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Review

Volatile Fatty Acid Production vs. Methane and Hydrogen in Anaerobic Digestion

by
Venko N. Beschkov
* and
Ivan K. Angelov
Institute of Chemical Engineering, Bulgarian Academy of Sciences, 1113 Sofia, Bulgaria
*
Author to whom correspondence should be addressed.
Fermentation 2025, 11(4), 172; https://doi.org/10.3390/fermentation11040172
Submission received: 13 February 2025 / Revised: 13 March 2025 / Accepted: 14 March 2025 / Published: 26 March 2025
(This article belongs to the Special Issue Fermentative Production of Valuable Chemicals from Lignocellulosic Biomass)
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Abstract

:
Volatile fatty acids (VFAs) are inevitable intermediates of biogas production during the anaerobic digestion of organic matter. The excessive accumulation of VFAs leads to a pH drop and the strong inhibition of methanogenesis. On the other hand, VFAs are useful commodities with different applications, and their fermentative production may compete with traditional production methods based on oil derivatives. The fermentation methods have commonalities with the biorefinery concept. The present review considers the methods of VFA fermentative production together with competitive simultaneous biogas and hydrogen production. Methods of the enhanced production of volatile fatty acids are presented, showing the option of integrated processes of product removal and energy production from the obtained biogas. On the basis of the present review, the following conclusion can be drawn. Volatile fatty acids (formic, acetic, propionic, and butyric ones) are useful commodities with various applications. That is why their targeted production with their desired production rate may shift the aims of the anaerobic digestion toward volatile fatty acids instead of biogas release. On the other hand, VFA production combined with biogas release can make the overall process self-consistent, with energy production sufficient to maintain the target processes using biogas for heating the digestor. The maintenance of optimum VFA concentrations can be accomplished by simultaneous VFA removal from the fermentation broth, thus integrating the product recovery with the maintenance of optimum operation conditions in the digester. The substrate preparation and the operating conditions (organic loading rate and hydraulic retention time) are of crucial importance for the successful fermentation process.

1. Introduction

The anaerobic biodegradation of organic waste is a well-known process in nature and is broadly utilized by mankind [1,2,3]. It is a common practice in agriculture, wastewater treatment, and landfill maintenance [4]. It is an excellent approach for simultaneous waste treatment and energy production and other applications, e.g., heating, transport, and electricity production [5,6]. The result of waste treatment by anaerobic digestion is biogas, containing methane and carbon dioxide with some impurities, like hydrogen sulfide, mercaptans, ethane, etc. The methane content in biogas varies from 55 to 90% (vol.), depending on the substrate nature, the pre-treatment method, the operation of the anaerobic digester, etc.
Biogas is broadly produced and utilized in countries with developed agriculture of large scale, like India, China, Brazil, etc.
Anaerobic digestion with biogas production is a complicated consecutive process of the hydrolysis of organic macromolecules (carbohydrates, lipids, and proteins) to oligosaccharides and peptides, the acidogenesis of these intermediates to fatty acids, mostly volatile ones (e.g., formic, acetic, propionic, etc.), acetogenesis, and finally methanogenesis [7,8]. A scheme of these consecutive and parallel processes is shown in Figure 1. Volatile fatty acids (VFAs) are short-chain aliphatic monocarboxylic acids that have 1–7 carbon atoms in each molecule as well as unsaturated fatty acids with more than one double bond.
The different processes in this set of reactions are carried out by different communities of microorganisms. Hydrolysis is accomplished by a large variety of bacteria, e.g., Pseudomonas, Ruminococcus, Bacillus, etc.), leading to the degradation of starch, cellulose, hemicellulose, and proteins to soluble compounds of lower molecular mass. Hydrolysis of starch (or cellulose) to glucose is shown below:
(C6H10O5)n + nH2O = nC6H12O6
Further, these compounds are converted in parallel to acetate and volatile fatty acids (VFA) by acidogenic (Acetobacterium, Desulfobulbus, and Clostridium) [9] and acetogenic microbes (Acetobacter and Clostridium) and hydrogen plus carbon dioxide.
The last step of methanogenesis is accomplished by methanogenic archaea and bacteria, leading to the production of methane and carbon dioxide. Methanogenic bacteria from the genera Methanosarcina and Methanosaeta convert acetic acid into CH4 and CO2 by decarboxylation [10]:
CH3COOH = CH4 + CO2
The strains Methanobacterium and Methanobrevibacter convert carbon dioxide to methane by reduction by hydrogen [11,12,13].
CO2 + 4H2 = CH4 + 2H2O
All methanogenic microbes are active in neutral media, i.e., for pH values between 6 and 8.
Acidogenesis is an important step in biogas production. The accumulation of fatty acids with deviations in pH lead to strong inhibition and even to microbial death. On one hand, methanogenesis is favored by fatty acid formation, but on the other hand, it could be strongly inhibited by fatty acid accumulation, resulting in a pH drop beyond the optimum pH range. Then, the produced gas is very rich in carbon dioxide, with a poor energy value because of the low methane content. That is why one must be very careful in feeding strategies using substrates and in selecting bioreactors and flow organization.
However, there is also another point of view. Volatile fatty acids can be considered as valuable products of this process of anaerobic digestion. Therefore, sometimes the accumulation of volatile fatty acids may be favorable from an economic point of view compared to methane production. Hence, the anaerobic digester can be considered as a biorefinery [14] and to contribute to the circular economy approach [15]. That is why there is keen interest toward this option where methane is not considered as the only target product and toward management that enhances VFA production. Recently, the production of medium-chain fatty acids (containing carbon atoms C8–C12 in their molecules) was also considered as practically interesting, and they can be obtained by the elongation of short-chain fatty acids, i.e., C1–C7.
This approach becomes attractive considering that the present methods for VFA bulk production are based on fossil oil processing. Volatile fatty acid production from residual organics by anaerobic digestion contributes to the concept of biorefinery and circular economy [16]. The fermentative methods allow avoiding petrochemical processes based on chemical syntheses with the complementary operations of oxidation and carboxylation, requiring excessive expenses of energy and the removal of by-products [16].
There are also efforts to utilize VFAs as products of anaerobic digestion by photosynthetic bacteria for further products like microbial proteins, polyhydroxyalkanoates, hydrogen, 5-aminolevulinic acid, etc. [17].
As outlined by Ramos-Suarez et al. [15], in situ recovery can help to remediate product-induced inhibition and keep an optimum pH level for further substrate utilization.
The present review considers the options of volatile acid production in anaerobic digestion compared to methane production. The strategies for boosting the volatile fatty acid production include methane utilization and VFA simultaneous recovery; in addition, utilization by an integrated process with in situ product removal is discussed as well.

2. Volatile Fatty Acids

Some of the intermediate reactions of acido- and acetogenesis are shown below. First, glucose is fermented to ethanol with the release of carbon dioxide.
C6H12O6 = 2CH3CH2OH + 2CO2
Further, glucose can be reduced to propionic acid:
C6H12O6 + 2H2 = 2CH3CH2COOH + 2H2O
The acetogenic reactions are
CH3CH2COO− + 3H2O = CH3COOH + HCO3− + 3H2
C6H12O6 + 2H2O = 2 CH3COOH + 2CO2 + 4H2
CH3CH2OH + O2 = CH3COOH + H2O
2HCO3− + 4H2 + H+ = CH3COO− + 4H2
As was mentioned above, volatile fatty acids have become valuable products. Their production by anaerobic digestion may compete with their present and traditional production from oil derivatives [18].
Acetic acid is used in organic syntheses for acetic anhydride, esters, vinyl acetate monomers, vinegar, and polymeric materials. Trivial applications are in the medicine and food industry [19]. Its production reaches 16 mln tons/year. Usually, for acetic acid production, Acetobacter and Gluconobacter have been commercially applied with very high conversion rates and high product concentration, namely up to 150 g/L [20]. The application of biomass such as lignocellulose as a potential alternative was found to produce 17 g/L and 30.98 g/L acetic acid by Clostridium lentocellum, respectively [20].
Propionic acid is used as precursor in herbicide production, rubbers, fine chemicals, synthetic cellulose fibers, food preservatives, etc. It is a significant product, with an annual growth rate of 2.7% of 470.0 kilotons in 2020 with a market price between 1250 and 1700 EUR/t. It is industrially produced by a catalytic reaction of carbon monoxide with ethylene or the oxidation of propanal. The Swedish company Perstorp Speciality Chemicals AB manufactures a market product consisting of propionic acid and propionic acid glycerol esters. It is applied as an animal feed preservative that inhibits the growth of molds and yeasts in stored grains [21].
There is growing interest toward the fermentative production of propionic acid. Most of the propionic acid-producing bacteria belong to the genus Propionibacterium. These strains show different yields and productivities depending on the substrate used. The productivity for propionic acid varies between 0.12 g/(L·h) for glucose and 3.69 g/(L·h) for mature Jerusalem artichoke tubercle roots [22]. The yield of propionic acid has been shown to reach 0.74 g/g when glycerol is used as the substrate. Glycerol seems to be a more cost-effective substrate than glucose, with a yield up to 0.74 g/g [23,24].
The next volatile fatty acid, i.e., butyric acid, is a product with an annual demand of 105 kilotons in 2020 [25]. It is applied in the production of plastics, pharmaceuticals, animal feed, and cosmetic products [8]. It is currently produced by chemical synthesis from oil-based substrates, e.g., butyraldehyde oxidation. The production of butyric acid by the fermentation of renewable feedstocks has received growing attention [26]. Various substrates have been tested (molasses, bagasse, wheat straw, sorghum stalks, etc.), reaching productivity up to 1.9 g/(L·h) [27].
The next two volatile fatty acids are valeric (C4H9COOH, used for perfumes, plastics, and lubricants) and caproic (C6H13COOH, for rubber and grease). These two acids are obtained at lower yields as by-products of lower fatty acids after chain elongation during the anaerobic digestion of organic substrates [26].
It seems that the substrate composition and the ratio of different components may impact the VFA contents and ratios. For example, a mixture of sewage sludge and organic waste yields more VFAs with a higher percentage of organic waste as the substrate, whereas the biogas generation decreases [28].
The production of medium-chain fatty acids (C8–C12) also deserves practical interest [29,30]. Medium-chain fatty acids can be obtained via a three-step chain elongation process of short-chain fatty acids. The continuous process can lead to reasonable conversion rates (25–50% w/w) with food waste as the substrate [31]. The medium-chain fatty acids serve as a precursor for the production of jet and diesel fuels, as well as for bio-polymer production, e.g., poly-hydroxyoctanoate and poly-hydroxydecanoate [32].
The strategies to accumulate more VFAs during fermentation depend on the differences between anaerobic digestion and acidogenic fermentation technology [31].

2.1. Strategies to Enhance the Production of Volatile Fatty Acids

Enhancing the production of VFAs can be achieved in different ways, depending on the substrate, inoculum, and the chosen technology.
Regarding substrate composition, usually, the substrates subjected to anaerobic digestion contain large amounts of proteins and polysaccharides like starch, oligosaccharides, cellulose, hemicellulose, etc. The hydrolysis of lignocellulosic substrates as a first step of anaerobic digestion is often hindered by the lignin structure and crystallinity of cellulose. During slow hydrolysis, the concentration of VFAs in the digestion system is too low to affect the methanogens’ activity and to be of interest as product yield.
Monomer-rich substrates (e.g., glucose, glycerol, etc.) can be rapidly converted into VFAs [23,24].
Lignocellulosic substrates, such as straw from barley, wheat, and rye, were tested for volatile fatty acid production. Barley straw was the most suitable as a substrate for methane production regarding the yield and production rate [27].
Regarding pre-treatment, in the case of VFA production as the target process, one of the main goals is deactivating the methanogens by overloading the broth with a substrate to attain high concentrations of VFAs and pH drop [27].
The pre-treatment of lignocellulosic materials aims to make the substrate molecules more accessible to hydrolytic microbes. This can be achieved by different processes: either by physical (mechanical grinding), thermal (steam explosion), chemical (by acidic or alkaline hydrolysis or oxidative treatment by ozone), or biological means (e.g., enzyme hydrolysis). A survey showed that mechanical pre-treatment has a modest effect on VFA production, i.e., 3–5% above the reference yield [32]. There are also studies on microwave or ultrasonic treatment. All these operations aim to destroy the substrate macromolecules, to remove lignin, and to change the crystallinity of cellulose for better hydrolysis and further digestion. When sludge biomass is used, the treatment helps to destroy the bacterial aggregates and flocs with further cell lysis. The survey showed that the yield of bio-methane is the highest when ultrasonic or microwave impact is exerted. In the case of ultrasonic treatment, there is an almost three-fold increase in VFA yield compared to the reference case [32].
Thermal pre-treatment decreases the thermal stability of biomass but leads to the formation of toxic derivatives at high temperatures. Inhibitors and secondary pollutants are also formed.
Once hydrolyzed, the monomer-rich substrates can be converted rapidly into volatile fatty acids. A neutral reaction at a pH between 6 and 8 can be more favorable for VFA production from glucose compared to acidic and alkaline conditions [33]. Acidic and alkaline treatments require further neutralization prior to anaerobic digestion.
pH level maintenance has a profound effect on VFA production and biogas fermentation. Controlling the pH value is very important because the production of VFAs can result in a sharp pH decrease and strong inhibition either of methane formation or VFA production. At pH levels below the dissociation constant (pKa) of VFAs, most of the acids are in undissociated form, and this can possibly harm the microorganisms.
The processes of acidification and methanogenesis can be slowed down because of product inhibition. Therefore, continuous removal of VFAs should be carried out, leading to an integrated process of fermentation/extraction.
Dahiya et al. [34] pointed out the correlation between pH and the volatile fatty acids obtained from acidogenic fermentation.
It was shown by Jiang et al. [35] that pH values between 6.0 and 7.0 lead to an approximately 20% increase in the hydrolysis rate, leading to very high chemical oxygen demand (COD). The VFA production was doubled because of higher hydrolytic enzyme activity and the avoidance of inhibitions. Furthermore, Zhang et al. [36] showed the effect of pH on continuous fermentation by adjusting the pH to a fixed value. When the pH was close to neutral, a better yield of VFAs was observed. The effect of neutral conditions of fermentative production of VFAs was demonstrated in another study [37]. At acidity maintained at pH 6.0, the VFA yield increased 7.5 times compared to at pH 4.0. In general, a pH value around 6.0 is considered as most suitable for the enhanced production of VFAs from a variety of organic substrates.
Temperature is a crucial parameter during acidogenic fermentation and methanogenesis due to its direct involvement in both microbial growth and metabolism.
There are three main temperature regimes for methanogenesis: psychrophilic (in the range of 15 and 20 °C), mesophilic (30–35 °C), and thermophilic (55–60 °C). The most exploited and convenient regime is the mesophilic one because of high product yields, it being a reasonable temperature to maintain, and minimizing heat losses.
The thermophilic pattern seems promising for higher conversion rates and for the sterilization and removal of parasites and pathogenic strains. However, the maintenance of such temperatures leads to big heat losses that cannot be repaid by the increased methane yield. In cases where VFAs are the target product, the heat balance for the thermophilic regime becomes unacceptable because the VFA concentrations are low, i.e., below 1 g/L [38]. Another drawback of the thermophilic regime is the high sensitivity of the thermophilic strains to the pH drop and product inhibition.
Moreover, variations in the operating temperature can alter the microbial components of the consortium involved in acidogenic fermentation, leading to different product yield.
The organic loading rate (OLR) indicates the amount of substrate fed into the bioreactor per unit of time per unit of working volume in terms of total solids (TSs), volatile solids (VSs), or chemical oxygen demand (COD).
Acetoclastic methanogenesis (acetic acid decarboxylation) is more sensitive to acetic acid than hydrogenotrophic methanogenesis (reduction of CO2 by hydrogen). The methanogenic microbial community can be changed significantly under these conditions with the enhancement of species that are vital at high organic loads.
Microbes using CO2/H2 cannot adapt well to excessive organic loads compared to those using acetic acid or ethanol. That is why an imbalance between the two methane metabolic pathways leads to low methane production.
One important factor of operating at a high OLR is the presence of inhibitors in the substrate [39]. High product yield can be achieved when the OLR is high enough to provide a suitable amount of carbon, but it must be balanced with other operating conditions such as the applied temperature, VFA production, and the inhibition effect. The inhibitors may affect not only methanogenesis but also hydrolysis and the acidifying bacteria. The accumulation of inhibitors also being target products (namely volatile fatty acids) may impede their production [35,40]. That is why an optimum OLR for VFA composition is required.
For example, a high OLR of 16 g TS/(L·d) leads to an increase in VFA production with a consequent decline in VFA concentration [26]. A lower OLR, ca. 10 g TS/(L·d), leads to a stable process of VFA production in time.
The hydraulic retention time (HRT) is defined as the average time that matter remains in a bioreactor. It is an important parameter because it specifies the feed flow rate and the residence time of the substrates and the products in the bioreactor. The HRT is also important because it is essential for the balance of the growth of slow-growing methanogens and relatively fast-growing acidogens and for their washout at a lower residence time in a bioreactor. The residence time should be long enough to allow the complete hydrolysis of the complex organic matter, thus favoring the acidogenic fermentation of the hydrolyzed substrate. On the other hand, a very long HRT reduces the quantity of the treated daily substrate and favors methanogenesis at suitable pH values.
That is why the selection of an appropriate hydraulic retention time in an anaerobic digester is a delicate task, depending on the target process—biogas or VFA production. If biogas is desired, longer HRTs are preferred to avoid the over-accumulation of volatile fatty acids and pH drop and allow their complete conversion to methane and carbon dioxide. When VFAs are the target products, shorter residence times at higher OLRs are recommended.
Dennehy et al. [41] studied the impact of changes in the HRT from 15 to 10.5 days on the anaerobic digestion of pig manure and food waste in a continuously stirred tank reactor. There was significant accumulation of VFAs at a lower HRT. Similar observations were made by Zhang et al. [42], who examined the influence of decreasing HRT on chicken manure fermentation at a constant OLR. Microbial washout and a high acetic acid concentration (>6000 mg/L) were observed when the HRT was decreased from 52 days to 5 days. Li et al. [43] noted that a short HRT was optimal for VFA production from proteins.
Appropriate retention times are very important when lignocellulosic substrates are used. In this case, a longer time for fermentation is necessary because of the lignin hindrance effect on the penetration of microbial cells.

2.2. Methane vs. VFA Formation

It is known that the accumulation of VFAs (acetic acid in particular) due to high organic loads inhibits anaerobic digestion (biogas production too) intensively [42].
It was mentioned above that the balance between VFA accumulation and methane production is too sensitive, depending on the target products. It is also sensitive to the substrate(s) and pre-treatments used [8,44].
The priority of the decision whether to focus on biogas or volatile fatty acids depends on some preconditions [45].
First is the amount of feedstock, its origin (agricultural waste or household waste), and its nature (manure, lignocellulose, or activated sludge). These input data will predetermine the yields of biogas or volatile fatty acids. The are different sources of information about those yields represented as produced amounts per unit volatile (total) solids, e.g., the energy content of biogas or the amount of produced fatty acids (totally or separately). The feedstock supply will help the choice and decision about the appropriate product.
Second is the actual market demand for these products. Biogas production is encouraged as a renewable source of energy and a source of methane for inclusion in the circular economy as well. Volatile fatty acids have various practical applications with considerable demand and prices (cf. Table 1).
Third is the comparison of the prices of both products depending on the market demand. Biogas is estimated as an energy source with a price per unit of energy (MWh, MBtu). The price of carbon footprints must be taken into account. The cost of biogas production varies between 6.8 and 68 USD/MWh depending on the region and the local conditions (USA, Europe, and South Asia). The cost of biogas electricity typically ranges from USD 50 to USD 150 per MWh, depending on the location, feedstock, and plant size.
Supposing the operational costs for both options are comparable for a single anaerobic digestion unit, one can conclude that volatile fatty acids are more favorable as final products than biogas production.
However, the heating value of the biogas released during anaerobic digestion can be used for the target process of VFA production even at a low methane content. Such an integrated process has been proposed, where the produced methane was used as an energy source for temperature maintenance [8].
Other options for the use of biogas as a feedstock for value-added chemicals are considered elsewhere [46]. The main process is to convert biogas into synthesis gas (CO + H2). The latter is used for the production of light hydrocarbon by the Fischer–Tropsch catalytical process.
Biogas with a low methane concentration can be used in other ways after upgrading it, i.e., via the separation of carbon dioxide and contaminants by pressure-swing adsorption, chemical scrubbing, membrane separation, etc. It can also be mixed with natural gas to attain a suitable methane content for heat, transport, and electricity.
Low-concentration biogas might be converted catalytically to carbon dioxide and hydrogen with their utilization.

2.3. Case Study: Glycerol as a Substrate in a Multi-Step Bioreactor

This case study is an illustration of the importance of a multi-step digester configuration and the operation mode to eliminate (or at least to lower) the inhibition effect of the intermediates, like the volatile fatty acids, accumulated in the broth [47]. Such bioreactor construction and flow organization enable to transform consecutive processes (i.e., hydrolysis, acidogenesis, acetogenesis, and methanogenesis) into simultaneous ones distributed spatially. Hence, fluctuations in the substrate or intermediate concentration and the associated pH drop will have less impact on the processes in the adjacent reactor compartments.
An example of a possible substrate for biogas and VFA production is waste glycerol from biodiesel production. This glycerol contains significant amounts of water, methanol, lipids, mono- and diglycerides, and alkaline residues, and its low quality makes it impossible to be sold on the market before purification.
One of the methods of glycerol utilization is the use of the AD process to produce biogas. One advantage of the method is that glycerol can be mixed with other wastes (such as cattle manure and straw) to boost the process. The result is more biogas with a higher content of methane and the simultaneous utilization of two wastes (glycerol and manure). It is well known that they are produced by different bacteria (from genera Klebsiella, Clostridium, and Enterobacter). They are capable of digesting glycerol to produce basic chemical compounds, which are different regarding their medium reactions and products, like 1,3-hydroxyacetone, 1,3-propanediol [48,49,50], formic acid, propionic acid [49,50], some polyesters [51,52], and 2,3-butanediol [52,53]. Figure 2 shows the metabolic scheme of glycerol transformation from the genus Klebsiella as suggested by da Silva et al. [54] and Zhang et al. [55].
As can be seen, two concurrent pathways, 1,3-propanediol and 2,3-butanediol, are produced. Formic acid, lactic acid, succinic acid, and acetic acid are also produced. The conversion of glycerol into valuable products by the genus Clostridium is similar to that by the genus Klebsiella [56]. Bacteria from the genus Clostridium mainly produces 1,3-propanediol and organic acids (formic, acetic, butyric, and lactic) but also n-butanediol. Bacteria from the genus Enterobacter produces mainly ethanol and hydrogen [57,58,59].
It is interesting to note the production of acetic and formic acid by those metabolic processes in relation to biogas production. The conversion of acetic and formic acid is possible in anaerobic conditions and in the presence of methanogenic bacteria.
It is a matter of interest whether it is possible to produce biogas by mixing cattle manure with waste glycerol in an anaerobic digestion reactor. We have carried out experiments on biogas production from mixtures of cattle manure and crude glycerol [60]. The experiments proved to be successful. We obtained combustible biogas, as well as other by-products such as propionic acid. It was observed that by mixing glycerol and cattle manure, not only was more biogas produced, but also by-products such as propionic acid. The quantity of the propionic acid was so high that it led to decreasing the pH level until it was too low for the AD process. When glycerol is used as the substrate, the final balance equation is
4C3H8O3 = 7CH4 + 2H2O + 5CO2
Intensifying the acidogenesis is desired because of the more effective treatment of the substrate, but it leads to the inhibition of methanogenesis due to a drop in pH levels. The slow digestion of the acetic and propionic acid combined with their quick accumulation leads to a drastic drop in pH levels, which in turn kills the methanogenic bacteria. It is also estimated that propionic acid and its anions are inhibitors of the methanogenic process.
Discussions on acidifying the reactor environment, which stops the methanogenic process, are very important, especially when a low-molecular-weight substrate is used, such as glycerol. This is due to its rapid conversion to organic acids (propionic, lactic, acetic, etc.) (cf. Figure 2). Therefore, it is not possible to work with high loadings of glycerol because of the rapid pH decrease.
Limitations on pH levels put the system in a very delicate condition so as to maintain the optimum conditions for each step. As an example, hydrolysis and acidogenesis require a low-pH environment, while the methanogenic process requires slightly higher pH values [60].
One possibility to avoid the quick pH drop is by the usage of alkali agents. That leads to a huge increase in the pH after the breakdown of the organic acids, which kills the methanogenic bacteria in the bioreactor. One suggested way to avoid that problem is to use ammonia (as a nitrogen source), which supports the growth of the bacteria. Unfortunately, ammonia is expensive, and the process of anaerobic digestion becomes more complex.
Another possibility to remove organic acids is to extract them by the use of ion-exchange resin or organic solvents [61,62,63] as well as reverse osmosis or ultrafiltration [64]. But in this case, the accumulation of hydroxylic anions in the environment could lead to an unwanted increase in pH levels after the breakdown of organic acids.
A mild and more effective way to remedy the effect of pH drop is the spatial separation of the inhibition zones from the other zones with a methanogenic process. A cascade reactor could be used for that purpose. It is separated into different reaction compartments (cf. Figure 3). It is known that this type of bioreactor is stable in relation to loading variation, pH, and temperatures changes. The main advantage of the proposed bioreactor type is the distribution of the different steps of anaerobic digestion in different parts (chambers) of the bioreactor.
When glycerol is used as a substrate, bacteria from Klebsiella and Clostridium are sufficient for the AD process. It can be seen from Figure 2 that the substrate is broken into anions of different organic acids, two of which (formic and acetic) serve as substrates for methane production. All other metabolic pathways lead to concurrent processes: the production of succinates, lactates, 1,3-propanediol, 2,3-propanediol, succinic acid, and lactic acid, as well as little amounts of ethanol. As can be observed from the metabolic scheme, the possibilities of producing biogas are mainly two: the breakdown of acetates to an equimolar mixture of methane and carbon dioxide [65,66] or the reduction of carbon dioxide with hydrogen. It is known that bacteria from the genus Methanosarcina breaks down acetate [67,68], while the reduction of carbon dioxide with hydrogen is conducted by bacteria from the genera Methanobacterium and Methanobrevibacter (cf. Fuchs et al.) [12].
The microbe distribution in the eight compartments of the cascade anaerobic digester is shown in Table 2. Aerobe microbes (such as fungi and Bacillus) are found in compartment 1. In all others, the aerobe strain Klebsiella was detected.
When glycerol is used as a substrate, representatives from the genera Methanobacteriales-Methanobrevibacter (Methanobrevibacter ruminantium) and Methanobacterium prevail in the bioreactor. The types of microbes present in the bioreactor could be explained by the different pathways of acetate breakdown—one of the main metabolites and a precursor for methane production. The acetate breakdown could happen in two ways—acetic and oxidative—to produce hydrogen and carbon dioxide. The first one is preferred by the Methanosarcina and Methanosaeta, while the second one is preferred by Methanobrevibacter и Methanobacterium.
The experimentally observed concentration profiles along the eight compartments of the baffled digester on the 12th day of anaerobic digestion are shown in Figure 4. The substrate (glycerol) is totally consumed in the fourth compartment. Acetic acid concentrations are negligible. There are observable concentrations of propionic acid in compartments 1–4 with the corresponding pH drop. The VFA concentrations in compartments 5–8 are negligible, and a pH increase to reasonable values for methanogenesis is observed.

2.4. Formation of Hydrogen vs. VFAs

The general similarities that exist between VFAs and hydrogen as metabolites of the anaerobic digestion process are that they are biosynthesized as intermediates and serve as precursors of biohydrogen and biogas production [14,69]. Due to the potential of obtaining these metabolites from organic substrates, many researchers have successfully limited the methanogen activities with the aim of optimizing the formation of either VFAs or hydrogen [69,70,71]. It can be concluded from these studies that bioprocesses that are favorable for the formation of the VFAs at high yields usually produce hydrogen as a by-product and vice versa. It has been observed that the presence of VFAs interferes with hydrogen production, either as a result of inhibition by undissociated acid molecules [72,73] or because of the consumption of hydrogen by homoacetogens [71].
One well-established metabolic pathway for biohydrogen during dark anaerobic fermentation [72] is shown in Figure 5.
The dissolved metabolites detected in the fermentation medium during the dark fermentation of hydrogen are mainly acetate and butyrate due to the established pathways mentioned earlier. On the other hand, other reduced metabolites such as propionate are associated with low hydrogen production [73]. Regarding the impact of hydrogen on VFAs, it has been suggested that increasing the hydrogen partial pressure in the bioreactor could possibly alter the product spectrum of the acids [74].
Many VFAs are observed in the broth, from formic acid (C1) to caproic acid (C5). Lactic acid is also detected. These two main streams (hydrogen or fatty acid production) are competitive. The question is, what is the target product, hydrogen or fatty acids? In the case that hydrogen is preferred, efforts to boost hydrogen production must be made, e.g., by the electro-fermentation process [75]. The constant electric field has a positive effect in hydrogen production, whereas the production rate of acetic and butyric acid decreases. Ethanol production is enhanced.

2.5. VFA Removal from Fermentation Broth During Anaerobic Digestion

It has been emphasized that the accumulation of volatile fatty acids may inhibit either methanogenesis or complete VFA conversion during anaerobic digestion. Moreover, acid accumulation can lower the pH and hence impede methanogen activity. In both cases, acid removal from the broth is recommended, both as a product recovery and inhibition remedy [15].
There are different ways to reach this aim. The first is in situ liquid–liquid extraction [63]. For this purpose, active solvents are used, namely tri-octylamine (TOA) and tributyl phosphate (TBP) diluted by different organic carriers, like n-hexane, kerosene, oleic alcohol [76], etc. There are also experiments with other solvents, i.e., tri-n-octylphosphine oxide (TOPO), Aliquat 336, trihexyl(tetradecyl) phosphonium, and bis(2,4,4-trimethylpentyl)-phosphinate [77]. A problem may occur because of the toxicity of the active extractant.
It was pointed out that this method is applicable both in batch and continuous mode [77,78]. However, the excessive removal of fatty acids may lead to pH increase and to loss of microbial activity or even death. Therefore, thorough pH control is recommended.
In the case of ionogenic products, like carboxylic acids, ion-exchange techniques can be applied. A principal scheme for continuous product removal by the anion-exchange process is shown in Figure 6. The solvent regeneration is accomplished by an alkaline solution to obtain the acids as their salts.
Another mode for organic acid removal from fermentation broth is the ion-exchange technique with the simultaneous maintenance of pH [79].
The in situ extraction approach is very promising from an economical point of view because the tedious process of downstream processing is considerably simplified. The selectivity of this process when a VFA mixture is produced depends on each acid pKa and the selected ion-exchange resin.
Another similar way for VFA removal from anaerobic digestors is membrane extraction [80]. Membranes have also been proposed to protect VFAs and biogas-producing bacteria from the inhibitory effect of medium- and long-chain fatty acids [81]. When microbes are encased by a membrane, the biogas production is better than in free culture.
There are more membrane methods for product separation (VFAs included) [82,83]. They are micro- and ultrafiltration, electrodialysis, reverse osmosis, membrane distillation, pervaporation, etc. For volatile products like VFAs, membrane distillation and pervaporation seem suitable. However, their practical application and effectiveness are strongly dependent on the solute concentration and its partial pressure in the broth.
One of these membrane-based methods is pervaporation. It was tested for VFA in situ separation from fermentation broth [84]. However, the efficiency of this process depends on the product volatility, i.e., its partial pressure. Since the concentrations of VFAs are not very high and the temperatures are medium, the applicability of this method for VFA separation is restricted.

3. Conclusions

On the basis of the presented review, the following conclusion can be drawn:
  • Volatile fatty acids are inevitable intermediate products of anaerobic digestion with biogas formation. Their excessive accumulation may lead to strong inhibition of methanogenic microbes and a lack of methane in the biogas.
  • However, volatile fatty acids (formic, acetic, propionic, and butyric ones) are useful commodities with various applications. That is why their targeted production with a desired production rate may shift the aims of the anaerobic digestion from biogas to volatile fatty acid production.
  • Combining VFA production with biogas release can be an integrated and self-consistent process with biogas production sufficient enough to supply the target processes with energy. The biogas can also be utilized as a feedstock for value-added chemicals or fuels (light hydrocarbons) and hydrogen.
  • The maintenance of optimum VFA concentrations can be accomplished by simultaneous VFA removal from the fermentation broth, thus integrating the product recovery with the maintenance of optimum operation conditions in the digester.
  • The substrate preparation and the operating conditions (organic loading rate and hydraulic retention time) are of crucial importance for a successful fermentation process.

Author Contributions

Conceptualization, V.N.B. and I.K.A.; methodology, V.N.B.; formal analysis, I.K.A.; writing, V.N.B. and I.K.A.; writing and editing, V.N.B.; supervision, V.N.B. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Fund for Scientific Research, Bulgaria, grant number KP-06-H67/3. The APC was funded by the Ministry of Education and Science, Republic of Bulgaria, project “Energy Storage and Hydrogen Energetics” (ESHER), grant agreement number DO1-160/28.08.18.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Own experimental data are available at the Institute of Chemical Engineering, Bulgarian Academy of Sciences.

Conflicts of Interest

The authors declare no conflicts of interest.

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Fermentation 11 00172 g001
Figure 1. Scheme of the consecutive processes of biogas formation with product recovery and utilization.
Figure 1. Scheme of the consecutive processes of biogas formation with product recovery and utilization.
Fermentation 11 00172 g001
Fermentation 11 00172 g002
Figure 2. Metabolic pathway of glycerol processing by Klebsiella genus according to ref. [55]. The reactions in the dotted rectangle include the participation of co-factors NAD+/NADH and FDH for CO2 release.
Figure 2. Metabolic pathway of glycerol processing by Klebsiella genus according to ref. [55]. The reactions in the dotted rectangle include the participation of co-factors NAD+/NADH and FDH for CO2 release.
Fermentation 11 00172 g002
Fermentation 11 00172 g003
Figure 3. A sketch of the multi-step bioreactor.
Figure 3. A sketch of the multi-step bioreactor.
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Fermentation 11 00172 g004
Figure 4. Concentrations of substrate, intermediates, and pH on the 12th day after feeding with glycerol [60].
Figure 4. Concentrations of substrate, intermediates, and pH on the 12th day after feeding with glycerol [60].
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Fermentation 11 00172 g005
Figure 5. Biochemical pathway for hydrogen production in mixed culture [72].
Figure 5. Biochemical pathway for hydrogen production in mixed culture [72].
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Figure 6. Scheme of continuous fermentation process with simultaneous product removal by ion exchange.
Figure 6. Scheme of continuous fermentation process with simultaneous product removal by ion exchange.
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Table 1. Some data about the market size and market prices for some volatile fatty acids, estimated for 2020. An excerpt from Table 6.1 in [26].
Table 1. Some data about the market size and market prices for some volatile fatty acids, estimated for 2020. An excerpt from Table 6.1 in [26].
Fatty AcidAreas of ApplicationsMarket Price,
EUR/ton		Annual Production
ktons/Year
AceticFood additives, plasticizers, and dyes400–80014,000–17,000
PropionicResins, pharmaceuticals, and paints1250–1700350–470
ButyricPerfumes, textiles, varnishes, and plastics1500–165090–105
Valeric *Perfumes, plasticizers, and lubricants1500–1650720 *
Caproic *Rubber, grease, and tobacco flavor1500–1650150 *
* Data for the annual production of valeric and caproic acids are valid for 2023.
Table 2. Content of different genera of bacteria in the separate compartments of the eight compartment bioreactor [60].
Table 2. Content of different genera of bacteria in the separate compartments of the eight compartment bioreactor [60].
CompartmentAerobes, Facultative Aerobes,
Count of Bacteria in 1 mL		Anaerobes,
Number of Bacteria in 1 mL		Methanogens,
Genera		Methane Production
1Fungi, Bacillus
~1 × 102		~1 × 101 -
2Klebsiella
~1 × 103		~1 × 105MethanosarcinaAcetate,
CO2 + H2
[67,68]
3Klebsiella
~1 × 103		~1 × 105MethanobacteriumCO2 + H2
[68]
4Klebsiella
~2 × 103		~1 × 106MethanobacteriumCO2 + H2
[12]
5Klebsiella
6–8 × 102		~4-5 × 106MethanobacteriumCO2 + H2
[12]
6Klebsiella
~4 × 102		~2 × 106MethanobrevibacterCO2 + H2
[68]
7Klebsiella
1–2 × 103		~1 × 106MethanobrevibacterCO2 + H2
[12]
8Bacillus, Klebsiella
~1 × 102		~1 × 105MethanobrevibacterCO2 + H2
[12]
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Beschkov, V.N.; Angelov, I.K. Volatile Fatty Acid Production vs. Methane and Hydrogen in Anaerobic Digestion. Fermentation 2025, 11, 172. https://doi.org/10.3390/fermentation11040172

AMA Style

Beschkov VN, Angelov IK. Volatile Fatty Acid Production vs. Methane and Hydrogen in Anaerobic Digestion. Fermentation. 2025; 11(4):172. https://doi.org/10.3390/fermentation11040172

Chicago/Turabian Style

Beschkov, Venko N., and Ivan K. Angelov. 2025. "Volatile Fatty Acid Production vs. Methane and Hydrogen in Anaerobic Digestion" Fermentation 11, no. 4: 172. https://doi.org/10.3390/fermentation11040172

APA Style

Beschkov, V. N., & Angelov, I. K. (2025). Volatile Fatty Acid Production vs. Methane and Hydrogen in Anaerobic Digestion. Fermentation, 11(4), 172. https://doi.org/10.3390/fermentation11040172

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