Anaerobic digestion (AD) is a biological process that breaks down organic materials (feedstocks) in the absence of oxygen (anaerobic conditions) into methane (CH₄) and carbon dioxide (CO₂). It is a process that occurs naturally in bogs, lake sediments, oceans, and digestive tracts. Cows contain one of the most well-known fermentation vats, the rumen, which is part of the stomach (in other animals as well). Fermentation takes place during digestion! The figure below shows a schematic of anaerobic digestion.

There are benefits to using an anaerobic digester, particularly when raising livestock. Biogas, which contains methane and hydrogen, will be produced that can be used as a fuel. From a waste treatment point of view, it reduces the volume and mass of the waste, as well as reduces organic content and biodegradability of waste so that the residual matter can be better used as soil amendment and fertilizer. There are also environmental benefits: 1) odors and emissions of greenhouse gases (i.e., methane) and volatile organic compounds are reduced, and 2) the digester will destroy pathogens in the waste.

So, what are the biological processes that occur during AD? The bacteria ferment and convert complex organic materials into acetate and hydrogen. There are four basic phases of anaerobic digestion, which is a synergistic process using anaerobic microorganisms: 1) hydrolysis, 2) acidogenesis, 3) acetogenesis, and 4) methanogenesis. The figure below shows the progression and types of products for each phase.

Hydrolysis Biochemistry

We have talked about hydrolysis in earlier lessons. Hydrolysis is a reaction with water. Acid and base can be used to accelerate the reaction. However, this occurs in enzymes as well. The figure below shows the hydrolysis reaction, and how cellulose, starch, and simple sugars can be broken down by water and enzymes. In anaerobic digestion, the enzymes are exoenzymes (cellulosome, protease, etc.) from a number of bacteria, protozoa, and fungi (see Reaction 1).

(1) $\text{ biomass } + {\mathrm{H} }_{2 }\mathrm{O} \to \text{ monomers } + {\mathrm{H} }_{2 }$

(Sources: cellulose, starch, sugars, fats, oils) (Products: mono-sugars [glucose, xylose, etc.], fatty acids)

Acidogenesis Biochemistry

During acidogenesis, soluble monomers are converted into small organic compounds, such as short-chain (volatile) acids (propionic, formic, lactic, butyric, succinic acids – see Reaction 2), ketones (glycerol, acetone), and alcohols (ethanol, methanol – see Reaction 3).

(2) ${\mathrm{C} }_{6 }{\mathrm{H} }_{12 }{\mathrm{O} }_{6 }+ 2 {\mathrm{H} }_{2 }\to 2 {\mathrm{CH} }_{3 }{\mathrm{CH} }_{2 }\mathrm{COOH} + 2 {\mathrm{H} }_{2 }\mathrm{O}$

(3) ${\mathrm{C} }_{6 }{\mathrm{H} }_{12 }{\mathrm{O} }_{6 }\to 2 {\mathrm{CH} }_{3 }{\mathrm{CH} }_{2 }\mathrm{OH} + 2 {\mathrm{CO} }_{2 }$

Acetogenesis Biochemistry

The acidogenesis intermediates are attacked by acetogenic bacteria; the products from acetogenesis include acetic acid, CO₂, and H₂. Reactions 4-7 show the reactions that occur during acetogenesis:

(4) ${\mathrm{CH} }_{3 }{\mathrm{CH} }_{2 }{\mathrm{COO} }^{- }+ 3 {\mathrm{H} }_{2 }\mathrm{O} \to {\mathrm{CH} }_{3 }{\mathrm{COO} }^{- }+ {\mathrm{H} }^{+ }+ {\mathrm{HCO} }_{3 }^{- }+ 3 {\mathrm{H} }_{2 }$

(5) ${\mathrm{C} }_{6 }{\mathrm{H} }_{12 }{\mathrm{O} }_{6 }+ 2 {\mathrm{H} }_{2 }\mathrm{O} \to 2 {\mathrm{CH} }_{3 }\mathrm{COOH} + 2 {\mathrm{CO} }_{2 }+ 4 {\mathrm{H} }_{2 }$

(6)${\mathrm{CH} }_{3 }{\mathrm{CH} }_{2 }\mathrm{OH} + 2 {\mathrm{H} }_{2 }\mathrm{O} \to {\mathrm{CH} }_{3 }{\mathrm{COO} }^{- }+ 2 {\mathrm{H} }_{2 }+ {\mathrm{H} }^{+ }$

(7) $2 {\mathrm{HCO} }_{3 }^{- }+ 4 {\mathrm{H} }_{2 }+ {\mathrm{H} }^{+ }\to {\mathrm{CH} }_{3 }{\mathrm{COO} }^{- }+ 4 {\mathrm{H} }_{2 }\mathrm{O}$

Several bacteria contribute to acetogenesis, including:

Syntrophobacter wolinii, propionate decomposer

Syntrophomonos wolfei, butyrate decomposer

Clostridium spp., peptococcus anaerobes, lactobacillus, and actinomyces are acid formers.

Methanogenesis Biochemistry

The last phase of anaerobic digestion is the methanogenesis phase. Several reactions take place using the intermediate products from the other phases, with the main product being methane. Reactions 8-13 show the common reactions that take place during methanogenesis:

(8)$2 {\mathrm{CH} }_{3 }{\mathrm{CH} }_{2 }\mathrm{OH} + {\mathrm{CO} }_{2 }\to 2 {\mathrm{CH} }_{3 }\mathrm{COOH} + {\mathrm{CH} }_{4 }$

(9) ${\mathrm{CH} }_{3 }\mathrm{COOH} \to {\mathrm{CH} }_{4 }+ {\mathrm{CO} }_{2 }$

(10) ${\mathrm{CH} }_{3 }\mathrm{OH} \to {\mathrm{CH} }_{4 }+ {\mathrm{H} }_{2 }\mathrm{O}$

(11)${\mathrm{CO} }_{2 }+ 4 {\mathrm{H} }_{2 }\to {\mathrm{CH} }_{4 }+ 2 {\mathrm{H} }_{2 }\mathrm{O}$

(12)${\mathrm{CH} }_{3 }{\mathrm{COO} }^{- }+ {\mathrm{SO} }_{4 }^{2 - }+ {\mathrm{H} }^{+ }\to 2 {\mathrm{HCO} }_{3 }+ {\mathrm{H} }_{2 } \mathrm{S}$

(13) ${\mathrm{CH} }_{3 }{\mathrm{COO} }^{- }+ {\mathrm{NO} }^{- }+ {\mathrm{H} }_{2 }\mathrm{O} + {\mathrm{H} }^{+ }\to 2 {\mathrm{HCO} }_{3 }+ {\mathrm{NH} }_{4 }^{+ }$

Several bacterial contribute to methanogenesis, including:

Methanobacterium, methanobacillus, methanococcus, and methanosarcina, etc.

As you can see, the bacteria for anaerobic digestion are different from other enzymes for making biofuels, and could even be in our own stomachs!

Any kind of organic matter can be fed to an anaerobic digester, including manure and litter, food wastes, green wastes, plant biomass, and wastewater sludge. The materials that compose these feedstocks include polysaccharides, proteins, and fats/oils. Some of the organic materials degrade at a slow rate; hydrolysis of cellulose and hemicellulose is rate limiting. There are some organic materials that do not biodegrade: lignin, peptidoglycan, and membrane-associated proteins. The organic residues contain water and biomass composed of volatile solids and fixed solids (minerals or ash after combustion). And it’s the volatile solids (VS) that can be non-biodegradable and biodegradable.

As we discussed regarding the pretreatment of biomass for making ethanol, the efficiency of anaerobic digestion improves with pretreatment. Hydrolysis of cellulose and hemicellulose (phase 1 in AD) is improved with pretreatment because it overcomes biomass recalcitrance. As discussed in a previous lesson, pretreatment options include treatments with acids, alkalines, steam explosion, size reduction, etc. Common alkaline agents include: NaOH, Ca(OH)₂, and NH₃.

Theoretical methane yield (Y_CH4, m³ STP/kg substrate converted) can be calculated from the elemental composition of a substrate:

${\mathrm{C} }_{d }{\mathrm{H} }_{h }{\mathrm{O} }_{x }{ \mathrm{N} }_{n }{ \mathrm{S} }_{s }$

$$Y_{CH4 }=\frac{22.4\left( \frac{c}{2}+\frac{h}{8}+\frac{x}{4}-\frac{3n }{8}-\frac{s}{4}\right) }{12c+h+16x+14n+16s }$$

The table below shows the substrate, a common elemental formula, and the theoretical methane yield for each.

The first figure below shows the biogas yield for several different feedstocks in m³/ton. Be aware that after digestion, there is a biogas yield, and the remainder of the digestion, is known as digestate. The biogas typically contains 50-60% CH₄, with the rest primarily composed of CO₂ and other trace gases. The digestate contains fiber, nutrients, and water, and these can be used for compost, animal bedding, and composite boards. The second figure below shows a schematic of the components of the digester.

There are several factors that will affect anaerobic digestion. Different feedstocks will degrade at different rates and produce different amounts of methane (as seen in the Biogas yields graph and table above). That depends on the biological degradability and methane potential, the carbon and nutrients available, and the moisture content of each feed material. As noted in the Biogas yields graph and table above, fats contain the highest volatile solids and can generate the greatest amount of biogas. Solids take a longer time to digest than feedstocks that are soluble. Nutrients are also important. A suitable carbon to nitrogen ratio (C/N) is less than 30, and the carbon to phosphorous ration (C/P) should be less than 50. For example, lignocellulosic biomass has a high C/N ratio, so nitrogen sources must be added. Nutrients also must be free of toxic components. Other factors that can influence digestion are the availability and location of feed materials (transportation costs involved here), logistics of how to get materials to certain sites, and whether size reduction is going to be necessary.

Digester performance will also depend on the microbial population in the digester. This means maintaining adequate quantities of fermenting bacteria and methanogens. A recycled stream is used to take a portion of the liquid digestate as inoculum (material used for inoculation of feed materials). Depending on feeds, there may be an acclimation period to reach acceptable conditions.

There are also variations in the operational factors and environmental conditions of the digester. It is important to know the total solids (TS) and volatile solids (VS) in the feeds, the best retention times, and to provide mixing. Operational factors include the amount and strength type of feedstocks added to the digester. The operation also depends on maintaining the microorganism population and organic loading in reactors, whether operating in a batch or continuous reactor. Mixing is also an important factor in any reaction. The goal of mixing is to keep the microorganisms in close interaction with the feed and nutrients. Mixing also prevents the formation of a floating crust layer, which can reduce the amount of biogas percolating out of the slurry. Mixing will benefit the breakdown of volatile solids and increase biogas production, but keep in mind mixing adds energy cost, so this must be balanced. The types of mixing in this system include gas bubbling and/or mechanical mixing.

Environmental conditions include the temperature and pH of the reactor, as well as concentrations of materials, including the volatile fatty acids, ammonia, salt, and cationic ions. Different methanogens react in temperature ranges. The type of methanogens that produce the most biogas are thermophiles, but the digester must be operating between 40-70 °C. Methanogens also prefer neutral pH conditions (6.5-8.2). Accumulation of volatile fatty acids (VFAs) can cause the digestion to stop producing gas – this happens when too much digestible organic material is added, a toxic compound is added, or there is a sudden temperature change. Toxic materials include: 1) oxygen, 2) antibiotics, 3) cleaning chemicals, 4) inorganic acids, 5) alkali and alkaline earth salt toxicity, 6) heavy metals, 7) sulfides, and 8) ammonia. An additional reason for AD process failure has to do with the reaction within being out of balance. In particular, the rate of acid formation and methane production should be equal. This is done by maintaining definite ranges and ratios of the following: solids loading, alkalinity, temperature, pH, mixing, and controlling VFA formation. When the methanogen microorganisms cannot keep up with the fermenting bacteria, the digester becomes acidic – also known as “sour.”

An ambient temperature liquid phase AD reactor is called a covered lagoon. The advantages of the covered lagoon are the low cost, ease of construction, and control of odor control with manure storage. Disadvantages include difficult sludge removal and only seasonal production. However, there are several designs that have controlled temperature, and are typical to different types of reactors: 1) complete mixing, 2) plug flow, 3) sequencing batch, and 4) fixed film. The table below shows a comparison of the variables for each type of anaerobic digester configuration.

There is an unusual process for liquids production from biomass: gasification followed by fermentation of gases into liquids. During gasification, the gases of CO, H₂, and CO₂ are formed (as we have learned in past lessons), but instead of using something like FT or MTG, this is formation of liquids fuels through a fermentation process using a microbial catalyst. Products are typically ethanol, acetone, and butanol. Gasification was discussed in depth in Lesson 4, but I will cover it briefly here to remind you of the various processing aspects. Gasification takes place at temperatures of 750-900°C under partial oxidation. It happens in the following steps: drying; pyrolysis in absence of O₂; gas-solid reactions to produce H₂, CO, and CH₄ from char; and gas-phase reactions that manage the amounts of H₂, CO, and CH₄. It is most often known as syngas, but if it contains N₂, then it is called producer gas. Syngas can be generated from any hydrocarbon feed. The main cost associated with gas-to-liquid technologies has to do with the syngas production, which is over half of the capital costs. Costs can be improved using improved thermal efficiency through better heat utilization and process integration and by decreasing capital costs.

There are advantages to using fermentation as part of liquids generation rather than using something like Fischer-Tropsch:

1. As with any gasification, it is independent of feedstock, and therefore, independent of biomass chemical composition.
2. Microorganisms are very specific to ethanol production, whereas with chemical catalysts, there are a wide range of reaction products.
3. No pretreatment is required as part of the biochemical platform.
4. Complete conversion of biomass is achieved, including lignin conversion. This can reduce the environmental impact of waste disposal.
5. Fermentation takes place at near ambient temperature and pressure, thus at a place where costs can be reduced significantly.
6. The requirement for CO/H₂ ratio is flexible.

Of course, there are disadvantages as well. These include:

1. Gas-liquid mass transfer limitations.
2. Low ethanol productivity, usually related to low cell density.
3. Impurities in syngas generated from biomass.
4. Sensitivity of microorganisms to environmental conditions (pH, oxygen concentration, and redox potential).

The microorganisms that are used for ethanol production from syngas are acetogens that can produce ethanol, acetic acid, and other products from CO and H₂ in the presence of CO₂. The organisms are: 1) Clostridium strain P11, 2) Clostridium ljungdahlii, 3) Clostridium woodii, 4) Clostridium thermoaceticum, and 5) Clostridium carboxidivorans P7. (Wilkens and Atiyeh, 2011) The bacteria are some of the same ones that occur during anaerobic digestion: acetogens and acidogens. I won’t go into great detail about the biochemistry, as it is a little beyond the scope of this class. The acetogens utilize the reductive acetyl-CoA (or Wood-Ljungdahl) pathway to grow carbons and hydrogens on single carbon substrates such as CO and CO₂. Clostridium bacteria use H₂ or organic compounds as the electron source for the reduction of CO₂ to acetyl-CoA, which are further converted into acids and alcohols. The process proceeds in two phases: acidogenic and solventogenic phases. In the acidogenic phase, mainly acids are produced (i.e., acetic acid and butyric acid). In the solventogenic phase, mainly solvents are produced (i.e., alcohols such as ethanol and butanol). Reactions 14 and 15 show the reaction chemistry for acetic acid formation, and reactions 16 and 17 show the reaction chemistry for ethanol formation:

Acetic acid formation:

(14)$\mathrm{ 4}\mathrm{CO}+2{\mathrm{H}}_2\mathrm{O}\to {\mathrm{CH}}_3\mathrm{COOH}+2{\mathrm{CO}}_2$

(15) $2{\mathrm{CO}}_2+4{\mathrm{H}}_2\to {\mathrm{CH}}_3\mathrm{COOH}+2{\mathrm{H}}_2\mathrm{O}$

Ethanol formation:

(16) $6\mathrm{CO}+3{\mathrm{H}}_2\mathrm{O}\to {\mathrm{C}}_2{\mathrm{H}}_5\mathrm{OH}+4{\mathrm{CO}}_2$

(17) $2{\mathrm{CO}}_2+6{\mathrm{H}}_2\to {\mathrm{C}}_2{\mathrm{H}}_5\mathrm{OH}+3{\mathrm{H}}_2\mathrm{O}$

In summation, gasification-fermentation alternative is a method for biofuel production utilizing syngas generated from gasification of biomass feedstocks. Because it is biologically based, it has the potential for reducing costs compared to other syngas to liquid technologies, but there are several challenges related to this technology. Challenges include low alcohol productivity, low syngas conversion efficiency, and limitations in gas-liquid mass transfer. These challenges must be solved if this technology is to become economically viable.

A microbial fuel cell is a bio-electrochemical device that can convert chemical energy directly into electrical energy. But first, let’s go over what a fuel cell is. A fuel cell is a battery of sorts. So, what is a battery? A battery is when two different types of metals are connected together through what is called an electrolyte. One metal is an anode, which is a metal that “wants” to give off electrons when under the right conditions. One metal is a cathode, which is a metal that “wants” to accept electrons when under the right conditions. When these two metals are in close proximity, and there is a fluid that will conduct the electrons (an electrolyte), then the flow of electrons from one metal to the other can occur. And we can capture that flow to extract electricity. Batteries that we use in remotes for televisions will eventually get used up and need to be replaced. This is an example of a primary battery. The figures below show a generic cell, a stack of cells using zinc and copper, and a picture of a Voltaic cell as created by Alessandro Volta (inventor).

We can also have secondary batteries, where the flow of electricity is established to provide electrical energy, but we can also apply electricity to the battery to reverse the flow of electrons and regenerate the life of the battery. While regenerated batteries don’t last forever, you can definitely get your money’s worth because you can regenerate them.

Fuel cells are also a sort of battery, but the materials are different and flow continuously to produce electricity. The figure below shows a generic fuel cell. As with a battery, it has an anode, cathode, and electrolyte. The anode typically uses hydrogen as the fuel, on the left side of the figure, and oxygen as the oxidant used in the cathode, on the right side of the figure. The electrolyte contains a fluid but also a membrane that removes the protons from the hydrogen, leaving the electrons to flow, and allowing oxygen to accept the protons to form water. Typically, these cells are run on hydrogen and oxygen, but we get electrical energy out rather than heat from burning hydrogen and oxygen.

At the anode, hydrogen reacts as shown in reaction 18:

(18) ${\mathrm{H} }_{2 }\to 2 \mathrm{H} + + 2 \mathrm{e} -$

This is an oxidation reaction that produces protons and electrons at the anode. The protons then migrate through an acidic electrolyte, and the electrons travel through an external circuit. Both arrive at the cathode to react with the oxidant, oxygen, as shown in reaction 19:

(19)$1 / 2 {\mathrm{O} }_{2 }+ 2 \mathrm{H} + + 2 \mathrm{e} - \to {\mathrm{H} }_{2 }\mathrm{O}$

This is the reduction reaction, where oxygen can be supplied purely or in the air. Essentially, the total circuit is completed through the mass transfer of protons in the electrolyte and external electrical circuit. There will be a small amount of heat lost through the electrodes. The overall reaction is shown in reaction 20:

(20) ${\mathrm{H} }_{2 }+ 1 / 2 {\mathrm{O} }_{2 }\to {\mathrm{H} }_{2 }\mathrm{O} + \text{ work } + \text{ waste heat }$

The water and waste heat are the by-products and must be removed on a continuous basis. An ideal voltage from this reaction is 1.22 volts, but less than that voltage will be realized. Other issues include the use of fuel different from hydrogen (methanol, hydrocarbons, etc.) and the fact that fuel cells produce direct current (DC) when most applications require alternating current. This little bit of background has been provided so a short discussion on microbial fuel cells can be had.

We are not going to go into great depth on microbial fuel cells, as some of the biochemistry can be complex. I will provide you with some generic information on how a microbial fuel cell is set up. These are bio-electro-chemical devices, which convert chemical energy directly into electrical energy. As we have discussed before, there are some steps that need to occur. Cellulose is hydrolyzed into sugars, i.e., glucose. The sugars are fermented into short-chain fatty acids, alcohols, hydrogen, and carbon dioxide. Finally, electricigenesis takes place, producing electricity, and carbon dioxide is carried through. Electricigenesis converts chemical energy to electrical energy by the catalytic reaction of microorganisms. The anode is anaerobic, and the anode chamber contains microbes and feedstock. The fuel is oxidized by microorganisms, which generate CO₂, electrons, and protons. The cathode is the aerobic chamber, and just like other fuel cells, a proton exchange membrane separates the two chambers and allows only protons (H+ ions) to pass.

There are two types of microbial fuel cells (MFCs): mediator or mediator-less. The mediator type was demonstrated in the early 20^th century and uses a mediator: a chemical that transfers electrons from the bacteria in the cell to the anode. Some of these chemicals include thionine, methyl viologen, methyl blue, humic acid, and neutral red. These chemicals are expensive and toxic. Mediator-less MFCs are a more recent development, from the 1970s. These types of cells have electrochemically active redox proteins such as cytochromes on their outer membrane that can transfer electrons directly to the anode. Some electrochemically active bacteria are Shewanella putrefaciens and Aeromomas hydrophila. Some bacteria have pili on the external membrane, which allows for electron production through the pili. They are beginning to find commercial use in the treatment of wastewater. I’ve included some YouTube videos explaining how the MFCs work. The first video is a brief but complete explanation of how an MFC works (2:03).

The next video is a little longer and goes into a little more depth of what was described above (7:23).

The next short video has Dr. Bruce Logan, a professor in the Civil Engineering Dept. at PSU providing a brief explanation on using these MFCs for wastewater treatment facilities (3:05).

The last video, put together by Dr. Logan’s group at PSU, shows how to construct three different types of MFCs (4:26).

MFCs can also be used in food processing plants and breweries, as well as being implanted as biomedical devices. There are technical challenges to MFCs. They have relatively low power densities, which means they don’t generate much power. Therefore research continues to improve power densities. These devices have an incredible future but still need more research to be commercialized at a large scale.

We have explored many uses of biomass to generate fuels, from use in electricity generation through combustion, to several conversion technologies to make ethanol, biodiesel, and fuels that are very similar to petroleum-based fuels like gasoline, jet fuel, and diesel fuel. I also wanted you to have the opportunity to see what is still being researched and what has been commercialized. While currently, due to the cost of crude oil being low, biofuels may not be as competitive economically, they show great benefit environmentally, especially when renewable methods are used to harvest and generate the fuels. You looked up several very interesting articles related to biofuel generation and use. Read articles on biofuels with a critical eye; you now know enough about biofuels so that you can be a more critical reader. I hope that you enjoyed the class.

Summary

This last lesson covered practical topics, such as anaerobic digestion (it is part of digestion), and also topics that are more research-oriented, such as syngas fermentation and microbial fuel cells. The best application for anaerobic digestion is on farms, as a way to utilize waste from animals. Syngas fermentation could be a lower energy-intensive process that could make gasification/syngas fermentation less expensive than using FT synthesis. And, finally, microbial fuel cells are a unique fuel cell that can take advantage of bacteria to make electricity or clean up wastewater inexpensively.

Lesson Objectives

By the end of this lesson, you should be able to:

• explain what anaerobic digestion is;
• explain what syngas fermentation is;
• utilize the information on each to supplement biofuel liquids conversion technologies;
• evaluate the best methods to date for biofuel liquids conversion technologies.

References

Jian Shi¹, Ruihong Zhang², Wei Liao ³, Conly L. Hansen⁴, Yebo Li^{1, *} 1. Department of Food, Agricultural, and Biological Engineering, The Ohio State University, 2. Department of Biological and Agricultural Engineering, University of California-Davis. 3. Department of Biosystems and Agricultural Engineering, Michigan State University, 4. Department of Nutrition and Food Sciences, Utah State University, BEEMS Module B7, Anaerobic Digestion, USDA Higher Education Challenger Program, 2009-38411-19761. Contact: Yebo Li, li.851@osu.edu

Frigon and Guiot, 2010, Biofuels, Bioproducts & Biorefining, 4, 447-458.

On-Farm Anaerobic Digester Operator Handbook. M.C. Gould and M.F. Crook. 2010. Modified by D.M. Kirk. January 2010.

Hasan K. Atiyeh, Department of Biosystems and Agricultural Engineering,  Oklahoma State University, Syngas Fermentation, BEEMS Module N1, USDA Higher Education Challenger Program, 2009-38411-19761, Contact: Hasan K. Atiyeh, hasan.atiyeh@okstate.edu.

Wilkins and Atiyeh. Current Opinion in Biotechnology 2011, 22:326–330.