidence level = 95%) was

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conducted using Minitab (Version 16, Minitab, Inc., State College, PA, USA) for assessing

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statistical significance.

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3. Results and discussion

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3.1. Enrichment of methanotrophic consortia from digestate

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Generally, methanotrophic consortia were obtained from digestate in mesophilic AD

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systems via enrichment at 37°C (Table 1). One exception was that enrichment of methanotrophic

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consortia failed using digestate from AD4 (Table 1). AD4 was conducted at a high F/I ratio, and

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was upset with a low pH of about 5 due to volatile fatty acid accumulation (data not shown).

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Methanotrophs in the AD system might not tolerate the low pH, which could have resulted in the

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failure of enrichment. Besides, no methanotrophic consortia were enriched at 55°C. These results

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indicated that the AD systems as well as the inoculum (AD effluent) likely lack acidophilic and

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thermophilic methanotrophs (Table 1).

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All the enriched methanotrophic consortia produced methanol. The highest methanol

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concentration (276 mg/L) was obtained with the methanotrophic consortium enriched from

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digestate in AD3 (Table 1). This consortium was named as MC-AD3 and used for further

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experiments.


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3.2. Effect of biogas to air ratio on cell growth of MC-AD3 When the MC-AD3 was cultivated at a biogas to air ratio of 1:2 (v/v), the methane content

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decreased slowly from 24% to 18% in 8 days (Figure 1a). A biogas to air ratio of 1:4 (v/v)

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resulted in a faster decrease (from 15% to 4%) of methane content than biogas to air ratio of 1:2

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(v/v) during 8 days of cell growth (Figure 1a). However, when the biogas to air ratio was

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decreased to 1:6 (v/v), the methane content decreased from 10% to 1% in 8 days, showing a

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slightly slower decrease of methane content than that obtained with a biogas to air ratio of 1:4

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(v/v) (Figure 1a). The cell yields from methane was 0.133, 0.234 and 0.202 g cells /g methane

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when biogas to air ratios were 1:2, 1:4, and 1:6 (v/v), respectively (Figure 1b). The biogas to air

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ratio of 1:4 (v/v) obtained the fastest decrease of methane content and highest cell yield from

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methane. However, there was no significant (p > 0.05) difference in cell yield between biogas to

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air ratios of 1:4 (v/v) and 1:6 (v/v), or initial methane contents of 15% and 10% (Figure 1b). This

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also supports the feasibility of using biogas sample B for further experiments (as mentioned in

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section 2.2) with an initial methane content of about 12-13% at the biogas to air ratio of 1:4 (v/v).

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The cell yield from methane with MC-AD3 was comparable to that of Methylocaldum 14B (0.2 g

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cells /g methane), a pure methanotrophic strain that was isolated from solid-state anaerobic

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digestate 25. However, the cell yields of MC-AD3 were still lower than those (0.5-0.7 g cells /g

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methane) of Methylosinus trichosporium OB3b, which indicates that MC-AD3 oxidized more

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methane for energy generation than for biomass production compared to M. trichosporium OB3b

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28,29

.

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3.3. Effect of temperature and pH on cell growth of MC-AD3


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Figure 2a shows methane content changes during cultivation of MC-AD3 at different

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temperatures. Interestingly, methane consumption increased as temperature increased from 30°C

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to 50°C, although methane consumption decreased when the temperature was further increased

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to 55°C (Figure 2a). For example, the methane content decreased from 12% to 3% in 144 h at

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37°C, while the same decrease of methane content only took 48 h at 50°C (Figure 2a). Cell

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yields from methane ranged from 0.22 to 0.40 g cells/ g methane with temperatures from 30°C to

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55°C (Figure 2b). Two methanotrophic strains, 14B and SAD2, which were also isolated from

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AD systems, grew effectively at 37-42°C and 30-37°C, respectively 25,30. Compared to

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methanotrophic strains 14B and SAD2, MC-AD3 had a much wider temperature range for cell

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growth. Furthermore, the highest cell yield (0.4 g cells/ g methane) of MC-AD3 was obtained at

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47°C, which indicates that MC-AD3 is a thermotolerant consortium (Figure 2b). According to

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Trotsenko et al. 22, a few thermophilic and thermotolerant methanotrophs were isolated from

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sources, such as bottom deposits of water bodies, activated sludge, soil, thermal spring silage,

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and manure. Most of these methanotrophs had optimum growth at 37-42°C, and only two

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showed optimum growth at 55-57°C 22. This study is the first time that a thermotolerant

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methanotrophic consortium was enriched from digestate in AD systems.

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Figure 3 further illustrates the effect of pH on methane consumption and cell yield of MC-

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AD3 at 47°C. When the initial pH was 5.0, there was minimal decrease of methane content

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during 6 days, indicating an inhibition of methane consumption with MC-AD3 (Figure 3a).

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Methane consumption was found to be consistent for initial pH levels of 5.5-7.5, with the

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methane content decreasing from 15% to 1.3-2.8% in 114 h (Figure 3a). In addition, cell yields

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from methane consumption were 0.20-0.33 g cells/ g methane at pH levels from 5.0 to 7.5

270

without significant (p > 0.05) difference (Figure 3b). The pH range (5.5-7.5) for cell growth was


Energy & Fuels

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271

comparable to that of 14B and SAD2, and to thermophilic/thermotolerant methanotrophic strains,

272

such as Methylococcus capsulatus, Methylocaldum szegediense, Methylothermus thermalis, and

273

Methylocystis sp. Se48 22,25,30.

274 275

3.4. Effect of culture conditions on methanol production from biogas with MC-AD3

276

Figure 4 shows methanol production performance of MC-AD3 at different temperatures.

277

Interestingly, methanol production with MC-AD3 also preferred a relatively high temperature

278

(47°C) (Figure 4). A methanol concentration of 0.33 g/L was obtained by MC-AD3 at 47°C,

279

which was 38% higher than that obtained at 37°C (Figure 4a). MC-AD3 accumulated a minimal

280

amount of methanol (0.04 g/L) at 55°C (Figure 4a), although it grew stably at this high

281

temperature (Figure 2). The methane to methanol conversion ratio (0.47 mol methanol/ mol

282

methane) at 47°C was also significantly (p < 0.05) higher than those at 37°C and 55°C (Figure

283

4b). According to Figures 2 and 4, MC-AD3 appeared to be more sensitive to high temperature

284

for methanol production than for cell growth. Methanol production with methanotrophs has

285

routinely been conducted at 25-37°C 4,16,25,30. Before this study, there have been no reports on

286

methanol production with methanotrophs at temperatures higher than 37°C, although

287

thermophilic/thermotolerant methanotrophs have been isolated and characterized in terms of cell

288

growth 22.

289

As shown in Figure 5a, different biogas to air ratios also resulted in different methanol

290

concentrations during 7 days of methanol production process. Relatively high methanol

291

concentrations of 0.33 g/L and 0.35 g/L were obtained with biogas to air ratios of 1:1 (v/v) and

292

1:2 (v/v), respectively (Figure 5a). The methanol conversion ratio (0.47 mol methanol/ mol

293

methane) achieved by using a biogas to air ratio of 1:1 (v/v) was significantly higher than those


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Energy & Fuels

294

obtained with other biogas to air ratios (Figure 5b). According to Figure 1 and 5, MC-AD3

295

preferred greater methane content for methanol production than that for cell growth.

296

As show in Table 2, methanol concentrations obtained by MC-AD3 at 47°C were

297

comparable to those achieved by methanotrophic strains 14B and SAD2, which were also

298

isolated from anaerobic digestate but grew and produced methanol at lower temperatures (around

299

37°C) (Table 2) 25,30. Besides, the methanol conversion ratio achieved by MC-AD3 at 47°C falls

300

within the range of those (0.23-0.80 mol methanol/ mol methane) obtained by other

301

methanotrophic strains/consortia at lower temperatures (25-37°C) (Table 2).

302

Currently, methanol concentrations obtained by methanotrophic bacteria are still low for

303

large scale production 4. A major reason is that methanol is toxic to cells at high concentrations,

304

which limits the final methanol concentration 25. Besides directly screening methanol tolerant

305

methanotrophic strains, it is also promising to investigate genes responsible for the methanol

306

tolerance, and further improve the methanol tolerance via genetic manipulations.

307 308 309

4. Conclusion The thermotolerant methanotrophic consortium, MC-AD3, was isolated from digestate in

310

AD systems. MC-AD3 grew stably for a wide range of temperature (30-55°C) and pH (5.5-7.5),

311

and achieved the highest cell yield (0.4 g cells/ g methane) at 47°C with a biogas to air ratio of

312

1:4 (v/v) and pH of 6.8. MC-AD3 produced 0.33 g/L of methanol at 47°C with a methanol

313

conversion ratio (0.47 mol methanol/ mol methane) that fall within the range of those obtained

314

by other strains at lower temperatures. As a result, the MC-AD3 is a promising candidate for

315

conversion of biogas to methanol at large scale.

316


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317

Acknowledgements

318

This material is based upon work that is supported by the National Institute of Food and

319

Agriculture, U.S. Department of Agriculture, under award number 2012-10008-20302, and by

320

state and federal funds appropriated to The Ohio State University, Ohio Agricultural Research

321

and Development Center. This research is also funded by China Scholarship Council. The

322

authors thank Mrs. Mary Wicks for her comprehensive review and thoughtful comments.

323 324

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376 377 378 379

Energy & Fuels

Table 1 Selection of methanotrophic consortia isolated from digestate from different anaerobic digestion (AD) systems AD conditions

C1

380 381 382 383 384 385

Cell growth

Methanol production (mg/L)

Feedstock

TS (%)a

F/Ib

T (ºC)

T (ºC) for enrichment

-

-

-

37c

37

+

192

c

55

-

-

No.

C2

-

-

-

55

AD1

Expired dog food

20

2

37

37

+

213

AD2

Expired dog food

20

2

55

55

-

-

AD3

Corn stover

20

3

37

37

+

276

AD4

Corn stover

20

4

37

37

-

-

AD5

Giant reed

20

2

37

37

+

147

AD6

Giant reed

8

1

37

37

+

205

a: total solids content; b: feedstock to inoculum ratio; c:AD effluent was activated at the temperature for one week.


Energy & Fuels

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386 387 388 389

Page 20 of 25

Table 2 Methanol production conditions and performance of methanotrophic strains and consortia

Sources

T (ºC)

Methanol concentration (g/L)

CH4 to methanol conversion ratio

Consortium

Landfill cover soil

30

0.19-0.22

0.43-0.80

19

M. trichosporium

Not reported

25-35

0.17-1.12

0.27-0.61

12,31,32

Strain 14B

Anaerobic digestate

37

0.43

0.26

25

Strain SAD2

Anaerobic digestate

37

0.34

0.34

30

MC-AD3

Anaerobic digestate

47

0.33

0.47

This study

Strains/ Consortia

390 391 392 393 394 395 396 397 398 399 400 401 402 403


References

Page 21 of 25

404 405 32

0.30 Cell yield from methane (g/g)

Biogas to Air = 1:2 Biogas to Air = 1:4 Biogas to Air = 1:6

28 Methane content (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Energy & Fuels

24 20 16 12 8 4 0

a 0.25

ab

0.20 b 0.15 0.10 0.05 0.00

0

24 48 72 96 120 144 168 192 Time (h)

1:6 1:4 1:2 Biogas to air ratio (v/v)

406 407 408 409

Figure 1. Effect of biogas to air ratio on (a) methane consumption and (b) cell yield from

410

methane during cultivation of MC-AD3 using biogas as carbon source at 37°C and an initial pH

411

of 6.8

412

Note: Means that do not share a letter are significantly different.

(a)

(b)

413


Energy & Fuels

414 415 0.50

12 10

Cell yield from methane (g/g)

30°C 37°C 42°C 47°C 50°C 55°C


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 22 of 25

8 6 4 2

a

a

0.40

ab cd

bc

0.30

d 0.20

0.10

0 0

24

48

72

96

120

25

144

30

Time (h)

35

40

45

50

55

60

Temperature (°C)

416 417 418 419

Figure 2. Effect of temperature on (a) methane consumption and (b) cell yield from methane

420

during cultivation of MC-AD3 using biogas as carbon source with a biogas to air ratio of 1:4 (v/v)

421

and an initial pH of 6.8

422


(a)

(b)

423


Page 23 of 25

424 425

16

0.4

pH 5.5 pH 6.8

Cell yield from methane (g/g)

pH 5.0 pH 6.0 pH 7.5


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Energy & Fuels

12 8 4 0 0

24

48

72

96

a a

a a

0.3

a

0.2

0.1

0

120 144

5.0

5.5

6.0

Time (h)

pH

(a)

(b)

6.8

7.5

426 427 428 429

Figure 3. Effect of initial pH on (a) methane consumption and (b) cell yield from methane during

430

cultivation of MC-AD3 using biogas as carbon source at 47°C and a biogas to air ratio of 1:4

431

(v/v)

432

a: There are no significant differences among means.

433 434


Energy & Fuels

435 436 CH4 to methanol conversion ratio (mol/mol)

0.4 Methanol concentration (g/L)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 24 of 25

37°C 47°C 55°C

0.3

0.2

0.1

0

0.7 0.6

a

0.5 0.4 0.3 b 0.2 0.1

b

0 0

24

48

72

96 120 144 168

37

Time (h)

47 Temperature (ºC)

(a)

(b)

55

437 438 439 440

Figure 4. Effect of temperature on (a) methanol accumulation and (b) methane to methanol

441

conversion ratio by MC-AD3 using biogas as carbon source at a biogas to air ratio of 1:1 (v/v)

442


443


Page 25 of 25

444 445 0.4

0.7 Biogas:Air = 2:1 Biogas:Air = 1:1 Biogas:Air = 1:2 Biogas:Air = 1:4 Biogas:Air = 1:6

0.3

CH4 to methanol conversion ratio (mol/mol)

Methanol concentration (g/L)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Energy & Fuels

0.2

0.1

a

0.6 0.5 0.4 0.3

b b b

0.2

b

0.1 0.0

0.0 0

24

48

2:1

72 96 120 144 168 Time (h)

1:1 1:2 1:4 Biogas to air ratio (v/v)

1:6

446 447 448 449

Figure 5. Effect of biogas to air ratio on (a) methanol accumulation and (b) methane to methanol

450

conversion ratio by MC-AD3 using biogas as carbon source at 47°C

451


(a)

(b)

452 453 454


Methanol Production from Biogas with a Thermotolerant

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