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Volatile fatty acid (VFA) yield from sludge anaerobic fermentation through a biotechnological approach
Volatile fatty acid (VFA) yield from sludge anaerobic fermentation through a biotechnological approach
29
Małgorzata Worwag, Anna Kwarciak-Kozłowska Faculty of Infrastructure and Environment, Institute of Environmental Engineering, Czestochowa University of Technology, Czestochowa, Poland
1
Characterization of volatile fatty acids (VFAs)
Volatile fatty acids (VFAs) are saturated aliphatic acids that contain from two to six carbon atoms in the particle, and sometimes even eight (Chwiałkowska and Oleskowicz-Popiel, 2016; Zielewicz, 2007). Their structure contains an acetyl group characterized by the presence of a carbon atom that is double-bonded to oxygen. Due to the electronegativity of oxygen present in the acetyl group, carboxyl acids belong to polar compounds. Despite their high boiling point, these acids can be removed from water due to distillation with water steam. These compounds are characterized by high vapour pressure, which is utilized during the determination of their concentration in environmental samples (Zielewicz, 2007). VFAs have strong hydrophilic properties. The nomenclature of VFAs is mostly based on customary names that suggest their plant or animal origins. These acids are referred to in the literature by various names, including low-weight carboxylic acid (LWCAs), short-chain fatty acids (SCFAs), low-molecular-weight aliphatic acids (LMWAs), and volatile organic acids (VOAs), as well as VFAs (Banel, 2010). Table 1 presents the names, chemical structures, and physicochemical properties of these compounds. Knowledge of the physicochemical properties of selected VFAs allows for prediction of their behavior in the environment and development of procedures for their determination (Banel and Zygmunt, 2009). Knowledge of the value of the negative log of the dissociation constant (pKa) allows for determination of the part of the compound present in the dissociated/nondissociated form in the sample. The balance of dissociation of three of the most important organic acids is presented in Fig. 1. It shows that reaction plays an important role. Analysis of the degree of dissociation of acetic acid reveals that at pH 6, there are 5% of nondissociated ethanoic acid (CH3COOH) particles, only 0.5% at pH 7, and 0.05% at pH 8. In general, it is accepted that in the range of pH from 6.0 to 8.0, the concentrations of nondissociated forms are reduced by two orders of power. The increase in the amount of nondissociated acids causes them to react with carbonates, with a simultaneous increase Industrial and Municipal Sludge. https://doi.org/10.1016/B978-0-12-815907-1.00029-5 © 2019 Elsevier Inc. All rights reserved.
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Table 1 Nomenclatures, chemical compositions, and physicochemical properties of VFAs (Banel, 2010; Chwiałkowska and Oleskowicz-Popiel, 2016)
Chemical formula
M (g/mol)
Methanoic acid (formic) Ethanoic acid (acetic) Propane acid (acid) Isobutane acid (isobutyric) Butanoic acid (butyric) Isopentanoic acid (isovaleric) Pentanoic acid (valeric) Hexane acid (caproic) Heptanoic acid (enanthic) Octane acid (caprylic)
HCOOH CH3COOH C2H5COOH (CH3)2CHCOOH C3H7COOH (CH3)2CHCH2COOH C4H9COOH C5H11COOH C6H13COOH C7H15COOH
46.03 60.1 74.1 88.1 88.1 102.1 102.1 116.2 130.2 144.2
1220 1049 990 960 964 925 938 927 918 910
Boiling point (°C)
Solubility in water at ambient temperatures (g/L)
pKa
100.8 117 141 154 164 177 186 206 223 235
Big Big Big 210 Average 25 40 10 2.6 0.7
3.75 4.75 4.87 4.85 4.81 4.78 4.82 4.88 4.89 4.89
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System name (custom)
Density at 25°C (g/L)
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Fig. 1 Percentage of nondissociated and dissociated forms of acids for pH values ranging from 6 to 8. Based on Sadecka, Z., 2010. Basics of Biological Wastewater Treatment, Seidel-Przywiecki Publishing.
in carbon dioxide (CO2) content, leading to a reduction in reaction. This results in the shift in balance between dissociated and nondissociated forms of VFAs toward nondissociated forms (Banel, 2010; Sadecka, 2010). With acidification of the reaction environment, dissociation is reversed, leading to easier transfer of nondissociated acid particles to the organic or headspace phase. In their dissociated form, VFAs are mostly soluble in water, whereas they are poorly soluble in organic solvents (nonmixable with water). Low concentrations of nondissociated forms of acetic acid and propionic acid have an inhibiting effect on the yield of the fermentation gas (Banel, 2010; Sadecka, 2010). VFAs are the compounds that are often observed in the environment. However, they rarely occur in a free state; rather, they mostly take the form of esters, salts, or amids. VFAs can be derived from both natural and anthropogenic sources. They are present in large amounts in sewage sludge, sludge generated during farming of breeding swine, and sludge from the food industry. Their sources are also organic waste and liquors generated during deposition of this waste in landfills or composting. In the liquors generated in young landfills, VFAs account for nearly 80% of all organic components, with their concentrations reaching as much as 6000mg/L. With the landfill aging, the amount of VFAs in liquor is substantially reduced, even to the level of 5 mg/L (Banel, 2010; Chwiałkowska and Oleskowicz-Popiel, 2016; Banel and Zygmunt, 2009). VFAs are also generated in human and animal intestines during digestion. In the environment, VFAs also can play a negative role. The compounds from this group may lead to acidification of the environment or increased mobility of heavy metals and radionuclides. Due to their chemical composition, VFAs play a significant role in biological treatment processes, as they represent an easily available source of carbon for microorganisms (Chwiałkowska and Oleskowicz-Popiel, 2016).
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Despite the substantial interest in the problems of acids in various environmental samples, much attention has been devoted to the methodology of determination of these compounds. The choice of adequate analytic methodology used for their determination depends mainly on the accuracy and precision of determinations, matrix composition, available apparatus, predicted level of their content in the sample, and time and cost of performing analysis. The methods based on titration, often preceded by the isolation of analytes from the primary matrix by means of distillation methods, have been used to evaluate total VFA content. Determination of individual acids is conducted using separation techniques, mostly chromatographic (Banel, 2010). The use of ion chromatography often requires a complex procedure of sample cleaning in order to reduce interference and reach an adequately low determination threshold. A useful tool for determination of VFAs in both water and gas samples is gas chromatography with flame ionization detector (GC-FID). This technique allows for the identification and quantitative determination of individual fatty acids (Banel and Zygmunt, 2009). One of the main steps before analysis of the sample from a medium is isolation of the compounds to the level that allows for detection and determination. Table 2 compared the methods used for separation of VFAs from the medium studied.
Table 2 Methods of VFA separation from the medium studied (Zacharof and Lovitt, 2013) Separation methods Precipitation
Distillation
Description
Benefits
Calcium salts are added to the purified medium, whereas the obtained solution of calcium carboxylane is solidified by vaporization and, through crystallization, separates it from the parent liquid. Ammonia is used to neutralize acids that react in order to generate ammonium carboxylate, which is mixed with alcohol in order to produce esters, followed by division through distillation.
l
l
l
l
l
Defects
High production coefficient. Low functional costs. High-purity products.
Solid waste production due to the use of sulfuric acid for the release of carboxylic acids from calcium carboxylates
High purity of products. By-products can be used as fertilizer.
High costs of energy connected with distillation, which is used for the separation of alcohol from carboxylic acids
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Table 2 Continued Separation methods Adsorption
Description
Benefits
Defects
Ion-exchange resins are used as adsorbents for exchange of carboxylate ions from the medium.
Easy operation
l
l
l
l
Electrodialysis
Extraction with solvent
With electric current in an electrodialyser, negatively charged carboxylate ions go through an anionexchange membrane to the anode.
Carboxylate is concentrated in water solution and does not require acidification.
Organic acids are used for extraction of carboxylic acids from the purified stream.
l
l
l
l
Low cost Opportunities for the production of carboxylate salts
l
l
Membrane processes
Utilization of semipermeable barriers where VFAs are selectively separated depending on the types of membranes (pore size). Transport is possible at the instant of using the driving force (pressure difference, concentrations, temperature and electrical potential).
High selectivity
High price of resins High demand for energy due to resin regeneration Low adsorption abilities Separation not highly selective Products are much contaminated, and it is necessary to continue their purification. High demand for energy. Initial acidification of the sample in order to obtain greater and efficient extraction. Extracts have to be regenerated through distillation or extraction.
l
Occurrence of concentration polarization
l
Reduction in the stream of permeate due to the phenomena of fouling and scaling High functional costs
l
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Table 3 Methods of production of carboxylic acids (Zacharof and Lovitt, 2013) Carboxylic acid
Chemical synthesis methods
Bioprocess
Formic acid
Alkene oxidation Hydrogenation of CO2 Methanol carbonylation Methanol carbonylation Acetaldehyde oxidation Ethylene oxidation Ethylene hydrocarboxylation Aerobic oxidation of propionaldehyde Chemical oxidation of butyraldehyde Ethylene oxidation Chemical synthesis
Glucose oxidation CH4 fermentation
Acetic acid
Propionic acid Butyric acid Caproic acid Lactic acid
Glucose oxidation CH4 fermentation CH4 fermentation Alcohol fermentation CH4 fermentation CH4 fermentation
Glucose Lactic acid
Lactic acid bacteria
Pyruvate
Propionic acid bacteria
Ethanol
Yeast
Coli-Arerogenes
Clostridium
Ethanol
Oxaloacetate
Acetate
Acetate Succinate
Acetylo-CoA
Acetoin Butanediol
Butyrate
Acetone
Propionate Formic acid Butanol
Isopropanol
Fig. 2 Diagram of hydrocarbon fermentation processes in bacteria. Based on Sadecka, Z., 2010. Basics of Biological Wastewater Treatment, Seidel-Przywiecki Publishing.
2
Production of VFAs
VFAs can be produced via the chemical processing of petroleum or anaerobic biological processes. Table 3 compares the most frequently used processes of VFA production. The diagram of initiated CH4 fermentation can be presented using the example of carbohydrate decomposition (Fig. 2). It shows that this process begins from glycolysis as it is the case in aerobic processes. The central derivative from which the pathways of aerobic and anaerobic biodegradation diverge is pyruvate. The fermentation process can be conducted depending on the orientation to a specific product. Different
Volatile fatty acid (VFA) yield from sludge
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Fig. 3 Biological pathways of carbohydrate conversion into VFAs during homolactic and heterolactic fermentation (A), alcohol fermentation (B), and during the cycle of tricarboxylic acids (C). Based on Zacharof (2013).
types of fermentation have been discovered, with their names taken from the dominant final product: alcoholic, lactic, propionic, formic, butyric, and acetic fermentation. In addition to the dominant final products, there are succinate, caproate, n-butanol, 2,3butanodiol, acetone, isopropanol, CO2, and hydrogen. All of the listed fermentation products can be generated separately or in different arrangements (Schlegel, 2003). Fig. 3 illustrates schematically biological pathways of carbohydrate conversion into VFAs during homolactic and heterolactic fermentation (in panel A), alcohol fermentation (B) and through the cycle of tricarboxylic acids (C). Extensive research has been conducted in recent years on the use of mixed cultures for production of carboxylic acids. With the high flexibility of these cultures, it is possible to process organic waste, sewage sludge, and agricultural and food industry waste (Chwiałkowska and Oleskowicz-Popiel, 2016). In anaerobic conditions, biological decomposition of organic matter occurs in several stages. The first stage of the CH4 fermentation process is hydrolysis; when insoluble, polymerized organic compounds are exposed to the effect of extracellular
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hydrolytic bacteria enzymes (e.g., amylases, lipases, or proteases), leading to their decomposition and liquidization. Polysaccharides are hydrolysed to monosaccharides, proteins to amino acids, and fats to polyols and fatty acids. The hydrolysis phase is a stage that limits the course and efficiency of fermentation (Zielewicz, 2007; Agler et al., 2011). Another stage is the phase of acidification or acidogenesis, which is also termed dark fermentation. The initial substrate for acid-forming bacteria is hydrolysis products. The bacteria that participate in this process are facultative bacteria, but there are also obligate anaerobes. The most frequently used of these include Aerobacter, Alcaligenes, Clostridium, Escherichia, Flavobacterium, Pseudomonas, Lactobacterium, Lactobacillus, Micrococcus, and Streptococcus. Bacteria of the acidification phase are relatively less susceptible to temperature or pH changes. They proliferate much faster than CH4 bacteria. With their activity, low molecular organic compounds are processed mainly (76%) into VFAs (e.g., formic acid, acetic acid, propionic acid, butyric acid, valeric acid, and caproic acid) and to alcohols (e.g., ethanol and methanol) and gaseous products (CO2 and H2). The remaining part is decomposed into acetates. In this phase, proteins are further decomposed into simple organic acids, thiols, and amines (Zielewicz, 2007; Rosik-Dulewska, 2007; Sadecka, 2010). In the third stage of CH4 fermentation, the so-called acetate phase (acetogenesis), ethanol and VFAs are processed into CO2, hydrogen, and acetic acid. These transitions occur with two groups of anaerobic bacteria of the Syntrophobacter and Syntrophomonas genuses (Michalska et al., 2014). The first group of acetogenic microorganisms is responsible for hydrolysis of organic polymers and lipids to basic structure-forming compounds such as monosaccharides or amino acids. The second group is involved in fermentation and destruction of products to simple organic acids, including acetic acid, propionic acid, and butyric acid (Zielewicz, 2007) Distribution of organic acids by bacteria is relatively specific because it requires the involvement of various bacteria species. For example, valeric acid is decomposed into acetic acid and propionic acid by Mbacterium suboxydans, but further decomposition of propionians into acetates, CO2, and CH4 is achieved with Mbacterium propionicum. The latter species is unable to absorb and decompose acetates; therefore, their further decomposition into CH4 requires the presence of Methanococcus mazei bacteria (Fig. 4).
Fig. 4 Activity of bacteria to produce CH4 from valeric acid. Based on Sadecka, Z., 2010. Basics of Biological Wastewater Treatment, Seidel-Przywiecki Publishing.
Volatile fatty acid (VFA) yield from sludge
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In addition to fermentation of carbohydrates and proteins, acetic acid is generated during the anaerobic oxidation of fats. Generation of propionic acid occurs first and foremost during the degradation of carbohydrates. Another product of the fermentation of proteins and fats is butyric acid. This acid also can be generated during the fermentation of carbohydrates from pyruvate. Oxidation of acids such as lactic acid, propionic acid, and lactic acid is possible if hydrogen produced by acetogens is received from the environment. Their oxidation is possible only at very low concentration of hydrogen and low redox potential (<–300 mV). An insignificant increase in hydrogen pressure results in an immediate decline in the rate of degradation of these acids (Błaszczyk, 2009). These transitions can occur if molecular hydrogen is removed from the reaction environment. With these environmental requirements, the two groups of microorganisms (i.e., acetogenic and methanogenic bacteria) live in symbiosis (Zalewski et al., 2012). Both types of bacteria can connect to various aggregates (e.g., flocs or granules), and their symbiosis results in the decomposition of propionic acid according to Eqs. (1)–(3) (Bitton, 2005; Rosik-Dulewska, 2007; Z˙ygadło, 1999). l
Acetogenic bacteria:
CH3 CH2 COO + 3H2 O ! HCO3 + CH3 COO + H + + 3H2 l
(1)
Methanogenic bacteria:
HCO3 + 4H2 + H + ! CH4 + 3H2 O
(2)
The total balancing reaction for these chemical reactions can be written as follows: 4CH3 CH2 COO + 3H2 O ! HCO3 + 4CH3 COO + 3CH4 + H +
(3)
The phenomenon of this symbiosis was discovered in the 1970s and is termed interspecies hydrogen transfer (IRT). Undisturbed hydrogen transfer guarantees the correct course of anaerobic distribution of organic substrates. Isolation of pure cultures of acetic bacteria is impossible, and growth of mixed cultures is very difficult and labour-consuming. Bacteria of the acetogenic phase are characterized by a long time of generation that depends on the type of available substrate. Fig. 5 presents the times of acetic bacteria generation at mesophilic temperature depending on the type of substrate (Sadecka, 2010). In addition to production of acetate from hydrogen and CO2, acetogenic bacteria can utilize other simple compounds (e.g., carbon dioxide, methanol, or glucosis), generating acetate as a final product of metabolism. Production of acetic acid by acetogenic microorganisms proceeds according to the reactions given as Eqs. (4)–(7) (Błaszczyk, 2009): CO2 + 4H2 ! CH3 COOH + 2 H2 O
(4)
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Fig. 5 Times of acetic bacteria generation at mesophilic temperatures (35–37°C) depending on the type of substrate. Based on Sadecka, Z., 2010. Basics of Biological Wastewater Treatment, Seidel-Przywiecki Publishing.
ΔG° ¼ 95 kJ/mol ethyl 4 CO2 + 2H2 O ! CH3 COOH + 2 CO2 + H +
(5)
ΔG° ¼ 175 kJ=mol ethyl 4CH3 OH + 2 CO2 ! 3CH3 COOH + 2 H2 O + 3H +
(6)
ΔG° ¼ 7 kJ=mol ethyl C6 H12 O6 ! 3CH3 COOH + 3H +
(7)
ΔG° ¼ 104 kJ=mol ethyl Disturbance to the process is observed when instead of a specific methanogenic bacteria, hydrogen starts to be consumed by another “partner.” This phenomenon is observed when sulfur bacteria from the Desulfovibrio genus start to be dominant in the environment. A large amount of sulfates in sewage sludge contributes to the growth of these bacteria. They oxidate hydrogen while reducing sulfates to hydrogen sulfide. Consequently, less hydrogen is supplied to CH4 bacteria, leading to lower CH4 production. The bacteria that reduce sulfates are active in two configurations: sometimes as acetogenic bacteria and sometimes as a competitor of acetogenic and methanogenic symbiosis (Fig. 6). The last phase of the fermentation process (methanogenesis) is dominated by methanogenic bacteria that belong to obligate anaerobes of the Methanobacterum,
Volatile fatty acid (VFA) yield from sludge
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Fig. 6 Sulfur bacteria as a competitor of acetogenic and methanogenic symbiosis. The bacteria that reduce sulfates are active in two configurations: sometimes as acetogenic bacteria (B) and sometimes as a competitor of aceto- and methanogenic symbiosis (A). Based on Sadecka, Z., 2010. Basics of Biological Wastewater Treatment, Seidel-Przywiecki Publishing.
Methanococcus, and Methanogenium genuses. They are characterized by high susceptibility to temperature and reaction changes. The time of generation ranges from 15 to 85 h. In addition to biocoenosis composition, its duration is affected by the temperature of the process and the type of substrate, which are presented in Fig. 7 (Sadecka, 2010). Two groups of bacteria can be distinguished, one of which generates CH4 from formic acid, methanol, hydrogen, and CO2. Energy yield in these reactions ranges from 106 to even 145 kJ/mol CH4. Time of generation of these bacteria is short, whereas their tolerance to changes in reaction is high. Reduction of CO2 with hydrogen is presented as follows: CO2 + 4H2 ! CH4 + 2H2 O
(8)
This reaction occurs with the participation of coenzymes and complex enzymatic systems. Synthesis of CH4 from methanol occurs according to the following reaction: CH3 OH + H2 ! CH4 + 2H2 O
(9)
The second group of bacteria is much poorer in type and generates CH4 from acetic acid according to the following reaction (Sadecka, 2010):
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Fig. 7 Times of CH4 bacteria generation at mesophilic temperatures (35–37°C) depending on the type of substrate. Based on Sadecka, Z., 2010. Basics of Biological Wastewater Treatment, Seidel-Przywiecki Publishing.
CH3 COOH ! CH4 + CO2 and ΔG ¼ 35:9 kJ=mol
(10)
Bacteria that decompose acetic acid to CH4 grow relatively slowly and are susceptible to environmental changes (Sadecka, 2010). The studies have shown that about 70% CH4 is generated during CH4 fermentation from acetic acid, whereas 30% is generated from hydrogen and CO2 ( Janosz-Rajczyk, 2008). A key factor in VFA yield is inhibition of the last phase (methanogenesis). This can be achieved by adding a specific inhibitor or changing the operating conditions of the process so that they are unfavorable to methanogens.
3
Effect of various factors on VFA yield
Fermentation using mixed bacteria cultures is characterized by the fact that with adequate control of such parameters as pH, temperature, and retention time, there are opportunities for obtaining products such as VFAs, biopolymers, and long-chain fatty acids (Shen et al., 2014; Arslan et al., 2013).
3.1 pH Another important factor in VFA yield is the value of maintained pH in the reactor. Many of the bacteria that generate these acids do not tolerate an environment that is strongly acidic environment (pH < 3) or strongly alkaline (pH > 12). The hydrolysis stage occurs most efficiently when the pH value is maintained between 5 and 7 (Arslan et al., 2013). Depending on the type of the substrate used, the most beneficial
Volatile fatty acid (VFA) yield from sludge
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pH for generation of VFA ranges from 5.5 to 11. At the time when fermentation is used for sewage sludge, it is indicated that the reaction should be alkaline, food waste–neutral, and sewage-acidic (Shen et al., 2014). CH4 bacteria develop most efficiently in an environment with pH similar to neutral (Carrillo-Reyes et al., 2014). Numerous studies have demonstrated that inhibition of the methanogenesis process occurs at pH 10–11. This causes a substantial increase in VFA yield compared to the process conducted at lower pH values. It was demonstrated that the VFA yield from activated sludge at pH 10 is over 6 times higher than for pH 4. Similar findings were observed during examinations performed by Liu et al. in 2012, who demonstrated that inhibition of methanogenesis occurs at pH 3, 11, and 12, whereas the VHA yield at pH 9 is nearly 10 times higher than for pH 3 (Khiewwijit et al., 2015). In addition to VFA generation rate, pH has an effect on their type. The most beneficial pH for generation of propionic acid during sludge fermentation ranges from 4 to 4.5, whereas for acetic acid and butyric acid, it ranges from 6 to 6.5. If the substrate is whey, an insignificant increase in pH (from 5.25 to 6) leads to an increase in the production of propionic acid and simultaneous lower production of butyric and acetic acid. Using a substrate rich in glucose, an increase in pH from 6 to 8 results in the increase in production of acetic and propionic acid, at the expense of butyric acid. This can be linked to the dominance of other groups of bacteria depending on the environment reaction (i.e., Clostridium butyricum (pH 6) and Propionibacterium (pH 8)) (Shen et al., 2014).
3.2 Temperature A number of studies have been devoted to the effect that the temperature of the CH4 fermentation process has on the level of production of VFAs. The examinations were performed at psychrophilic temperatures (4–20°C), mesophilic temperatures (20–50°C), thermophilic temperatures (50–60°C) and extremely thermophilic temperatures (60–80°C). It was found that the increase in temperatures in the first two ranges affects the increase in VFA concentration, as well as the speed and efficiency of their production. Increasing the temperature from 10°C to 35°C during fermentation of activated sludge results in the increase in VFA concentration by 300%, whereas the increase in temperature from 8°C to 25°C during fermentation of initial sludge can even lead to a sixfold increase in the VFA production index. Numerous examinations have demonstrated that VFA generation at thermophilic temperature (55°C) is lower than at mesophilic temperature (37°C). The amount of VFA obtained at a temperature of 55°C is up to 40% lower than at 37°C (Shen et al., 2014). Most of CH4 bacteria grow optimally in the mesophilic range of temperatures. The increase in temperature of 60°C or its reduction below 20°C results in higher efficiency of production of hydrogen, hydrogen dioxide, and ethanol during CH4 fermentation. Biogas maintained during fermentation of sewage sludge in these temperatures is characterized by lower CH4 content (Ledakowicz and Krzystek, 2005; Shen et al., 2014). Compared to pH, the effect of temperature on VFA production is low and
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changes the composition of generated acids to an insignificant degree (Shen et al., 2014; Chwiałkowska and Oleskowicz-Popiel, 2016).
3.3 Retention time Important parameters of monitoring of anaerobic bioreactors include hydraulic retention time (HRT) and time of retention of mixed cultures of bacteria (i.e., sludge retention time (SRT)). HRT is connected with bioreactor volume, whereas SRT has an effect on the bacteria species that live in the bioreactor (Shen et al., 2014). Sufficiently long hydraulic retention times allow for longer contact of microorganisms with the substrate, resulting in greater production of VFAs. However, excessive HRT times may lead to the stagnation of carboxylic acid production. It should be also remembered that longer HRT times require bigger reactors, which is linked to higher functional costs (Shen et al., 2014). Similar to pH, control of HRT allows for the production of a specific type of carboxylic acids. The studies have shown that the increase in HRT from 20 h to 95 h is conducive to the production of propionic acid, but it inhibits the generation of butyric acid. Similarly, extending the HRT time during fermentation of sewage from the paper industry from 11 h to 24 h results in more intensive generation of propionic acid and lower generation of butyric acid. However, the changes in HRT from 1 day to 4 days do not affect VFA composition during cofermentation of sewage sludge with the waste from fruit and vegetable industry (Shen et al., 2014). The effect of SRT on the composition of VFA generated has not been fully examined to date, while it has been found that SRT should be long enough to allow for substrate hydrolysis. Available results from a number of studies have demonstrated that shorter SRT times are beneficial to VFA generation from sewage sludge. This involves a lower rate of growth of methanogenic bacteria than acidogenic bacteria. It was found that elongation of SRT from 4 to 12 days during fermentation of activated sludge yielded a greater amount of VFA. Another extension of SRT to 16 days, however, caused a reduction in the amount of VFA in the reactor due to their gradual use by methanogenic bacteria (Shen et al., 2014). Table 4 shows selected parameters and factors that disturb the anaerobic process of biodegradation of organic compounds. A frequent case is washing methanogenic bacteria from anaerobic bioreactors (Miksch, 2010). During inhibition of methanogenesis, it is possible to increase the production of carboxylic acids and continue elongation of their carbon chain while yielding products that are easier to separate from the mixture of fermentation residue (Oleskowicz-Popiel and Jankowska, 2014).
4
Substrates used for VFA production
Due to factors such as high content of organic matter, sewage sludge generated in wastewater treatment plants (WWTPs) is a valuable substrate for the generation of VFAs. The chemical oxygen demand (COD) value of this sludge ranges from 14,800–23,000 mg/L. VFAs account for 3.5%–6.4% d.m. of initial sludge
Volatile fatty acid (VFA) yield from sludge
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Table 4 Disturbances in the process of anaerobic biodegradation of organic compounds Change size
Parameter or disturbing factor Increase in substrate load with solved organisms
Increase in substrate load with organisms in the suspension Temperature fluctuations
Increase in pH > 8 Decline in pH < 6 Toxic substances in the inlet
Biological effect
Gas production
VFA
pH
Disturbed balance between process phases Washing the CH4 bacteria Disturbed balance between process phases Inhibition of methanogenesis Inhibition of methanogenesis Inhibition of methanogenesis
+++
+++
--
++
++
---
+++
--
---
+++
+++
---
+++
---
---
+++
--
- - - Fast decline; +++ Fast increase. - - Moderate decline; ++ Moderate increase. Based on Miksch, K., 2010. Wastewater Biotechnology. Warsaw, PWN.
(Malej, 2001). Greater amounts of VFAs are obtained at higher concentrations of organic dry matter. The problem occurs with the cellular structure of the initial sludge and excess sludge, which limits the stage of hydrolysis. This sludge requires initial processing that intensifies this fermentation stage (i.e., chemical, biological, and thermal processing) using microwaves, ultrasound, or combinations of these methods. The presence of VFA is not observed in fresh activated sludge because using organic acids as easily available substrates takes place quickly in biochemical transitions. The soluble part of COD in this sludge is smaller, which delays their hydrolysis. At the moment of performing cofermentation of the mixture of initial sludge and activated sludge, hydrolysis occurs most effectively. Maximal production of VFAs from initial sludge is on average 85 mg COD/gd.m and can even increase by 40% (to 119 mg COD/gs) when the activated sludge is added with the ratio of 1:1. The increase in VFA production from initial sludge can be obtained by adding sludge that is rich in starch (Shen et al., 2014; Chwiałkowska and Oleskowicz-Popiel, 2016). There is a huge potential of VFA production from biomass and biological waste in Europe. The amount of waste and by-products of the food industry is 250 million Mt/year ( Jankowska et al., 2017). Food waste and kitchen waste comprise a group that accounts for 22%–54% of municipal waste, which can be used successfully for VFA production. Their content of COD is very high, ranging from 91,900–166,180 mg/L. In the case of this substrate, the problem arises with separation of the usable part of organic waste from nonbiodegradable fraction (i.e., glass, aluminum, or plastics)
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(Shen et al., 2014). Production of VFAs also can use liquid waste and wastewater from agriculture and the dairy, cellulose, and paper industries. The whey permeate and waste from paper and cellulose industry are suitable for VFA generation due to their high content of easily fermenting organic matter. Despite the high COD (<10,000 mg/L), petrochemical waste is not a good substrate for fermentation, as it contains compounds that can have a toxic effect on microorganisms. CH4 fermentation is also not used for municipal waste due to its low COD content, ranging from 450 to 1200 mg/L (Shen et al., 2014; Sadecka, 2010; Janosz-Rajczyk, 2008). The lignocellulose biomass can be also used as a substrate for production of VFAs, with substantial resources on Earth. Its global production in 2009 was 916 million tons. Lignocellulose belongs to materials that are especially resistant to degradation processes. Consequently, with insufficient initial processing, its distribution in the phase of hydrolytic CH4 fermentation is slow and incomplete (Oleskowicz-Popiel and Jankowska, 2014; Ghimire et al., 2015). Disintegration of lignocellulose biomass allows, through permeating the layer of lignin, for hydrolysis of cellulose and hemicellulose to simple sugars. The generated carbohydrates can be converted to organic acids or biohydrogen during dark fermentation (Ghimire et al., 2015). Biological initial processing can be performed by means of fungi such as Phanerichataete chrysosporium, Phlebia radiata, Dichmitus squalens, Rigidosporus lognosus, and Jungua separabilima (Owczuk et al., 2014). Lignin is not decomposed during hydrolysis (Mulka and Szlachta, 2013). It is generally accepted that the best substrate for VFA production must be characterized by a high content of organic matter (COD > 4000 mg/L) and the content of ammonium nitrogen should be <5000 mg/L. Apart from properties of substrates used for fermentation processes, one should take into consideration their availability (Shen et al., 2014). The substrates used for production of VFAs include activated sludge, primary sludge, food waste, kitchen waste, and palm oil. Higher concentrations of VFAs can be obtained with the addition of cosubstrate (Shen et al., 2014).
5
Technological systems used for VFA production
Two technologies of bioreactors are used for production of VFAs from waste or industrial wastewater. With the first type, the growth of microorganisms occurs on porous material (e.g., activated carbon or zeolites), whereas with the second type, it occurs in tiny suspended particles (e.g., sand). For industrial or semi-industrial purposes, production of VFAs is performed by means of various types of reactors, filled with porous bed, fluidized-bed, UASB reactors, and CSTR (Fig. 8). Sludge used in UASB reactors has a granulated form characterized by a specific composition of biocenosis. Bacteria that reduce sulfates (acidogenesis) and homoacetate bacteria that produce acetates and bind water to CO2 develop on the external part of the granule. A dominant type of colonizing bacteria in the granule is bacteria of the Methanosarcina genus. They are present in both external and central parts of the granule. Homoacetate bacteria and CH4 bacteria that consume hydrogen and CO2 in biochemical processes develop under the external layer The use of the sludge in the
Gas
Gas
Effluent
Effluent
Distilation unit Carrier particles with biomass
Packed bed
Recycle
Air bubble
Pump
Distilation unit
Pump
Diffuser
Influent
Influent
Continuous stirred tank reactor (CSTR)
Upflow anaerobic sludge blanket reactor (UASB) To biogas chamber Gas-liquid soild separator
Effluent Baffle
Volatile fatty acid (VFA) yield from sludge
Fluidized bed reactor
Packed bed reactor
Distilation unit
Pump Influent
Motor Cooling jacked
Gas bubbles Sludge granule
Influent
Sludge bed
Pump
Air bubble
Stirrer
Effluent
Distilation unit
697
Fig. 8 Types of reactors for VFA production. Based on Lee, W.S., Chua, A.S.M., Yeoh, H.K., Ngoh, G.C., 2014. A review of the production and applications of waste-derived volatile fatty acids. Chem. Eng. J. 235, 83–99; Bhatia, S.K., Yang, Y.H., 2017. Microbial production of volatile fatty acids: current status and future perspectives. Rev. Environ. Sci. Biotechnol. 16, 327–345.
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Industrial and Municipal Sludge
form of granules in the anaerobic process allows for the use of higher loads of reactor with the load of contaminants, while maintaining a long retention time (KwarciakKozlowska and Krzywicka, 2015). Reactors of the UASB type have opportunities for working at a high load of contaminants (from 5 to 15 kgCOD/m3d) and sludge concentration (from 10 to 50 g d.m.o/L). CSTRs operate at a lower load of contaminants to the sludge compared to UASB reactors, not higher than 5 kgCOD/m3d. The abovementioned anaerobic bioreactors operate mostly in the flow mode, which results in a short retention time. To accommodate reactions that need a longer time to occur, one should use reactors operating in the periodical mode (i.e., the breaks should be made during the supply and return of their content) (Shen et al., 2014).
6
Intensification of hydrolysis in order to increase VFA yields
Generation of VFAs from initial sludge has been used virtually in many WWTPs. However, there are numerous examinations of intensification of hydrolysis by initial conditioning of wastewater, sewage sludge, or waste using physical, chemical, biological, or hybrid methods (Zielewicz, 2007; Zawieja et al., 2016). The example of this method is ultrasonic disintegration, which significantly affects the intensification of the phase of hydrolysis of organic compounds, consequently leading to increased production of VFAs. The increased VFA production in the fermentation process results from both biological hydrolysis, representing the first stage of the process, and the phenomenon of sonochemical hydrolysis, taking place during sludge conditioning with ultrasound fields. Such compounds as nucleic acids, proteins, polysaccharides, and lipoproteids are decomposed when exposed to the effect of ultrasonic vibration. These processes are similar to the reaction of enzymatic hydrolysis, but disintegration of particles occurs irregularly during exposure to ultrasounds (Zielewicz, 2007; Zawieja and Wolny, 2014; Zawieja et al., 2015). Depending on the amount of energy supplied and sonication time, the first phase of conditioning with an ultrasound field leads to the disintegration of flocculent structures. However, this stage does not lead to the destruction of cellular walls of microorganisms. This occurs in the next phase, where free hydroxyl radicals generated during sonochemical reactions react with molecules of various organic substances, leading to their chemical decomposition and transformation of soluble forms (Zawieja and Wolny, 2014; Kwarciak-Kozlowska and Krzywicka, 2015). In a study published in 2009, Zawieja et al. demonstrated that with sonochemical processes occurring during conditioning of sewage sludge with an ultrasonic field, extension of sonication time and increase in vibration amplitude lead to a substantial increase in VFAs. Conditioning of sewage sludge with ultrasound fields with a vibration amplitude of 36.6 μm allows for an eightfold increase in VFA over their initial value. In the case of fermentation not supported with the effect of ultrasonic fields, the increase in VFA yield was 2.5 times. The frequency of sonication in that study was 40 kHz. (Zawieja et al., 2009). A somewhat worse effect in VFA generation (i.e., only 3.2-fold increase during CH4 fermentation of sewage sludge) was observed
Volatile fatty acid (VFA) yield from sludge
699
if the sonication frequency was 25 kHz. This study also showed that the increase in VFA for nonconditioned tests was about 2.6 times (Kuszmider and Kudela, 2015). Another physical method that affects the increase in disintegration of organic compounds is the thermal method. It leads to the destruction of cells of microorganisms and acceleration or shortening of hydrolysis. The use of the thermal method before CH4 fermentation leads to an increase in the speed of VFA generation and increase in its yield on consecutive days of the process. With initiation of thermal hydrolysis, a higher degree of transformation of VFAs into CH4 is obtained during the anaerobic stabilization process (Zawieja and Wolski, 2013). After thermal conditioning at 50°C and 70°C, an increase in the VFA yield on the second day of the process compared to the initial value is observed, at 22% and 30%, respectively. Other studies have shown that the best effects of acceleration of hydrolysis are obtained by storing sludge at temperature of 165–180°C for 10–30 min. Conducting the process at lower temperatures (60–80°C) has a beneficial effect on the composition of the obtained hydrolysate, but it requires longer reaction times (60–120 min). The hydrolysis phase also can be accelerated by alternate freezing and defrosting of the biomass. The experiments performed by Franceschini in 2010 demonstrated that the best effects are obtained for a single cycle of freezing and defrosting at freezing temperatures (below 10°C). The process is most effective if the freezing temperature is reduced at the rate of 1–3°C per min (Owczuk et al., 2014). An alternative solution is offered by the use of microwave radiation. The effect of microwave radiation is of selective character, which means that it is effective only for substances with specific dielectric properties. Numerous studies have shown an increase in activity of enzymatic processes in systems subjected to the effect of microwave radiation compared to a study conducted conventionally (Dębowski et al., 2010). It was demonstrated that the highest increase in COD and VFAs occurs in the initial period of exposure of samples to the effect of microwaves (i.e., from 0 to 3 min) (Owczuk et al., 2014). However, microwave radiation may stimulate the formation of hardly biodegradable compounds such as melanoidins or humic acids (Shen et al., 2014). Popular chemical methods include conditioning with bases or acids. The use of acids or bases as an initial method of processing is aimed to increase the solubility of extracellular polymeric substances and then release extracellular organic matter through the destruction of cellular walls. Another option is initial processing using strong oxidizers such as ozone (O3) and hydrogen peroxide (H2O2). The use of O3 involves higher costs than H2O2. However, O3 has a higher oxidation potential (2.1 V) compared to the cheaper alternative of H2O2 (1.8 V). The zone dose used solubilization of solid waste is 0.16 g/gs.m, whereas this value for H2O2 is about 1 g/gs.m (Shen et al., 2014). VFA generation from sewage sludge also can be based on peracetic acid. The use of this reactant with the amount of 0.1 cm3 CH3COOOH/L leads to a 15% increase in VFAs. The increase in its dose to 0.5 cm3 CH3COOOH/L and 2.5 cm3 CH3COOOH/L results in a further increase in their amounts by 41% and 60%, respectively (Zawieja and Wolski, 2013). Another method of intensification of hydrolysis is adding biological agents such as hydrolytic enzymes, pure cultures of Cellulomonasuda, Cellulomonas biazotea,
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Industrial and Municipal Sludge
Aspergillus awamori, activated sludge, and mature compost. The dose added to the reactor is mostly a mixture of enzymes, which allows for a simultaneous hydrolysis of various organic compounds that are contained in the fermented substrate. The initial processing using enzymes and pure cultures is expensive, but a cheaper alternative can be a mature compost or activated sludge (Shen et al., 2014). Numerous examinations that combine various methods of intensification of the hydrolysis stage have been conducted. When choosing a combination of the methods of initial support for hydrolysis, the amount of waste or sewage sludge for biodegradation and investment and functional costs also should be taken into consideration. Methods of intensification of hydrolysis contribute to the generation of VFAs. Single pretreatment methods include acid, alkaline, O3, H2O2, biological, microwave, ultrasound, and thermal, and combined pretreatments include slkaline + ultrasound, O3 + ultrasound, thermal + biological, and ultrasound + thermal (Shen et al., 2014).
7
The use of VFAs in industry
VFAs are mainly used in the food, pharmaceutical, petrochemical, chemical and cosmetics industries (Fig. 9). The increase in interest in the CH4 fermentation process in recent years has led to research into the production of chemicals from a variety of biomass types. It is possible to combine CH4 fermentation with other processes based on the so-called carboxylic platforms, where VFAs are intermediates. The main advantages of the biological acquisition of VFA are its efficiency, which does not require Volatile fatty acids
O
The industrial use of VFA
O C
OH
H
Acetic acid
OH
Formic acid Rubber processing Tanning Pharmaceutical manufacturing Pesticides
Formic acid
Butyric acid Ester used food industry as aroma additive Food additive flavoring Pharmaceuticals Animal food supplement Fishing balt additive
O OH Butyric acid
O
O OH Propionic acid
Acetic acid Vinyl acetate monomer (polymers, adhensives, dyes) Chemicals Ester production Solvent Food additive Vinegar
OH OH Lactic acid
Fig. 9 The industrial use of VFAs. Based on Zacharof (2013).
Propionic acid Chemical Monomer for biodegradable polymers Buffering agent Anti acne agents Skin lightoing agents Moisurizers
Lactic acid Animal and human food additive Chemical intermediate Solvent Flavouring agent
Volatile fatty acid (VFA) yield from sludge
701
sterilization, low costs, low CO2 production, and coproduction of hydrogen (Zacharof and Lovitt, 2013). In recent years, many studies have been carried out to maximize the production of VFA from various types of waste, along with the methods of preconditioning. Adjusting the conditions of an anaerobic reactor and proper process control allow for the production of the appropriate type of carboxylic acid. The use of VFAs in industrial processes requires the use of integrated methods of processing, and the appropriate place for this to occur is called a biorefinery (Oleskowicz-Popiel and Jankowska, 2014). A wide range of chemical and biochemical processes are used here, including chemical and/or enzymatic hydrolysis, dehydration, fermentation involving microorganisms, pyrolysis, thermal deoxidation, and hydrogenation (Szwach and Kulesza, 2014). The VFAs obtained in the anaerobic process as intermediates can be used for the production of biopolymers (PHA), biohydrogen, biogas, bioplastics, and biodiesel, among other products (Grzelak et al., 2015; Shen et al., 2014; Chen and Wu, 2005). VFAs also have been used in the technology of microbial fuel cells (MFCs). The type of VFA used in MFCs has a significant impact on electricity generation efficiency. The average amount of current produced in MFC supplied with acetate is twice as high as with higher-molecular-weight carboxylic acids (Freguia et al., 2010; Markowska et al., 2013).
References Agler, M.T., Wrenn, B.A., Zinder, S.H., Angenent, L.T., 2011. Waste to bioproduct conversion with undefined mixed cultures: the carboxylate platform. Trends Biotechnol. 29, 70–78. Arslan, D., Steinbusch, K.J.J., Diels, L., De Wever, H., Hamelers, H.V.M., Buisman, C.J.N., 2013. Selective carboxylate production by controlling hydrogen, carbon dioxide and substrate concentrations in mixed culture fermentation. Bioresour. Technol. 136, 452–460. Banel, A., 2010. Development and comparison of chromatographic methodologies for determination of short-chain alkan monocarboxylic acids in water samples and permanent. Doctoral Dissertation, Gdansk. Banel, A., Zygmunt, B., 2009. Volatile fatty acids in the landfill—occurrence and determination. Ecol. Chem. Eng. 16 (S2), 192–206. Bitton, G., 2005. Wastewater Microbiology. John Wiley & Sons, Inc. Błaszczyk, M., 2009. Microorganisms in Environmental Protection. PWN Scientific Publisher. Carrillo-Reyes, J., Celis, L.B., Alatriste-Mondrago´n, F., Razo-Flores, E., 2014. Decreasing methane production in hydrogenogenic UASB reactors fed with cheese whey. Biomass Bioenergy 63, 101–108. Chen, G.Q., Wu, Q., 2005. The application of polyhydroxyalkanoates as tissue engineering materials. Biomaterials 26 (33), 6565–6578. Chwiałkowska, J., Oleskowicz-Popiel, P., 2016. The carboxylic platform. Production and use of volatile fatty acids. Chem. Ind. 95 (4), 800–808. Dębowski, M., Zielinski, M., Krzemieniewski, M., 2010. Influence of microwave radiation on methane fermentation of post-slaughter waste in thermophilic conditions. Annu. Set Environ. Protect. 12, 381–392. Freguia, S., The, E.H., Boon, N., Rabaey, K., 2010. Microbial fuel cells operating on mixed fatty acids. Bioresour. Technol. 101 (4), 1233–1238.
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Ghimire, A., Frunzo, L., Pirozzi, Trably, E., Escudie, R., Lens, P., Esposito, G., 2015. A review on dark fermentative biohydrogen production from organic biomass: process parameters and use of by-products. Appl. Energy 144, 73–95. Grzelak, J., S´lęzak, R., Krzystek, L., 2015. Obtaining volatile fatty acids and hydrogen from the organic fraction of municipal solid waste. Eng. Chem. Apparatus 4, 157–158. Jankowska, E., Chwialkowska, J., Stodolny, M., Oleskowicz-Popiel, P., 2017. Volatile fatty acids production during mixed culture fermentation—the impact of substrate complexity and pH. Chem. Eng. J. 326, 901–910. Janosz-Rajczyk, M., 2008. Research on Selected Wastewater Treatment Processes. Technical University of Czestochowa. Khiewwijit, R., Temmink, H., Labanda, A., Rijnaarts, H., Keesman, K.J., 2015. Production of volatile fatty acids from sewage organic matter by combined bioflocculation and alkaline fermentation. Bioresour. Technol. 197, 295–301. Kuszmider, G., Kudela, P., 2015. Impact of sonification time on VFA generation and changes of total solids during sewage sludge anaerobic digestion process. Ecol. Eng. 41, 148–152. Kwarciak-Kozlowska, A., Krzywicka, A., 2015. Influence of ultrasonic field on the increase of biodegradability of coke wastewater. Arch. Waste Manage. Environ. Protect. 17 (3), 133–142. Ledakowicz, S., Krzystek, L., 2005. The use of methane fermentation in the utilization of waste from the agri-food industry. Biotechnology 70, 165–183. Malej, J., 2001. The generation of volatile fatty acids from the raw sewage stream and some problems of sewage brought by the slurry tanker. Annu. Set Environ. Protect. 3, 103–128. Markowska, K., Grudniak, A.M., Wolska, K., 2013. Microbial fuel cells: the basics of technology, its limitations and potential applications. Microbiol. Adv. 52 (1), 29–40. Michalska, K., Pazera, A., Bizukojc, M., 2014. Innovations for the dairy industry—plant biogas installations. Electron. Doc. 1–19. Miksch, K., 2010. Wastewater Biotechnology. PWN, Warsaw. Mulka, R., Szlachta, J., 2013. State of knowledge regarding modeling of anaerobic digestion processes. Agric. Eng. 145, 281–290. Oleskowicz-Popiel, P., Jankowska, E., 2014. The use of fermentation processes in biorefinery systems. Chem. Ind. 93/3 (39), 51–354. Owczuk, M., Matuszewska, A., Filip, A., Prachnio, P., 2014. Investigation of the efficiency of biomass disintegration for the methane fermentation process. Automotive Arch. 66 (4), 143–151. Rosik-Dulewska, C., 2007. Basics of Waste Management. PWN Scientific Publisher. Sadecka, Z., 2010. Basics of Biological Wastewater Treatment. Seidel-Przywiecki Publishing. Schlegel, H.G., 2003. General Microbiology. PWN Scientific Publisher. Shen, L.W., Seak, M., Chua, A., Koon, Y.H., Cheng, N.G., 2014. A review of the production and applications of waste-derived volatile fatty acids. Chem. Eng. J. 235, 83–99. Szwach, I., Kulesza, R., 2014. Potential of biomass in the aspect of obtaining selected raw materials and chemical products. Chemist 68 (10), 893–900. Zacharof, M.P., Lovitt, R.W., 2013. Complex effluent streams as a potential source of volatile fatty acids. Waste Biomass Valor 4, 557–581. Zalewski, M., Chmielewski, A., Palige, J., Roubinek, O., Wierzchnicki, R., Usidus, J., 2012. Technological solutions for installations for biogas production from vegetable raw materials. Instal 12, 41–44. Zawieja, I., Wolny, L., 2014. Influence of degree of disintegration of sewage sludge subjected to alkaline modification to the value of unitary biogas production. Eng. Environ. Protect. 17 (3), 503–512.
Volatile fatty acid (VFA) yield from sludge
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Zawieja, I., Wolny, L., Pro´ba, M., 2016. Effectiveness of generating volatile fatty acids during mesophilic and thermophilic methane fermentation of excessive sludge. Ecol. Eng. 48, 226–232. Zawieja, I., Wolny, L., Wolski, P., 2009. Influence of the ultrasonic hydrolysis process on the generation of volatile fatty acids in the acid fermentation of excessive sludge. Eng. Environ. Protect. 12 (3), 207–217. Zawieja, I., Wolny, L., Wolski, P., 2015. Influence of ultrasonic pretreatment on anaerobic digestion of excess sludge from the food industry. Ann. Set Environ. Protect. 17 (1), 351–366. Zawieja, I., Wolski, P., 2013. The influence of chemical-thermal modification of excessive sludge on the generation of volatile fatty acids in the methane fermentation process. Annu. Set Environ. Protect. 15 (3), 2054–2070. Zielewicz, E., 2007. Ultrasonic Disintegration of Excess Sludge in Obtaining Volatile Fatty Acids. Scientific Papers Silesian University of Technology, Gliwice. Z˙ygadło, M., 1999. Municipal Waste Management. Kielce University of Technology Publishing.
Further reading Bhatia, S.K., Yang, Y.H., 2017. Microbial production of volatile fatty acids: current status and future perspectives. Rev. Environ. Sci. Biotechnol. 16, 327–345. Kwarciak-Kozlowska, A., 2007. The process of purification of leachate from municipal landfills in anaerobic membrane bioreactor supported by an ultrasonic field. Ph.D. dissertation, Czestochowa. Lee, W.S., Chua, A.S.M., Yeoh, H.K., Ngoh, G.C., 2014. A review of the production and applications of waste-derived volatile fatty acids. Chem. Eng. J. 235, 83–99.
29
Małgorzata Worwag, Anna Kwarciak-Kozłowska Faculty of Infrastructure and Environment, Institute of Environmental Engineering, Czestochowa University of Technology, Czestochowa, Poland
1
Characterization of volatile fatty acids (VFAs)
Volatile fatty acids (VFAs) are saturated aliphatic acids that contain from two to six carbon atoms in the particle, and sometimes even eight (Chwiałkowska and Oleskowicz-Popiel, 2016; Zielewicz, 2007). Their structure contains an acetyl group characterized by the presence of a carbon atom that is double-bonded to oxygen. Due to the electronegativity of oxygen present in the acetyl group, carboxyl acids belong to polar compounds. Despite their high boiling point, these acids can be removed from water due to distillation with water steam. These compounds are characterized by high vapour pressure, which is utilized during the determination of their concentration in environmental samples (Zielewicz, 2007). VFAs have strong hydrophilic properties. The nomenclature of VFAs is mostly based on customary names that suggest their plant or animal origins. These acids are referred to in the literature by various names, including low-weight carboxylic acid (LWCAs), short-chain fatty acids (SCFAs), low-molecular-weight aliphatic acids (LMWAs), and volatile organic acids (VOAs), as well as VFAs (Banel, 2010). Table 1 presents the names, chemical structures, and physicochemical properties of these compounds. Knowledge of the physicochemical properties of selected VFAs allows for prediction of their behavior in the environment and development of procedures for their determination (Banel and Zygmunt, 2009). Knowledge of the value of the negative log of the dissociation constant (pKa) allows for determination of the part of the compound present in the dissociated/nondissociated form in the sample. The balance of dissociation of three of the most important organic acids is presented in Fig. 1. It shows that reaction plays an important role. Analysis of the degree of dissociation of acetic acid reveals that at pH 6, there are 5% of nondissociated ethanoic acid (CH3COOH) particles, only 0.5% at pH 7, and 0.05% at pH 8. In general, it is accepted that in the range of pH from 6.0 to 8.0, the concentrations of nondissociated forms are reduced by two orders of power. The increase in the amount of nondissociated acids causes them to react with carbonates, with a simultaneous increase Industrial and Municipal Sludge. https://doi.org/10.1016/B978-0-12-815907-1.00029-5 © 2019 Elsevier Inc. All rights reserved.
682
Table 1 Nomenclatures, chemical compositions, and physicochemical properties of VFAs (Banel, 2010; Chwiałkowska and Oleskowicz-Popiel, 2016)
Chemical formula
M (g/mol)
Methanoic acid (formic) Ethanoic acid (acetic) Propane acid (acid) Isobutane acid (isobutyric) Butanoic acid (butyric) Isopentanoic acid (isovaleric) Pentanoic acid (valeric) Hexane acid (caproic) Heptanoic acid (enanthic) Octane acid (caprylic)
HCOOH CH3COOH C2H5COOH (CH3)2CHCOOH C3H7COOH (CH3)2CHCH2COOH C4H9COOH C5H11COOH C6H13COOH C7H15COOH
46.03 60.1 74.1 88.1 88.1 102.1 102.1 116.2 130.2 144.2
1220 1049 990 960 964 925 938 927 918 910
Boiling point (°C)
Solubility in water at ambient temperatures (g/L)
pKa
100.8 117 141 154 164 177 186 206 223 235
Big Big Big 210 Average 25 40 10 2.6 0.7
3.75 4.75 4.87 4.85 4.81 4.78 4.82 4.88 4.89 4.89
Industrial and Municipal Sludge
System name (custom)
Density at 25°C (g/L)
Volatile fatty acid (VFA) yield from sludge
683
Fig. 1 Percentage of nondissociated and dissociated forms of acids for pH values ranging from 6 to 8. Based on Sadecka, Z., 2010. Basics of Biological Wastewater Treatment, Seidel-Przywiecki Publishing.
in carbon dioxide (CO2) content, leading to a reduction in reaction. This results in the shift in balance between dissociated and nondissociated forms of VFAs toward nondissociated forms (Banel, 2010; Sadecka, 2010). With acidification of the reaction environment, dissociation is reversed, leading to easier transfer of nondissociated acid particles to the organic or headspace phase. In their dissociated form, VFAs are mostly soluble in water, whereas they are poorly soluble in organic solvents (nonmixable with water). Low concentrations of nondissociated forms of acetic acid and propionic acid have an inhibiting effect on the yield of the fermentation gas (Banel, 2010; Sadecka, 2010). VFAs are the compounds that are often observed in the environment. However, they rarely occur in a free state; rather, they mostly take the form of esters, salts, or amids. VFAs can be derived from both natural and anthropogenic sources. They are present in large amounts in sewage sludge, sludge generated during farming of breeding swine, and sludge from the food industry. Their sources are also organic waste and liquors generated during deposition of this waste in landfills or composting. In the liquors generated in young landfills, VFAs account for nearly 80% of all organic components, with their concentrations reaching as much as 6000mg/L. With the landfill aging, the amount of VFAs in liquor is substantially reduced, even to the level of 5 mg/L (Banel, 2010; Chwiałkowska and Oleskowicz-Popiel, 2016; Banel and Zygmunt, 2009). VFAs are also generated in human and animal intestines during digestion. In the environment, VFAs also can play a negative role. The compounds from this group may lead to acidification of the environment or increased mobility of heavy metals and radionuclides. Due to their chemical composition, VFAs play a significant role in biological treatment processes, as they represent an easily available source of carbon for microorganisms (Chwiałkowska and Oleskowicz-Popiel, 2016).
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Despite the substantial interest in the problems of acids in various environmental samples, much attention has been devoted to the methodology of determination of these compounds. The choice of adequate analytic methodology used for their determination depends mainly on the accuracy and precision of determinations, matrix composition, available apparatus, predicted level of their content in the sample, and time and cost of performing analysis. The methods based on titration, often preceded by the isolation of analytes from the primary matrix by means of distillation methods, have been used to evaluate total VFA content. Determination of individual acids is conducted using separation techniques, mostly chromatographic (Banel, 2010). The use of ion chromatography often requires a complex procedure of sample cleaning in order to reduce interference and reach an adequately low determination threshold. A useful tool for determination of VFAs in both water and gas samples is gas chromatography with flame ionization detector (GC-FID). This technique allows for the identification and quantitative determination of individual fatty acids (Banel and Zygmunt, 2009). One of the main steps before analysis of the sample from a medium is isolation of the compounds to the level that allows for detection and determination. Table 2 compared the methods used for separation of VFAs from the medium studied.
Table 2 Methods of VFA separation from the medium studied (Zacharof and Lovitt, 2013) Separation methods Precipitation
Distillation
Description
Benefits
Calcium salts are added to the purified medium, whereas the obtained solution of calcium carboxylane is solidified by vaporization and, through crystallization, separates it from the parent liquid. Ammonia is used to neutralize acids that react in order to generate ammonium carboxylate, which is mixed with alcohol in order to produce esters, followed by division through distillation.
l
l
l
l
l
Defects
High production coefficient. Low functional costs. High-purity products.
Solid waste production due to the use of sulfuric acid for the release of carboxylic acids from calcium carboxylates
High purity of products. By-products can be used as fertilizer.
High costs of energy connected with distillation, which is used for the separation of alcohol from carboxylic acids
Volatile fatty acid (VFA) yield from sludge
685
Table 2 Continued Separation methods Adsorption
Description
Benefits
Defects
Ion-exchange resins are used as adsorbents for exchange of carboxylate ions from the medium.
Easy operation
l
l
l
l
Electrodialysis
Extraction with solvent
With electric current in an electrodialyser, negatively charged carboxylate ions go through an anionexchange membrane to the anode.
Carboxylate is concentrated in water solution and does not require acidification.
Organic acids are used for extraction of carboxylic acids from the purified stream.
l
l
l
l
Low cost Opportunities for the production of carboxylate salts
l
l
Membrane processes
Utilization of semipermeable barriers where VFAs are selectively separated depending on the types of membranes (pore size). Transport is possible at the instant of using the driving force (pressure difference, concentrations, temperature and electrical potential).
High selectivity
High price of resins High demand for energy due to resin regeneration Low adsorption abilities Separation not highly selective Products are much contaminated, and it is necessary to continue their purification. High demand for energy. Initial acidification of the sample in order to obtain greater and efficient extraction. Extracts have to be regenerated through distillation or extraction.
l
Occurrence of concentration polarization
l
Reduction in the stream of permeate due to the phenomena of fouling and scaling High functional costs
l
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Industrial and Municipal Sludge
Table 3 Methods of production of carboxylic acids (Zacharof and Lovitt, 2013) Carboxylic acid
Chemical synthesis methods
Bioprocess
Formic acid
Alkene oxidation Hydrogenation of CO2 Methanol carbonylation Methanol carbonylation Acetaldehyde oxidation Ethylene oxidation Ethylene hydrocarboxylation Aerobic oxidation of propionaldehyde Chemical oxidation of butyraldehyde Ethylene oxidation Chemical synthesis
Glucose oxidation CH4 fermentation
Acetic acid
Propionic acid Butyric acid Caproic acid Lactic acid
Glucose oxidation CH4 fermentation CH4 fermentation Alcohol fermentation CH4 fermentation CH4 fermentation
Glucose Lactic acid
Lactic acid bacteria
Pyruvate
Propionic acid bacteria
Ethanol
Yeast
Coli-Arerogenes
Clostridium
Ethanol
Oxaloacetate
Acetate
Acetate Succinate
Acetylo-CoA
Acetoin Butanediol
Butyrate
Acetone
Propionate Formic acid Butanol
Isopropanol
Fig. 2 Diagram of hydrocarbon fermentation processes in bacteria. Based on Sadecka, Z., 2010. Basics of Biological Wastewater Treatment, Seidel-Przywiecki Publishing.
2
Production of VFAs
VFAs can be produced via the chemical processing of petroleum or anaerobic biological processes. Table 3 compares the most frequently used processes of VFA production. The diagram of initiated CH4 fermentation can be presented using the example of carbohydrate decomposition (Fig. 2). It shows that this process begins from glycolysis as it is the case in aerobic processes. The central derivative from which the pathways of aerobic and anaerobic biodegradation diverge is pyruvate. The fermentation process can be conducted depending on the orientation to a specific product. Different
Volatile fatty acid (VFA) yield from sludge
687
Fig. 3 Biological pathways of carbohydrate conversion into VFAs during homolactic and heterolactic fermentation (A), alcohol fermentation (B), and during the cycle of tricarboxylic acids (C). Based on Zacharof (2013).
types of fermentation have been discovered, with their names taken from the dominant final product: alcoholic, lactic, propionic, formic, butyric, and acetic fermentation. In addition to the dominant final products, there are succinate, caproate, n-butanol, 2,3butanodiol, acetone, isopropanol, CO2, and hydrogen. All of the listed fermentation products can be generated separately or in different arrangements (Schlegel, 2003). Fig. 3 illustrates schematically biological pathways of carbohydrate conversion into VFAs during homolactic and heterolactic fermentation (in panel A), alcohol fermentation (B) and through the cycle of tricarboxylic acids (C). Extensive research has been conducted in recent years on the use of mixed cultures for production of carboxylic acids. With the high flexibility of these cultures, it is possible to process organic waste, sewage sludge, and agricultural and food industry waste (Chwiałkowska and Oleskowicz-Popiel, 2016). In anaerobic conditions, biological decomposition of organic matter occurs in several stages. The first stage of the CH4 fermentation process is hydrolysis; when insoluble, polymerized organic compounds are exposed to the effect of extracellular
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Industrial and Municipal Sludge
hydrolytic bacteria enzymes (e.g., amylases, lipases, or proteases), leading to their decomposition and liquidization. Polysaccharides are hydrolysed to monosaccharides, proteins to amino acids, and fats to polyols and fatty acids. The hydrolysis phase is a stage that limits the course and efficiency of fermentation (Zielewicz, 2007; Agler et al., 2011). Another stage is the phase of acidification or acidogenesis, which is also termed dark fermentation. The initial substrate for acid-forming bacteria is hydrolysis products. The bacteria that participate in this process are facultative bacteria, but there are also obligate anaerobes. The most frequently used of these include Aerobacter, Alcaligenes, Clostridium, Escherichia, Flavobacterium, Pseudomonas, Lactobacterium, Lactobacillus, Micrococcus, and Streptococcus. Bacteria of the acidification phase are relatively less susceptible to temperature or pH changes. They proliferate much faster than CH4 bacteria. With their activity, low molecular organic compounds are processed mainly (76%) into VFAs (e.g., formic acid, acetic acid, propionic acid, butyric acid, valeric acid, and caproic acid) and to alcohols (e.g., ethanol and methanol) and gaseous products (CO2 and H2). The remaining part is decomposed into acetates. In this phase, proteins are further decomposed into simple organic acids, thiols, and amines (Zielewicz, 2007; Rosik-Dulewska, 2007; Sadecka, 2010). In the third stage of CH4 fermentation, the so-called acetate phase (acetogenesis), ethanol and VFAs are processed into CO2, hydrogen, and acetic acid. These transitions occur with two groups of anaerobic bacteria of the Syntrophobacter and Syntrophomonas genuses (Michalska et al., 2014). The first group of acetogenic microorganisms is responsible for hydrolysis of organic polymers and lipids to basic structure-forming compounds such as monosaccharides or amino acids. The second group is involved in fermentation and destruction of products to simple organic acids, including acetic acid, propionic acid, and butyric acid (Zielewicz, 2007) Distribution of organic acids by bacteria is relatively specific because it requires the involvement of various bacteria species. For example, valeric acid is decomposed into acetic acid and propionic acid by Mbacterium suboxydans, but further decomposition of propionians into acetates, CO2, and CH4 is achieved with Mbacterium propionicum. The latter species is unable to absorb and decompose acetates; therefore, their further decomposition into CH4 requires the presence of Methanococcus mazei bacteria (Fig. 4).
Fig. 4 Activity of bacteria to produce CH4 from valeric acid. Based on Sadecka, Z., 2010. Basics of Biological Wastewater Treatment, Seidel-Przywiecki Publishing.
Volatile fatty acid (VFA) yield from sludge
689
In addition to fermentation of carbohydrates and proteins, acetic acid is generated during the anaerobic oxidation of fats. Generation of propionic acid occurs first and foremost during the degradation of carbohydrates. Another product of the fermentation of proteins and fats is butyric acid. This acid also can be generated during the fermentation of carbohydrates from pyruvate. Oxidation of acids such as lactic acid, propionic acid, and lactic acid is possible if hydrogen produced by acetogens is received from the environment. Their oxidation is possible only at very low concentration of hydrogen and low redox potential (<–300 mV). An insignificant increase in hydrogen pressure results in an immediate decline in the rate of degradation of these acids (Błaszczyk, 2009). These transitions can occur if molecular hydrogen is removed from the reaction environment. With these environmental requirements, the two groups of microorganisms (i.e., acetogenic and methanogenic bacteria) live in symbiosis (Zalewski et al., 2012). Both types of bacteria can connect to various aggregates (e.g., flocs or granules), and their symbiosis results in the decomposition of propionic acid according to Eqs. (1)–(3) (Bitton, 2005; Rosik-Dulewska, 2007; Z˙ygadło, 1999). l
Acetogenic bacteria:
CH3 CH2 COO + 3H2 O ! HCO3 + CH3 COO + H + + 3H2 l
(1)
Methanogenic bacteria:
HCO3 + 4H2 + H + ! CH4 + 3H2 O
(2)
The total balancing reaction for these chemical reactions can be written as follows: 4CH3 CH2 COO + 3H2 O ! HCO3 + 4CH3 COO + 3CH4 + H +
(3)
The phenomenon of this symbiosis was discovered in the 1970s and is termed interspecies hydrogen transfer (IRT). Undisturbed hydrogen transfer guarantees the correct course of anaerobic distribution of organic substrates. Isolation of pure cultures of acetic bacteria is impossible, and growth of mixed cultures is very difficult and labour-consuming. Bacteria of the acetogenic phase are characterized by a long time of generation that depends on the type of available substrate. Fig. 5 presents the times of acetic bacteria generation at mesophilic temperature depending on the type of substrate (Sadecka, 2010). In addition to production of acetate from hydrogen and CO2, acetogenic bacteria can utilize other simple compounds (e.g., carbon dioxide, methanol, or glucosis), generating acetate as a final product of metabolism. Production of acetic acid by acetogenic microorganisms proceeds according to the reactions given as Eqs. (4)–(7) (Błaszczyk, 2009): CO2 + 4H2 ! CH3 COOH + 2 H2 O
(4)
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Industrial and Municipal Sludge
Fig. 5 Times of acetic bacteria generation at mesophilic temperatures (35–37°C) depending on the type of substrate. Based on Sadecka, Z., 2010. Basics of Biological Wastewater Treatment, Seidel-Przywiecki Publishing.
ΔG° ¼ 95 kJ/mol ethyl 4 CO2 + 2H2 O ! CH3 COOH + 2 CO2 + H +
(5)
ΔG° ¼ 175 kJ=mol ethyl 4CH3 OH + 2 CO2 ! 3CH3 COOH + 2 H2 O + 3H +
(6)
ΔG° ¼ 7 kJ=mol ethyl C6 H12 O6 ! 3CH3 COOH + 3H +
(7)
ΔG° ¼ 104 kJ=mol ethyl Disturbance to the process is observed when instead of a specific methanogenic bacteria, hydrogen starts to be consumed by another “partner.” This phenomenon is observed when sulfur bacteria from the Desulfovibrio genus start to be dominant in the environment. A large amount of sulfates in sewage sludge contributes to the growth of these bacteria. They oxidate hydrogen while reducing sulfates to hydrogen sulfide. Consequently, less hydrogen is supplied to CH4 bacteria, leading to lower CH4 production. The bacteria that reduce sulfates are active in two configurations: sometimes as acetogenic bacteria and sometimes as a competitor of acetogenic and methanogenic symbiosis (Fig. 6). The last phase of the fermentation process (methanogenesis) is dominated by methanogenic bacteria that belong to obligate anaerobes of the Methanobacterum,
Volatile fatty acid (VFA) yield from sludge
691
Fig. 6 Sulfur bacteria as a competitor of acetogenic and methanogenic symbiosis. The bacteria that reduce sulfates are active in two configurations: sometimes as acetogenic bacteria (B) and sometimes as a competitor of aceto- and methanogenic symbiosis (A). Based on Sadecka, Z., 2010. Basics of Biological Wastewater Treatment, Seidel-Przywiecki Publishing.
Methanococcus, and Methanogenium genuses. They are characterized by high susceptibility to temperature and reaction changes. The time of generation ranges from 15 to 85 h. In addition to biocoenosis composition, its duration is affected by the temperature of the process and the type of substrate, which are presented in Fig. 7 (Sadecka, 2010). Two groups of bacteria can be distinguished, one of which generates CH4 from formic acid, methanol, hydrogen, and CO2. Energy yield in these reactions ranges from 106 to even 145 kJ/mol CH4. Time of generation of these bacteria is short, whereas their tolerance to changes in reaction is high. Reduction of CO2 with hydrogen is presented as follows: CO2 + 4H2 ! CH4 + 2H2 O
(8)
This reaction occurs with the participation of coenzymes and complex enzymatic systems. Synthesis of CH4 from methanol occurs according to the following reaction: CH3 OH + H2 ! CH4 + 2H2 O
(9)
The second group of bacteria is much poorer in type and generates CH4 from acetic acid according to the following reaction (Sadecka, 2010):
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Industrial and Municipal Sludge
Fig. 7 Times of CH4 bacteria generation at mesophilic temperatures (35–37°C) depending on the type of substrate. Based on Sadecka, Z., 2010. Basics of Biological Wastewater Treatment, Seidel-Przywiecki Publishing.
CH3 COOH ! CH4 + CO2 and ΔG ¼ 35:9 kJ=mol
(10)
Bacteria that decompose acetic acid to CH4 grow relatively slowly and are susceptible to environmental changes (Sadecka, 2010). The studies have shown that about 70% CH4 is generated during CH4 fermentation from acetic acid, whereas 30% is generated from hydrogen and CO2 ( Janosz-Rajczyk, 2008). A key factor in VFA yield is inhibition of the last phase (methanogenesis). This can be achieved by adding a specific inhibitor or changing the operating conditions of the process so that they are unfavorable to methanogens.
3
Effect of various factors on VFA yield
Fermentation using mixed bacteria cultures is characterized by the fact that with adequate control of such parameters as pH, temperature, and retention time, there are opportunities for obtaining products such as VFAs, biopolymers, and long-chain fatty acids (Shen et al., 2014; Arslan et al., 2013).
3.1 pH Another important factor in VFA yield is the value of maintained pH in the reactor. Many of the bacteria that generate these acids do not tolerate an environment that is strongly acidic environment (pH < 3) or strongly alkaline (pH > 12). The hydrolysis stage occurs most efficiently when the pH value is maintained between 5 and 7 (Arslan et al., 2013). Depending on the type of the substrate used, the most beneficial
Volatile fatty acid (VFA) yield from sludge
693
pH for generation of VFA ranges from 5.5 to 11. At the time when fermentation is used for sewage sludge, it is indicated that the reaction should be alkaline, food waste–neutral, and sewage-acidic (Shen et al., 2014). CH4 bacteria develop most efficiently in an environment with pH similar to neutral (Carrillo-Reyes et al., 2014). Numerous studies have demonstrated that inhibition of the methanogenesis process occurs at pH 10–11. This causes a substantial increase in VFA yield compared to the process conducted at lower pH values. It was demonstrated that the VFA yield from activated sludge at pH 10 is over 6 times higher than for pH 4. Similar findings were observed during examinations performed by Liu et al. in 2012, who demonstrated that inhibition of methanogenesis occurs at pH 3, 11, and 12, whereas the VHA yield at pH 9 is nearly 10 times higher than for pH 3 (Khiewwijit et al., 2015). In addition to VFA generation rate, pH has an effect on their type. The most beneficial pH for generation of propionic acid during sludge fermentation ranges from 4 to 4.5, whereas for acetic acid and butyric acid, it ranges from 6 to 6.5. If the substrate is whey, an insignificant increase in pH (from 5.25 to 6) leads to an increase in the production of propionic acid and simultaneous lower production of butyric and acetic acid. Using a substrate rich in glucose, an increase in pH from 6 to 8 results in the increase in production of acetic and propionic acid, at the expense of butyric acid. This can be linked to the dominance of other groups of bacteria depending on the environment reaction (i.e., Clostridium butyricum (pH 6) and Propionibacterium (pH 8)) (Shen et al., 2014).
3.2 Temperature A number of studies have been devoted to the effect that the temperature of the CH4 fermentation process has on the level of production of VFAs. The examinations were performed at psychrophilic temperatures (4–20°C), mesophilic temperatures (20–50°C), thermophilic temperatures (50–60°C) and extremely thermophilic temperatures (60–80°C). It was found that the increase in temperatures in the first two ranges affects the increase in VFA concentration, as well as the speed and efficiency of their production. Increasing the temperature from 10°C to 35°C during fermentation of activated sludge results in the increase in VFA concentration by 300%, whereas the increase in temperature from 8°C to 25°C during fermentation of initial sludge can even lead to a sixfold increase in the VFA production index. Numerous examinations have demonstrated that VFA generation at thermophilic temperature (55°C) is lower than at mesophilic temperature (37°C). The amount of VFA obtained at a temperature of 55°C is up to 40% lower than at 37°C (Shen et al., 2014). Most of CH4 bacteria grow optimally in the mesophilic range of temperatures. The increase in temperature of 60°C or its reduction below 20°C results in higher efficiency of production of hydrogen, hydrogen dioxide, and ethanol during CH4 fermentation. Biogas maintained during fermentation of sewage sludge in these temperatures is characterized by lower CH4 content (Ledakowicz and Krzystek, 2005; Shen et al., 2014). Compared to pH, the effect of temperature on VFA production is low and
694
Industrial and Municipal Sludge
changes the composition of generated acids to an insignificant degree (Shen et al., 2014; Chwiałkowska and Oleskowicz-Popiel, 2016).
3.3 Retention time Important parameters of monitoring of anaerobic bioreactors include hydraulic retention time (HRT) and time of retention of mixed cultures of bacteria (i.e., sludge retention time (SRT)). HRT is connected with bioreactor volume, whereas SRT has an effect on the bacteria species that live in the bioreactor (Shen et al., 2014). Sufficiently long hydraulic retention times allow for longer contact of microorganisms with the substrate, resulting in greater production of VFAs. However, excessive HRT times may lead to the stagnation of carboxylic acid production. It should be also remembered that longer HRT times require bigger reactors, which is linked to higher functional costs (Shen et al., 2014). Similar to pH, control of HRT allows for the production of a specific type of carboxylic acids. The studies have shown that the increase in HRT from 20 h to 95 h is conducive to the production of propionic acid, but it inhibits the generation of butyric acid. Similarly, extending the HRT time during fermentation of sewage from the paper industry from 11 h to 24 h results in more intensive generation of propionic acid and lower generation of butyric acid. However, the changes in HRT from 1 day to 4 days do not affect VFA composition during cofermentation of sewage sludge with the waste from fruit and vegetable industry (Shen et al., 2014). The effect of SRT on the composition of VFA generated has not been fully examined to date, while it has been found that SRT should be long enough to allow for substrate hydrolysis. Available results from a number of studies have demonstrated that shorter SRT times are beneficial to VFA generation from sewage sludge. This involves a lower rate of growth of methanogenic bacteria than acidogenic bacteria. It was found that elongation of SRT from 4 to 12 days during fermentation of activated sludge yielded a greater amount of VFA. Another extension of SRT to 16 days, however, caused a reduction in the amount of VFA in the reactor due to their gradual use by methanogenic bacteria (Shen et al., 2014). Table 4 shows selected parameters and factors that disturb the anaerobic process of biodegradation of organic compounds. A frequent case is washing methanogenic bacteria from anaerobic bioreactors (Miksch, 2010). During inhibition of methanogenesis, it is possible to increase the production of carboxylic acids and continue elongation of their carbon chain while yielding products that are easier to separate from the mixture of fermentation residue (Oleskowicz-Popiel and Jankowska, 2014).
4
Substrates used for VFA production
Due to factors such as high content of organic matter, sewage sludge generated in wastewater treatment plants (WWTPs) is a valuable substrate for the generation of VFAs. The chemical oxygen demand (COD) value of this sludge ranges from 14,800–23,000 mg/L. VFAs account for 3.5%–6.4% d.m. of initial sludge
Volatile fatty acid (VFA) yield from sludge
695
Table 4 Disturbances in the process of anaerobic biodegradation of organic compounds Change size
Parameter or disturbing factor Increase in substrate load with solved organisms
Increase in substrate load with organisms in the suspension Temperature fluctuations
Increase in pH > 8 Decline in pH < 6 Toxic substances in the inlet
Biological effect
Gas production
VFA
pH
Disturbed balance between process phases Washing the CH4 bacteria Disturbed balance between process phases Inhibition of methanogenesis Inhibition of methanogenesis Inhibition of methanogenesis
+++
+++
--
++
++
---
+++
--
---
+++
+++
---
+++
---
---
+++
--
- - - Fast decline; +++ Fast increase. - - Moderate decline; ++ Moderate increase. Based on Miksch, K., 2010. Wastewater Biotechnology. Warsaw, PWN.
(Malej, 2001). Greater amounts of VFAs are obtained at higher concentrations of organic dry matter. The problem occurs with the cellular structure of the initial sludge and excess sludge, which limits the stage of hydrolysis. This sludge requires initial processing that intensifies this fermentation stage (i.e., chemical, biological, and thermal processing) using microwaves, ultrasound, or combinations of these methods. The presence of VFA is not observed in fresh activated sludge because using organic acids as easily available substrates takes place quickly in biochemical transitions. The soluble part of COD in this sludge is smaller, which delays their hydrolysis. At the moment of performing cofermentation of the mixture of initial sludge and activated sludge, hydrolysis occurs most effectively. Maximal production of VFAs from initial sludge is on average 85 mg COD/gd.m and can even increase by 40% (to 119 mg COD/gs) when the activated sludge is added with the ratio of 1:1. The increase in VFA production from initial sludge can be obtained by adding sludge that is rich in starch (Shen et al., 2014; Chwiałkowska and Oleskowicz-Popiel, 2016). There is a huge potential of VFA production from biomass and biological waste in Europe. The amount of waste and by-products of the food industry is 250 million Mt/year ( Jankowska et al., 2017). Food waste and kitchen waste comprise a group that accounts for 22%–54% of municipal waste, which can be used successfully for VFA production. Their content of COD is very high, ranging from 91,900–166,180 mg/L. In the case of this substrate, the problem arises with separation of the usable part of organic waste from nonbiodegradable fraction (i.e., glass, aluminum, or plastics)
696
Industrial and Municipal Sludge
(Shen et al., 2014). Production of VFAs also can use liquid waste and wastewater from agriculture and the dairy, cellulose, and paper industries. The whey permeate and waste from paper and cellulose industry are suitable for VFA generation due to their high content of easily fermenting organic matter. Despite the high COD (<10,000 mg/L), petrochemical waste is not a good substrate for fermentation, as it contains compounds that can have a toxic effect on microorganisms. CH4 fermentation is also not used for municipal waste due to its low COD content, ranging from 450 to 1200 mg/L (Shen et al., 2014; Sadecka, 2010; Janosz-Rajczyk, 2008). The lignocellulose biomass can be also used as a substrate for production of VFAs, with substantial resources on Earth. Its global production in 2009 was 916 million tons. Lignocellulose belongs to materials that are especially resistant to degradation processes. Consequently, with insufficient initial processing, its distribution in the phase of hydrolytic CH4 fermentation is slow and incomplete (Oleskowicz-Popiel and Jankowska, 2014; Ghimire et al., 2015). Disintegration of lignocellulose biomass allows, through permeating the layer of lignin, for hydrolysis of cellulose and hemicellulose to simple sugars. The generated carbohydrates can be converted to organic acids or biohydrogen during dark fermentation (Ghimire et al., 2015). Biological initial processing can be performed by means of fungi such as Phanerichataete chrysosporium, Phlebia radiata, Dichmitus squalens, Rigidosporus lognosus, and Jungua separabilima (Owczuk et al., 2014). Lignin is not decomposed during hydrolysis (Mulka and Szlachta, 2013). It is generally accepted that the best substrate for VFA production must be characterized by a high content of organic matter (COD > 4000 mg/L) and the content of ammonium nitrogen should be <5000 mg/L. Apart from properties of substrates used for fermentation processes, one should take into consideration their availability (Shen et al., 2014). The substrates used for production of VFAs include activated sludge, primary sludge, food waste, kitchen waste, and palm oil. Higher concentrations of VFAs can be obtained with the addition of cosubstrate (Shen et al., 2014).
5
Technological systems used for VFA production
Two technologies of bioreactors are used for production of VFAs from waste or industrial wastewater. With the first type, the growth of microorganisms occurs on porous material (e.g., activated carbon or zeolites), whereas with the second type, it occurs in tiny suspended particles (e.g., sand). For industrial or semi-industrial purposes, production of VFAs is performed by means of various types of reactors, filled with porous bed, fluidized-bed, UASB reactors, and CSTR (Fig. 8). Sludge used in UASB reactors has a granulated form characterized by a specific composition of biocenosis. Bacteria that reduce sulfates (acidogenesis) and homoacetate bacteria that produce acetates and bind water to CO2 develop on the external part of the granule. A dominant type of colonizing bacteria in the granule is bacteria of the Methanosarcina genus. They are present in both external and central parts of the granule. Homoacetate bacteria and CH4 bacteria that consume hydrogen and CO2 in biochemical processes develop under the external layer The use of the sludge in the
Gas
Gas
Effluent
Effluent
Distilation unit Carrier particles with biomass
Packed bed
Recycle
Air bubble
Pump
Distilation unit
Pump
Diffuser
Influent
Influent
Continuous stirred tank reactor (CSTR)
Upflow anaerobic sludge blanket reactor (UASB) To biogas chamber Gas-liquid soild separator
Effluent Baffle
Volatile fatty acid (VFA) yield from sludge
Fluidized bed reactor
Packed bed reactor
Distilation unit
Pump Influent
Motor Cooling jacked
Gas bubbles Sludge granule
Influent
Sludge bed
Pump
Air bubble
Stirrer
Effluent
Distilation unit
697
Fig. 8 Types of reactors for VFA production. Based on Lee, W.S., Chua, A.S.M., Yeoh, H.K., Ngoh, G.C., 2014. A review of the production and applications of waste-derived volatile fatty acids. Chem. Eng. J. 235, 83–99; Bhatia, S.K., Yang, Y.H., 2017. Microbial production of volatile fatty acids: current status and future perspectives. Rev. Environ. Sci. Biotechnol. 16, 327–345.
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Industrial and Municipal Sludge
form of granules in the anaerobic process allows for the use of higher loads of reactor with the load of contaminants, while maintaining a long retention time (KwarciakKozlowska and Krzywicka, 2015). Reactors of the UASB type have opportunities for working at a high load of contaminants (from 5 to 15 kgCOD/m3d) and sludge concentration (from 10 to 50 g d.m.o/L). CSTRs operate at a lower load of contaminants to the sludge compared to UASB reactors, not higher than 5 kgCOD/m3d. The abovementioned anaerobic bioreactors operate mostly in the flow mode, which results in a short retention time. To accommodate reactions that need a longer time to occur, one should use reactors operating in the periodical mode (i.e., the breaks should be made during the supply and return of their content) (Shen et al., 2014).
6
Intensification of hydrolysis in order to increase VFA yields
Generation of VFAs from initial sludge has been used virtually in many WWTPs. However, there are numerous examinations of intensification of hydrolysis by initial conditioning of wastewater, sewage sludge, or waste using physical, chemical, biological, or hybrid methods (Zielewicz, 2007; Zawieja et al., 2016). The example of this method is ultrasonic disintegration, which significantly affects the intensification of the phase of hydrolysis of organic compounds, consequently leading to increased production of VFAs. The increased VFA production in the fermentation process results from both biological hydrolysis, representing the first stage of the process, and the phenomenon of sonochemical hydrolysis, taking place during sludge conditioning with ultrasound fields. Such compounds as nucleic acids, proteins, polysaccharides, and lipoproteids are decomposed when exposed to the effect of ultrasonic vibration. These processes are similar to the reaction of enzymatic hydrolysis, but disintegration of particles occurs irregularly during exposure to ultrasounds (Zielewicz, 2007; Zawieja and Wolny, 2014; Zawieja et al., 2015). Depending on the amount of energy supplied and sonication time, the first phase of conditioning with an ultrasound field leads to the disintegration of flocculent structures. However, this stage does not lead to the destruction of cellular walls of microorganisms. This occurs in the next phase, where free hydroxyl radicals generated during sonochemical reactions react with molecules of various organic substances, leading to their chemical decomposition and transformation of soluble forms (Zawieja and Wolny, 2014; Kwarciak-Kozlowska and Krzywicka, 2015). In a study published in 2009, Zawieja et al. demonstrated that with sonochemical processes occurring during conditioning of sewage sludge with an ultrasonic field, extension of sonication time and increase in vibration amplitude lead to a substantial increase in VFAs. Conditioning of sewage sludge with ultrasound fields with a vibration amplitude of 36.6 μm allows for an eightfold increase in VFA over their initial value. In the case of fermentation not supported with the effect of ultrasonic fields, the increase in VFA yield was 2.5 times. The frequency of sonication in that study was 40 kHz. (Zawieja et al., 2009). A somewhat worse effect in VFA generation (i.e., only 3.2-fold increase during CH4 fermentation of sewage sludge) was observed
Volatile fatty acid (VFA) yield from sludge
699
if the sonication frequency was 25 kHz. This study also showed that the increase in VFA for nonconditioned tests was about 2.6 times (Kuszmider and Kudela, 2015). Another physical method that affects the increase in disintegration of organic compounds is the thermal method. It leads to the destruction of cells of microorganisms and acceleration or shortening of hydrolysis. The use of the thermal method before CH4 fermentation leads to an increase in the speed of VFA generation and increase in its yield on consecutive days of the process. With initiation of thermal hydrolysis, a higher degree of transformation of VFAs into CH4 is obtained during the anaerobic stabilization process (Zawieja and Wolski, 2013). After thermal conditioning at 50°C and 70°C, an increase in the VFA yield on the second day of the process compared to the initial value is observed, at 22% and 30%, respectively. Other studies have shown that the best effects of acceleration of hydrolysis are obtained by storing sludge at temperature of 165–180°C for 10–30 min. Conducting the process at lower temperatures (60–80°C) has a beneficial effect on the composition of the obtained hydrolysate, but it requires longer reaction times (60–120 min). The hydrolysis phase also can be accelerated by alternate freezing and defrosting of the biomass. The experiments performed by Franceschini in 2010 demonstrated that the best effects are obtained for a single cycle of freezing and defrosting at freezing temperatures (below 10°C). The process is most effective if the freezing temperature is reduced at the rate of 1–3°C per min (Owczuk et al., 2014). An alternative solution is offered by the use of microwave radiation. The effect of microwave radiation is of selective character, which means that it is effective only for substances with specific dielectric properties. Numerous studies have shown an increase in activity of enzymatic processes in systems subjected to the effect of microwave radiation compared to a study conducted conventionally (Dębowski et al., 2010). It was demonstrated that the highest increase in COD and VFAs occurs in the initial period of exposure of samples to the effect of microwaves (i.e., from 0 to 3 min) (Owczuk et al., 2014). However, microwave radiation may stimulate the formation of hardly biodegradable compounds such as melanoidins or humic acids (Shen et al., 2014). Popular chemical methods include conditioning with bases or acids. The use of acids or bases as an initial method of processing is aimed to increase the solubility of extracellular polymeric substances and then release extracellular organic matter through the destruction of cellular walls. Another option is initial processing using strong oxidizers such as ozone (O3) and hydrogen peroxide (H2O2). The use of O3 involves higher costs than H2O2. However, O3 has a higher oxidation potential (2.1 V) compared to the cheaper alternative of H2O2 (1.8 V). The zone dose used solubilization of solid waste is 0.16 g/gs.m, whereas this value for H2O2 is about 1 g/gs.m (Shen et al., 2014). VFA generation from sewage sludge also can be based on peracetic acid. The use of this reactant with the amount of 0.1 cm3 CH3COOOH/L leads to a 15% increase in VFAs. The increase in its dose to 0.5 cm3 CH3COOOH/L and 2.5 cm3 CH3COOOH/L results in a further increase in their amounts by 41% and 60%, respectively (Zawieja and Wolski, 2013). Another method of intensification of hydrolysis is adding biological agents such as hydrolytic enzymes, pure cultures of Cellulomonasuda, Cellulomonas biazotea,
700
Industrial and Municipal Sludge
Aspergillus awamori, activated sludge, and mature compost. The dose added to the reactor is mostly a mixture of enzymes, which allows for a simultaneous hydrolysis of various organic compounds that are contained in the fermented substrate. The initial processing using enzymes and pure cultures is expensive, but a cheaper alternative can be a mature compost or activated sludge (Shen et al., 2014). Numerous examinations that combine various methods of intensification of the hydrolysis stage have been conducted. When choosing a combination of the methods of initial support for hydrolysis, the amount of waste or sewage sludge for biodegradation and investment and functional costs also should be taken into consideration. Methods of intensification of hydrolysis contribute to the generation of VFAs. Single pretreatment methods include acid, alkaline, O3, H2O2, biological, microwave, ultrasound, and thermal, and combined pretreatments include slkaline + ultrasound, O3 + ultrasound, thermal + biological, and ultrasound + thermal (Shen et al., 2014).
7
The use of VFAs in industry
VFAs are mainly used in the food, pharmaceutical, petrochemical, chemical and cosmetics industries (Fig. 9). The increase in interest in the CH4 fermentation process in recent years has led to research into the production of chemicals from a variety of biomass types. It is possible to combine CH4 fermentation with other processes based on the so-called carboxylic platforms, where VFAs are intermediates. The main advantages of the biological acquisition of VFA are its efficiency, which does not require Volatile fatty acids
O
The industrial use of VFA
O C
OH
H
Acetic acid
OH
Formic acid Rubber processing Tanning Pharmaceutical manufacturing Pesticides
Formic acid
Butyric acid Ester used food industry as aroma additive Food additive flavoring Pharmaceuticals Animal food supplement Fishing balt additive
O OH Butyric acid
O
O OH Propionic acid
Acetic acid Vinyl acetate monomer (polymers, adhensives, dyes) Chemicals Ester production Solvent Food additive Vinegar
OH OH Lactic acid
Fig. 9 The industrial use of VFAs. Based on Zacharof (2013).
Propionic acid Chemical Monomer for biodegradable polymers Buffering agent Anti acne agents Skin lightoing agents Moisurizers
Lactic acid Animal and human food additive Chemical intermediate Solvent Flavouring agent
Volatile fatty acid (VFA) yield from sludge
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sterilization, low costs, low CO2 production, and coproduction of hydrogen (Zacharof and Lovitt, 2013). In recent years, many studies have been carried out to maximize the production of VFA from various types of waste, along with the methods of preconditioning. Adjusting the conditions of an anaerobic reactor and proper process control allow for the production of the appropriate type of carboxylic acid. The use of VFAs in industrial processes requires the use of integrated methods of processing, and the appropriate place for this to occur is called a biorefinery (Oleskowicz-Popiel and Jankowska, 2014). A wide range of chemical and biochemical processes are used here, including chemical and/or enzymatic hydrolysis, dehydration, fermentation involving microorganisms, pyrolysis, thermal deoxidation, and hydrogenation (Szwach and Kulesza, 2014). The VFAs obtained in the anaerobic process as intermediates can be used for the production of biopolymers (PHA), biohydrogen, biogas, bioplastics, and biodiesel, among other products (Grzelak et al., 2015; Shen et al., 2014; Chen and Wu, 2005). VFAs also have been used in the technology of microbial fuel cells (MFCs). The type of VFA used in MFCs has a significant impact on electricity generation efficiency. The average amount of current produced in MFC supplied with acetate is twice as high as with higher-molecular-weight carboxylic acids (Freguia et al., 2010; Markowska et al., 2013).
References Agler, M.T., Wrenn, B.A., Zinder, S.H., Angenent, L.T., 2011. Waste to bioproduct conversion with undefined mixed cultures: the carboxylate platform. Trends Biotechnol. 29, 70–78. Arslan, D., Steinbusch, K.J.J., Diels, L., De Wever, H., Hamelers, H.V.M., Buisman, C.J.N., 2013. Selective carboxylate production by controlling hydrogen, carbon dioxide and substrate concentrations in mixed culture fermentation. Bioresour. Technol. 136, 452–460. Banel, A., 2010. Development and comparison of chromatographic methodologies for determination of short-chain alkan monocarboxylic acids in water samples and permanent. Doctoral Dissertation, Gdansk. Banel, A., Zygmunt, B., 2009. Volatile fatty acids in the landfill—occurrence and determination. Ecol. Chem. Eng. 16 (S2), 192–206. Bitton, G., 2005. Wastewater Microbiology. John Wiley & Sons, Inc. Błaszczyk, M., 2009. Microorganisms in Environmental Protection. PWN Scientific Publisher. Carrillo-Reyes, J., Celis, L.B., Alatriste-Mondrago´n, F., Razo-Flores, E., 2014. Decreasing methane production in hydrogenogenic UASB reactors fed with cheese whey. Biomass Bioenergy 63, 101–108. Chen, G.Q., Wu, Q., 2005. The application of polyhydroxyalkanoates as tissue engineering materials. Biomaterials 26 (33), 6565–6578. Chwiałkowska, J., Oleskowicz-Popiel, P., 2016. The carboxylic platform. Production and use of volatile fatty acids. Chem. Ind. 95 (4), 800–808. Dębowski, M., Zielinski, M., Krzemieniewski, M., 2010. Influence of microwave radiation on methane fermentation of post-slaughter waste in thermophilic conditions. Annu. Set Environ. Protect. 12, 381–392. Freguia, S., The, E.H., Boon, N., Rabaey, K., 2010. Microbial fuel cells operating on mixed fatty acids. Bioresour. Technol. 101 (4), 1233–1238.
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Zawieja, I., Wolny, L., Pro´ba, M., 2016. Effectiveness of generating volatile fatty acids during mesophilic and thermophilic methane fermentation of excessive sludge. Ecol. Eng. 48, 226–232. Zawieja, I., Wolny, L., Wolski, P., 2009. Influence of the ultrasonic hydrolysis process on the generation of volatile fatty acids in the acid fermentation of excessive sludge. Eng. Environ. Protect. 12 (3), 207–217. Zawieja, I., Wolny, L., Wolski, P., 2015. Influence of ultrasonic pretreatment on anaerobic digestion of excess sludge from the food industry. Ann. Set Environ. Protect. 17 (1), 351–366. Zawieja, I., Wolski, P., 2013. The influence of chemical-thermal modification of excessive sludge on the generation of volatile fatty acids in the methane fermentation process. Annu. Set Environ. Protect. 15 (3), 2054–2070. Zielewicz, E., 2007. Ultrasonic Disintegration of Excess Sludge in Obtaining Volatile Fatty Acids. Scientific Papers Silesian University of Technology, Gliwice. Z˙ygadło, M., 1999. Municipal Waste Management. Kielce University of Technology Publishing.
Further reading Bhatia, S.K., Yang, Y.H., 2017. Microbial production of volatile fatty acids: current status and future perspectives. Rev. Environ. Sci. Biotechnol. 16, 327–345. Kwarciak-Kozlowska, A., 2007. The process of purification of leachate from municipal landfills in anaerobic membrane bioreactor supported by an ultrasonic field. Ph.D. dissertation, Czestochowa. Lee, W.S., Chua, A.S.M., Yeoh, H.K., Ngoh, G.C., 2014. A review of the production and applications of waste-derived volatile fatty acids. Chem. Eng. J. 235, 83–99.