BIOLOGICAL CONVERSION OF METHANE

Matthew Scott Cantu, Erin Loth, Miranda Reinhard, Jane Ooi, Ellen Jun Liu, Kiersten Wiley, Danny Lou, Vianney Trujillo, Carolina Castro

WHAT IS METHANE?
Main component of natural gas Most abundant organic compound on earth Relative abundance = attractive fuel Transportation difficult due to gaseous state

OVERVIEW
Anaerobic Fermentation for Methane Methane Production Non-biological Conversion Biological Conversion Problems to Consider Microorganisms Production Industry Use Future Prospects

WHERE DO WE GET METHANE?

Numerous purification means Biological: Anaerobic Digestion Primarily a resource from wastewater treatment

In use since late 1800s septic systems

Widely popular in Europe, yet to take off in US

METHANE PRODUCTION

Four steps:

Hydrolysis Acidogenesis Acetogenesis Methanogenesis

HYDROLYSIS
Breaking complex organic matter into simpler compounds
Proteins to amino acids, fats to acids, etc.

Can be the limiting step in methane production for time

ACIDOGENESIS

Partial step found with some bacteria Break down of nutrients to volatile fatty acids

Propionate Butyrate Lactate Many others

ACETOGENESIS

A step that provides further food sources for bacteria Break down of volatile fatty acids to form acetate

Hydrogen gas and carbon dioxide byproducts

Can bypass acidogenesis by using monomers from fats

METHANOGENESIS

Primary process of concern in methane production Food sources for methanogens fermented to form biogas

~60/35/5 split of Methane, Carbon Dioxide, and various gases Can be highly limited by hydrogen and potentially oxygen produced form earlier steps

PRODUCING METHANE
Anaerobic digestion naturally occurs where no oxygen is found Can be contained in reactors, gas harvested Heavily dependent on feedstock compositions

FEEDSTOCK

Carbon/Nitrogen ratio

Ratio of organic carbon to nitrogen found in feedstock

C:N ratio must be ideal for efficient methane production

Too low, no methanogenesis production will occur Too high, extremely slow methanogenesis will occur Aim for about 20-30:1 C:N

FEEDSTOCK

Natural wastewater may not be the most efficient

Anaerobic digestion can still work and be effective, but methanogenesis can be improved

Improve methane production through co-digestion

CO-DIGESTION

Addition of material to influent, balancing out composition

Can add high C:N material to low C:N material, reaching a middle ground

Virtually any organic compound can be used, within reason

CO-DIGESTION

Various feedstocks in research include

Crop residues

Can be used on farm-scale plants Useful in manure treatment

Straws and grasses

Fruit and vegetable waste

Typically low in cellulose, hydrolysis not rate-limiting; high in acids

Very rich in carbon

Paper waste

Wastewater sludge

Extension of composting method

Low C:N ratio, use other digestrates; can offset biodiesel costs

Algal sludge

PRETREATMENT

Can modify influent or co-digestrates to improve process

Can lower time for hydrolysis Can provide more efficient conditions for reactions Mechanical, chemical, thermal pretreatment strategies

METHANE PRODUCTION

With feedstock in mind, how do we contain the process?

PRODUCTION OF METHANE

Anaerobic Digestion 4 main types of reactors:

Covered Lagoon Digester Complete Mix Digester Plug Flow Digester Upflow Anaerobic Sludge Blanket

COVERED LAGOON DIGESTER

Passive system First cell covered, second cell uncovered

COMPLETE MIX DIGESTER

Low rate Large tank Manure and microorganisms mixed

PLUG FLOW DIGESTER

Similar to complete mix Manure moves through system as a plug

UPFLOW ANAEROBIC SLUDGE BLANKET

Suspended media digester Microbes suspended Liquid flow constantly upward Works best with more dilute waste

WHAT DO THEY HAVE IN COMMON?

Microorganisms break down organic matter No oxygen in system Biogas produced Share attachable units that convert methane to electricity to power reactor, typically used as an electric power generator

TRADITIONAL CONVERSION OF METHANE TO METHANOL

Nonbiological Thermal conversion process Lignocellulosics undergo:

Gasification Fischer-Tropsch Hydrocarbons/Bio-oil Biofuels

GASIFICATION

Converts lignocellulosic biomass into syngas (CO and H2) through reactions at high temperatures (>700C) with limited oxygen and steam This high temperature distinguishes gasification from biological processes like anaerobic digestion that produce syngas. Syngas energy can serve as a feedstock for electricity generation or a building block for chemicals

GASIFICATION
Steps: 1. Biomass
2.

Lignin, polysaccharides syngas

Intermediates

3.

Fuel Products

Alkanes (jet fuel), ethanol, FischerTropsch liquids, DME

GASIFICATION

GASIFICATION CHAMBER

FISCHER-TROPSCH PROCESS

1. 2.

3.

AKA Fischer-Tropsch Synthesis A catalyzed chemical reaction where synthesis gas or syngas is converted into liquid hydrocarbons as biofuels Steps: Liquid synthesis Preparation of synthetic hydrocarbons (oils and waxes) Product separation and refining (fuels, lubricants, waxes)

GASIFICATION AND FISCHERTROPSCH PROCESS

THERMAL CONVERSION

Can accept a wide range of feedstocks Fewer steps are involved Technology might be more suitable for small scale fuel production There are fewer product and byproduct streams

OF METHANE USING

ARCHAEA

Archaea can consume methane and produce cellular carbon and energy

anoxic environment Archaea and sulfate-reducing bacteria can couple methane oxidation with sulfate reduction Methane is converted by methanotrophic archeae to carbon dioxide and reduced by-products which are consumed by sulfate-reducing bacteria

METHANOTROPHS
Unique group of bacteria capable of growth using methane as sole carbon and energy source Key component in methane cycle Methane monooxygenase:

Cytoplasmic soluble methane monooxygenase (sMMO) Membrane-bound particulate methane monooxygenase (pMMO)

shares similarities with AMO

METHANOTROPHS

Methylococcus capsulatus

Responsible for the oxidation of biologically generated methane Methane oxidation occur both aerobically and anaerobically, in both terrestrial and aquatic habitats 11 genera are found

BIOLOGICAL CONVERSION OF METHANE USING AOB

AOB Ammonia oxidizing bacteria AOB oxidizes CH4 to CH3OH via nonspecific action of the enzyme ammonia monooxygenase (AMO). AOB utlizes the CO2 in gas mixtures for cell synthesis

METABOLISM OF AOB

Primary source of energy: Oxidation of ammonianitrogen (NH3-N) to nitrite-nitrogen (NO2--N)

NH3-N NH2OH by means of ammonia monooxygenase (AMO) NH2OH NO2-N Enzyme: hydroxylamine oxidoreductase (HAO)

Oxidation of NH3-N and NH2OH are linked

The reducing power generated in HAO-mediated NH2OH oxidation is for AMO-driven NH3-N oxidation

METABOLIC PATHWAY

PATHWAYS TO OBTAIN ENERGY

From the oxidation of NH2OH to NO2 N3 and subsequent electron transfer phosphorylation
Dominant mode of energy is generated through aerobic metabolic pathways. Under anaerobic environment, AOB can utilize alternate electron acceptors such as NO2-, dimeric nitrogen dioxide (N2O4) and produce N2O and NO

BENEFITS OF USING AOB

AOB

can utilize AMO which has broad substrate base; thus they are capable of oxidizing a variety of hydrocarbon compounds such as methane, methanol, ethylene and methyl bromide

Nitrosomonas europaen Nitrosococcus oceani

BENEFITS OF USING AOB (CONT.)

AOB partially oxidize CH4 to CH3OH

MOB oxidize methane completely to CO2, which cannot be used as a fuel

AOB produce trace amounts of formaldehyde (HCHO) which is toxic to AOB and subsequently improve CH3OH conversion rate

Any further oxidation of CH3OH to HCHO will be inhibited.

the genes coding for conversion of formate (HCOOH) to CO2 are missing in AOB

COMPETITION BETWEEN
SUBSTRATES
Competition

between CH4 and NH3 oxidation

Proposed usage of alternate reducing power source such as NH2OH Separate the oxidation process of NH3 and CH4 To avoid competition between the two substrates

PROBLEMS
Desired product further metabolized Productivities are extremely low

USE OF MOS FOR HIGH METHANOL PRODUCTION

Methylosinus trichosporium OB3b

Oddball strain 3b Also well known for its ability to biodegrade recalcitrant hydrocarbons, such as trichloroethylene (TCE). Possible, high methane-to-methanol conversion rate of >60% using high cell density process in the presence of high concentration of phosphate buffer.
Other method of methanol production:

Use of syngas

High-energy consumption, low conversion rates, and high capital costs. Biotransformation of methane to methanol require less energy input and is more selective and productive.

USE OF MOS FOR HIGH METHANOL PRODUCTION

Main challenge in methanol production

Methanol dehydrogenase (MDH)

In order to achieve high methanol yields, we need to increase cell density with increased concentrations of phosphate buffer to inhibit MDH.

USE OF MOS FOR HIGH METHANOL P RODUCTION A 2011 study from Graduate University of Chinese Academy of

Sciences, Beijing. Silicone (bubble free) membrane aerated reactor was used for methane conversion to methanol:

USE OF MOS FOR HIGH METHANOL PRODUCTION

(Preliminary) Results of using different cell & phosphate concentrations: Methanol accumulation decreased with increasing rxn time No difference in methanol accumulation by using higher cell concentrations ranging from 1.3 to 3.9 g dry cell/L if phosphate concentration was less than 120 mmol/L. Higher concentration of methanol could be accumulated using high cell densities in presence of higher phosphate concentration (see Fig. 2).

So for all subsequent experiments, they used 17 g dry cell/L as the optimal cell concentration in order to produce a higher methanol concentration.

USE OF MOS FOR HIGH METHANOL PRODUCTION

USE OF MOS FOR HIGH METHANOL PRODUCTION

Results of using different phosphate conc.

Effects of using MgCl2

Maximum methanol production was observed to be 0.96 g/L with a phosphate concentration of 400-500 mmol/L and a methane-to-methanol conversion rate of 53%.

Optimal conc. of MgCl2 was found to be 10 mmol/L, in which methanol conc. reached 1.03 g/L after 50 hours of rxn time. This corresponds to a methane-tomethanol conversion rate of 56%.

USE OF MOS FOR HIGH METHANOL PRODUCTION

Effects of using sodium formate

With 10 to 80 mmol/L of sodium formate, 0.69 g/L methanol could be accumulated after 50 hour period.

Methanol could also accumulate in the absence of sodium formate but at a lower concentration.

USE OF MOS FOR HIGH METHANOL PRODUCTION

Conclusions of this study:

Optimal rxn conditions for methanol production:

17.3 g dry cell/L 400 mmol/L phosphate 10 mmol/L MgCl2 Incubation at 30C and pH 6.3 In the presence or absence of 20 mmol/L sodium formate.

Maximum methanol production was 1.13 g/L after 40-hour rxn time in presence of 20 mmol/L sodium formate. = Conversion rate of 64%

Indicates the significant potential of MOs to produce high conc. of methanol at a high methane conversion rate. Emphasizes that the application of the silicone tube membrane aerated bioreactor mentioned earlier is a highly efficient and safe methane-to-methanol conversion method.

APPLICATIONS OF METHANOL
Transportation Fuel Fuel Cell Hydrogen Carrier Biodiesel Transesterification Electricity Generation

TRANSPORTATION FUEL

FUEL CELL HYDROGEN CARRIER

BIODIESEL TRANSESTERIFICATION

ELECTRICITY GENERATION

RECAP

Anaerobic Fermentation of Methane Occurs in four Steps: Hydrolysis Acidogenesis Acetogenesis Methanogenesis Methane Production Four types of reactors Covered Lagoon Digester Complete Mix Digester Plug Flow Digester Upflow Anaerobic Sludge Blanket Non-biological Conversion Thermal conversion process Lignocellulosics undergo: Gasification Fischer- Tropsch Hydrocarbons/Bio-oil Biofuels Biological Conversion

Methanotrophs Methane monooxygenases (MMOs)

Issues to Consider Low productivity, continuous metabolization Microorganisms

Production

Address inhibiting methanol dehydrogenase (MDH) Transportation Fuel Fuel Cell Hydrogen Carrier Biodiesel Transesterification Electricity Generation

Industry Use

FUTURE PROSPECTS

Energy industry seeking proposals for development of biological techniques to produce methanol from methane

Approaches include, but not limited to:

Adding chemicals to: Inhibit methanol dehydrogenase (MDH) Activate MMO and inhibit MDH Constructing genetically modified microorganisms Using E. coli and yeast (S. cerevisiae)

FUTURE PROSPECTS

Department of Energys Advanced Research Projects Agency Energy issued a Funding Opportunity Announcement for up to $20 Million to fund development of bioconversion technologies to convert methane into liquid fuels

Vision: economically viable bioconversion alternatives of liquid fuels from natural gas at < $2/gallon to meet US demand Three technical categories considered:
High-efficiency biological methane activation High-efficiency biological synthesis of liquid fuels Process intensification approaches for biological methane conversion

RESOURCES
1. 2.

3.

4. 5. 6.

7.

8.

"Anaerobic Digesters and Methane Production ."University of Wisconsin Extension Services . University of Wisconsin Extension Services . Web. 28 Apr 2013. <http://bio.uwex.edu/library/documents/methanepubbw.pdf>. Duan, C., M. Luo , and X. Xing. "High-rate conversion of methane to methanol by Methylosinus trichosporium OB3b." NCBI. N.p.. Web. 28 Apr 2013.Hallam SJ, et al. Reverse methanogenesis: testing the hypothesis with environmental genetics. Science. 2004. 305. 1457. Holmes AJ, Costello A, Lidstrom ME, Murrel JC. Evidence that particulate methane monooxygenase and ammonia monooxygenase may be evolutionarily related. FEMS Microbiology Letters. 1995. 132. 203-208. Production of Methanol from Methane by Methanotrophic Bacteria Biocatalysis and Biotransformation, 2004 VOL. 22 (3). pp. 225/229 Researchers develop high-rate, high-yield bacterial process to convert methane to methanol http://www.greencarcongress.com/2013/03/taher-20130322.html Taher E, Chandran K. High-rate, high-yield production of methane oxidizing bacteria. Environmental Science and Technology . "Types of Anaerobic Digesters." AgriLife Extension . Texas A&M AgriLife Extension , 2 4 2012. Web. 28 Apr 2013. <http://www.extension.org/pages/30307/types-of-anaerobic-digesters>. Ward NM, Larsen O, et al. Genomics insights into methanotrophy: the complete genome sequence of Methylococcus capsulatus . PLoS Biology. 2004. 2.10.e303.