Journal of Environmental Chemical Engineering 10 (2022) 107917 Contents lists available at ScienceDirect Journal of Environmental Chemical Engineering journal homepage: www.elsevier.com/locate/jece Review about bioproduction of Volatile Fatty Acids from wastes and wastewaters: Influence of operating conditions and organic composition of the substrate Ana Vázquez-Fernández, María Eugenia Suárez-Ojeda *, Julián Carrera GENOCOV Research Group, Department of Chemical, Biological and Environmental Engineering, School of Engineering, Universitat Autònoma de Barcelona, Edifici Q, Bellaterra, Barcelona 08193, Spain A R T I C L E I N F O A B S T R A C T Editor: Apostolos Giannis Volatile fatty acids (VFAs) are a group of carboxylic acids considered as building block chemicals. Nowadays, commercial production of VFAs is performed using fossil fuel sources. As an alternative, acidogenic fermentation Keywords: of wastes by mixed microbial cultures (MMC) is starting to be considered as a potential bioproduction process Acidogenic Fermentation that would replace conventional production processes and contribute to the circular economy. Nevertheless, Review more research is needed to control the VFA production yields and to precisely drive the fermentation process to Polyhydroxyalkanoates the production of a certain VFA or a mixture of VFAs, either by modifying the operational parameters or by Mixed Microbial Cultures Energy-based Metabolic Models appropriately tunning the substrate composition. Following this gap, this review starts screening the metabolic Co-fermentation routes that yield VFAs by anaerobic fermentation. Subsequently, the effect of different operational parameters on VFA production yield and VFA composition distribution is extensively discussed depending on the organic composition of the waste in terms of proteins, carbohydrates and lipids. To the best of our knowledge, previous review articles analyzed the impact of these parameters for different types of wastes, but without specifically considering their organic composition in terms of proteins, carbohydrates and lipids. Afterwards, energy-based metabolic models are presented as the one of the best modelling approaches to predict VFA composition. Then, polyhydroxyalkanoates (PHAs) production by MMC is described since it is one of the most promising applications of waste derived VFAs. Finally, we highlight the research gaps that should be further investigated to develop a large scale VFA bioprocess based on MMC platform from waste streams. 1. Introduction acetic, propionic and butyric acids was estimated in 18.5 Mt for 2020 [1]. In food industry, acetic acid is used as vinegar as well as food ad­ Volatile fatty acids (VFAs) are a subgroup of fatty acids, ranging from ditive and preservative; propionic acid is utilized in the preservation of two to five carbon atoms, including acetic, propionic, isobutyric, food grains and in animal feed while butyric acid is employed as butyric, iso-valeric and valeric acids. VFAs have a wide range of appli­ flavoring [2–5]. Other applications of acetic acid include terephthalic cations in numerous areas, thus they are considered building block acid production, which is then used in the manufacture of polyethylene chemicals. Among the VFAs, acetic, propionic, and butyric acids are terephthalate (PET), and the production of acetate esters [6]. Propionic those most industrially produced. The annual global market demand of acid is also a building block in the pharmaceutical industry, and it is Abbreviations: COD, Chemical Oxigen Demand; ED, Entner-Doudoroff pathway (ED); EMP, Embden-Meyerhof pathway; HAc, Acetic Acid; HBt, Butyric Acid; HPr, Propionic Acid; HRT, Hydraulic Retention Time; HV, Valeric Acid; mcl-PHAs, Medium Chain Length Polyhydroxyalkanoates; LCFA, Long Chain Fatty Acid; MMC, Mixed Microbial Cultures; OFMSW, Organic Fraction of Municipal Solid Waste; OLRs, Specific Organic Loading Rate; OLRv, Volumetric Organic Loading Rate; PHA, Polyhydroxyalkanoate; PHB, Polyhydroxybutyrate; PHB-co-PHV, Poly (3-hydroxybutyrate-co-3-hydroxyvalerate); PK, Phosphoketolase pathway; PPP, Pentose Phosphate pathway; scl-PHAs, Short Chain Length Polyhydroxyalkanoates; VFA, Volatile Fatty Acid; VS, Volatile Solids; VSS, Volatile Suspended Solids; WAS, Waste Activated Sludge; WWTP, Wastewater Treatment Plant; 3HB, 3-Hydroxybutyrate; 3HHx, 3-Hydroxyhexanoate; 3H2MB, 3-Hydroxy-2-methylbutyrate; 3H2MV, 3- Hydroxy-2-methylvalerate; 3HV, 3-Hydroxyvalerate. * Corresponding author. E-mail addresses: ana.vazquez.fernandez@uab.cat (A. Vázquez-Fernández), mariaeugenia.suarez@uab.cat (M.E. Suárez-Ojeda), julian.carrera@uab.cat (J. Carrera). https://doi.org/10.1016/j.jece.2022.107917 Received 30 November 2021; Received in revised form 19 April 2022; Accepted 12 May 2022 Available online 16 May 2022 2213-3437/© 2022 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by- nc-nd/4.0/). A. Vázquez-Fernández et al. Journal of Environmental Chemical Engineering 10 (2022) 107917 employed in the production of herbicides and propionate esters for mixture of VFAs by tuning the operational conditions or design pa­ perfumes [4]. Butyric acid is widely used in the chemical industry in the rameters; (2) to improve the efficiency of the downstream purification production of cellulose acetate butyrate for the synthesis of thermo­ methods. plastics and in the pharmaceutical industry for the manufacture of drugs Regarding the downstream purification methods for recovering VFAs with several therapeutic effects [5]. VFAs also have potential applica­ like gas stripping with absorption, adsorption, solvent extraction, elec­ tions as renewable feedstock such as for polyhydroxyalkanoate (PHA) trodialysis, microfiltration, reverse osmosis and nanofiltration, mem­ synthesis [7], production of electricity in microbial fuel cells (MFCs) [8], brane contactor and in-line recovery have been extensively discussed in production of hydrogen [9], production of biofuels [10] or even as a the literature [33–43] and are out the scope of this review. source of organic matter for biological nutrient removal [11]. Finally, In this sense, this review only covers the VFA production by anaer­ there are further products that can be obtained from VFAs, such as fatty obic fermentation of waste streams by MMC, focusing on the first alcohols, hydrocarbons, rhamnolipids, sophorolipids, N-acylethanol­ mentioned challenge. Therefore, first of all, the metabolic routes for the amines or lycopene [12,13]. anaerobic production of VFAs are summed up according to a detailed Commercial production of VFAs is normally based on chemical literature review. Subsequently, the operational parameters affecting synthesis starting from petrochemical raw sources [14]. Acetic acid is VFA yield and VFA composition distribution are discussed considering mainly obtained by methanol carbonylation, followed by other pro­ the organic composition of the waste streams in terms of proteins, car­ cesses such as the catalytic oxidation of acetaldehyde, ethylene, or bohydrates and lipids. Following that, energy-based metabolic models butane [15,16]. Propionic acid is traditionally produced as a by-product for the prediction of VFA distribution are reviewed. After that, PHA of acetic acid production or by the hydroxycarboxylation of ethylene in production by MMC from waste-derived VFAs is presented to exemplify the presence of a catalyst of rhodium or nickel carbonyl [17]. Butyric how to link the VFA bioproduction process with a real potential appli­ acid is usually produced by oxidation of butyraldehyde, that is, syn­ cation. Finally, conclusions about the state-of-the-art of VFA bio­ thesized by oxosynthesis of propylene obtained from crude oil. Another production by MMC and research gaps are pointed out. conventional process is the extraction of butyric acid from butter, but To the best of our knowledge, previous review articles analyzed the this method is very expensive [18]. However, due to climate change influence of the operational parameters on VFA production yield and concerns and fossil fuel depletion, VFA bioproduction via microbial VFA composition for different ‘types of waste’ without considering the fermentation is gaining interest. Moreover, the final cost of the VFAs particular organic composition the different types of wastes can have. produced from petrochemical resources depends on the price of fossil Since these works found contradictory results, especially when fuels. analyzing the VFA composition, this review article wants to consider the In the last years, several studies related to fermentative bio­ waste attending to its organic composition (carbohydrate, protein and production of VFAs from different carbon sources have been published. lipid content) instead of its classification as a certain ‘type of waste’. Most of the VFA bioproduction by fermentation is carried out by using pure substrates, such as glucose, xylose, or glycerol [19–21]. However, 2. Metabolic routes for anaerobic production of VFAs even though the use of pure carbon sources should lead to high yields and productivities, the cost of this kind of substrates is high and raises Volatile Fatty Acids are produced during anaerobic digestion of the overall cost of the process. To reduce the costs for making compet­ organic matter. Anaerobic digestion is a well-known technology that is itive the fermentative bioproduction process compared to the implemented at industrial scale for biogas production [44]. Nonetheless, fossil-based one, different types of waste, such as lignocellulosic lately, anaerobic digestion is gaining interest in the production of other biomass, waste activated sludge (WAS), food waste, dairy wastewater, value-added products, such as VFAs and hydrogen. The anaerobic paper mill wastewater or even olive mill wastewater have been proposed digestion process consists in the biological reduction of organic matter as substrates [22–28]. In addition, the use of wastes to bioproduce VFAs in absence of oxygen or nitrate/nitrite and takes place in four interde­ contributes to the implementation of a circular economy model and pendent steps: hydrolysis, acidogenesis, acetogenesis and methano­ avoids the use of edible raw materials to produce chemicals [29]. The genesis (Fig. 1) [45]. When biological process is stopped on the main disadvantage of using waste as a raw material of the fermentative acidogenesis and acetogenesis and the methanogenesis does not take bioproduction is the cost of the downstream purification process of the place, the process is usually named anaerobic or acidogenic fermenta­ final products. Yet, when using pure carbon substrates, side products are tion instead of anaerobic digestion. Hydrolysis consists in the conversion not normally produced, so the purification step is inexpensive [15]. of hardly soluble compounds such as complex carbohydrates, proteins Moreover, some types of waste need pretreatment before fermentation. and lipids into monosaccharides, amino acids, and long-chain fatty For example, anaerobic microorganisms are not able to directly digest acids. In the acidogenic step, the products of the hydrolysis are trans­ solid waste, such as lignocellulosic biomass or WAS so physical, chem­ formed by several fermentative reactions performed by facultative and ical or enzymatic pretreatments might be needed [30]. obligatory anaerobes into CO2, hydrogen, VFAs and other products, such Hence, in the design of a VFA bioproduction process, the following as ethanol and lactic acid. During acetogenesis, VFAs which cannot be items must be considered: VFA production yield, productivity, raw transformed into methane by methanogenic microorganisms, are con­ material costs, need of a pretreatment step and downstream processing verted into acetate, hydrogen, and CO2. Finally, methanogenesis is costs [15,31]. One alternative to increase the productivity, avoid the carried out by two different groups of Archaea: the acetoclastic metha­ formation of side products, and use different carbon sources is the uti­ nogens produce methane and CO2 from acetate while the hydro­ lization of engineered strains [12]. Nevertheless, in this case, the cost of genotrophic methanogens produce methane from hydrogen and CO2 the process will also increase due to the requirement of sterile condi­ [46,47]. tions. For that reason, many researchers are focusing their work on VFA Since anaerobic digestion is performed by MMC, different pathways biosynthesis through anaerobic fermentation by mixed microbial cul­ would take place and several by-products and intermediates can be tures (MMC), which do not require sterile conditions and are considered formed. Hydrolytic bacteria are strict anaerobes that include Bacteroides, more robust systems [32]. Clostridium, Micrococcus, Butiryvibrio, Selenomonas and Streptococcus Thus, VFA bioproduction by fermentation of wastes using MMC is a [46]. The acid-forming bacteria, including acetogens and homoace­ potential and true alternative to the conventional processes based on togens, are responsible for VFA synthesis [48]. Acetogens can help petrochemical resources or to the biological processes based on the use fermentative bacteria to perform the hydrolysis step by transforming of pure organic feedstocks and pure bacterial strains. Nevertheless, non-biodegradable organic macromolecules into smaller biodegradable research in this field must overcome two challenges: (1) to understand molecules. After hydrolysis, acetogenic bacteria consume the obtained how to drive the fermentation process to a targeted VFA or to a specific monosaccharides, amino acids and long-chain fatty acids and store them 2 A. Vázquez-Fernández et al. Journal of Environmental Chemical Engineering 10 (2022) 107917 Fig. 1. Steps in Anaerobic Digestion Process. (adapted from Ersahin et al. [59]). intracellularly as pyruvates, which later are converted into acetate or specific substrates [57]. The competition between methanogens and other VFAs [49,50]. Polysaccharides are broken into monosaccharides acetogens needs further study and it is of great interest for the industrial and then, converted to pyruvate by following different pathways such use of anaerobic fermentation for VFA bioproduction [58]. Strategies as: Embden-Meyerhof pathway (EMP), Entner-Doudoroff pathway (ED), promoting the acidogenic step and inhibiting the methanogenesis step Pentose Phosphate pathway (PPP), Bifidus pathway and Phosphoketo­ must be followed to maximize VFA bioproduction. Most of these stra­ lase pathway (PK) [51,52]. Finally, pyruvate is transformed into VFAs, tegies can be satisfactorily implemented by tuning the operational ethanol, lactic, H2 and CO2 [51]. Proteins are broken into polypeptides conditions of the process, which will be discussed in Section 3. and amino acids. Subsequently, the latter can be converted into VFAs by three possible pathways: oxidation-reduction of pairs of amino acids 3. Operational parameters affecting VFA production yield (Stickland reaction), oxidative deamination from an individual amino acid or reductive deamination of an individual amino acid [50,53]. There are several operational parameters that affect the VFA bio­ Lastly, hydrolysis of lipids produces glycerol and long-chain fatty acids. production by acidogenic fermentation of waste. But, before starting the The resulting glycerol can be transformed into pyruvate and then, form analysis, it is important to mention how to quantify the performance of a acetyl-CoA, acetic acid and other VFAs, while long-chain fatty acids are VFA bioproduction process. In this sense, to quantify the amount of VFAs converted into acetic acid by β-oxidation [49]. Moreover, homoace­ produced, the literature describes two different parameters: VFA pro­ togens can produce acetic acid from carbon dioxide and hydrogen duction yield and the degree of acidification. The VFA production yield through the Wood-Ljungdahl pathway [45,48]. (expressed in terms of g COD⋅g− 1 COD, where COD stands for chemical Previous studies have particularly focused on the pathways of oxygen demand) can be defined as the amount of VFAs produced divided methanogens more than that of acetogens and acidogens due to the by the substrate consumed [60]. Other authors, especially when the larger interest in biogas production. Until now, the number of species substrate is a solid waste, define the VFA production yield (expressed in identified as acidogenic microorganisms is very limited. The most terms of g COD⋅g− 1 VSS or g COD⋅g− 1 VS) as the amount of VFAs pro­ studied phyla able to degrade complex substrates and transform them duced divided by the organic matter contained in the solid waste [61, into VFAs are Firmicutes, Proteobacteria and Bacteroidetes [54]. The va­ 62] expressed in terms of volatile suspended solids (VSS) or volatile riety of species that can perform the acidogenesis step demonstrates that solids (VS). Regarding the degree of acidification, this parameter is different fermentation products can be obtained depending on the calculated by dividing the total VFA concentration in the product strains present. However, the leading microorganisms will change effluent and the total initial COD of the substrate [60]. depending mainly on the substrate, so the substrate would a key element Recently, many researchers have focused their efforts on studying in a bioproduction process of VFAs [44]. the effect of several operational parameters on VFA production, such as: During methanogenesis, approximately two thirds of the methane substrate composition [53,63], pH [64,65], temperature [66,67], volu­ generated comes from acetate. Thus, methanogens are considered metric organic loading rate (ORLv) and hydraulic retention time (HRT) competitors of acetogens since they use hydrogen, formate and acetate [68,69]. The aforementioned parameters affect all the steps of the for growing [55]. The use of acetate as a substrate for growth and for anaerobic digestion, so they can impact on VFA, hydrogen and methane methane formation competes with the accumulation of acids and are production and hence it is necessary to understand their influence to carried out by strains that belong to the genera Methanosarcina and maximize VFA accumulation. Most of the mentioned studies have Methanosaeta [54]. Accumulation of acids implies a drop in pH that separately analyzed each parameter. Nevertheless, the influence of each could lead to a reduction of the methane produced, as methanogens one of these parameters on VFA production yield might change require pH over 6.5 [56]. This reduction, in turn, comes with a greater depending on the type of carbon source used as substrate regarded in accumulation of acids, as acetate is not consumed [45]. When using terms of its composition defined as the proportion between carbohy­ easily biodegradable feedstocks, methanogenesis is the rate-limiting drates, proteins, or lipids [70]. In the current section, we consider the step of the process, as methanogens grow very slowly and need very impact of the organic composition of the waste on the VFA production 3 A. Vázquez-Fernández et al. Journal of Environmental Chemical Engineering 10 (2022) 107917 yield, and then, we sum up the combined influence of some operational influence VFA production yield. Shen et al. [82] compared the hydro­ parameters: pH, temperature, organic loading rate and HRT according to lysis and acidification of tofu (vegetable protein) and white egg (animal the organic composition of substrate. protein). They found that white egg conducted to larger VFA yield than that of tofu [82]. 3.1. Organic composition of the waste A parameter that can be used to study the impact on the substrate composition on VFA production yield is the Carbon to Nitrogen (C/N) There are several types of wastes that can be used as substrates in ratio. For example, carbohydrates and lipids contribute to increase the acidogenic fermentation, including: (i) solid wastes, such as tuna waste C/N ratio of a substrate since they have a higher content in carbon than [71], mushroom compost [72], apple pomace and winterization oil cake nitrogen, while proteins tend to decrease the C/N ratio because of their [28], (ii) slurry-like wastes, such as primary sludge from a wastewater high nitrogen content. An optimal C/N ratio is needed in any anaerobic treatment plant (WWTP) [73], WAS from a WWTP [28,74], food waste digestion process to ensure that nutrients are balanced for the mainte­ [75], organic fraction of municipal solid waste (OFMSW) [76], maize nance and growth of the bacteria. Moreover, a low C/N ratio could be silage [68], and; (iii) liquid wastes or wastewaters, such as dairy related to the release of free ammonia or ammonium, which causes wastewater [24], palm oil mill wastewater [77], sugar industry waste­ acidogenesis inhibition [83]. The optimal range of the C/N ratio for water [78], olive mill wastewater [26,28,60], glycerol [28,79] and anaerobic digestion is 20–30 [84]. paper mill effluent [64]. The difference between solid and slurry-like Finally, the combination of different kinds of substrates (co- waste lies in the solids content. In this work, we consider slurry-like fermentation) generally leads to higher VFA yields in comparison to the waste to have less than 50% in solids content. fermentation of different substrates separately. Table 1 presents studies Besides, it makes no sense to compare VFA production yields ob­ showing that VFA production yield is increased by mixing different tained from different substrates unless the experiments are carried out in substrates. For example, a mixture of carbohydrates and proteins boosts exactly the same conditions since several operational parameters might VFA yield, rather than using these two substrates individually: Feng influence VFA yield. Montiel-Jarillo et al. [28] compared different waste et al. [85] found that adding rice (carbohydrate-rich substrate) to WAS streams to determine their acidogenic potential under mesophilic con­ (protein-rich substrate) enhances VFA production in comparison with ditions after pretreating the biomass to inhibit methanogenic activity. the fermentation of only WAS [85]. Co-fermentation of more than one The larger VFA production yields were obtained when using WAS, olive kind of substrate can be used to enhance the VFA production yield by mill wastewater, winterization oil cake, apple pomace and glycerol (in balancing the C/N ratio. Besides, co-fermentation processes involve descending order) [28]. Similarly, Silva et al. [79] previously performed other advantages [86,87]: (i) balancing of micronutrients and moisture, experiments to study the VFA production of several substrates under (ii) dilution of inhibitory compounds, (iii) enhancing of the pH-buffer mesophilic conditions using an inhibitor of methanogenic bacteria. In capacity and (iv) providing an active inoculum adapted to the sub­ this case, the substrates that yielded more VFA production (in strate when one or more of the substrates used are waste. descending order) were cheese whey, sugarcane molasses, OFMSW, waste glycerol, winery effluent, olive mill wastewater, soapy slurry 3.2. pH and organic composition of the waste waste and landfill leachate [79]. As stated in the introduction of Section 3, the organic composition of pH is one of the most important key factors affecting the VFA pro­ the waste would affect VFA production yield. Therefore, in this review, duction yield because it affects the prevalence of acidogenic or meth­ the organic matter would be studied in terms of its composition into anogenic microorganisms [88,89]. The accumulation of VFA would lead three different kinds of macromolecules: lipids, carbohydrates and to a decrease in pH, which can inhibit the methanogenic microorganisms proteins. Very few studies have analyzed VFA production yield from the [90]. Acidogenic microorganisms can also be inhibited in acidic envi­ main organic matter components [53,63]. Yin et al. [63], for example, ronments (pH < 5.0) or extremely alkaline conditions (pH>12.0) [91]. found that glucose (carbohydrate) led to higher VFA yields in compar­ Apart from the acidogenesis step, the working pH also has a notable ison to peptone (protein) and glycerol (lipid hydrolysate) and that a effect on the hydrolysis step [92]. Since the hydrolysis step efficiency is mixture of glucose, peptone and glycerol led to an even larger VFAs yield subjected to the complexity of the substrate, optimal pH strongly de­ than glucose alone [63]. pends on the nature of the substrate used. In general, lipidic substrates are less preferred than carbohydrates Table 2 shows the pHs reported as optimal to lead to high VFA and proteins in fermentation processes. First, hydrolysis of lipids is production yields depending on the substrate used and its organic slower than hydrolysis of carbohydrates and proteins [63]. Secondly, composition. In general, it can be concluded that acidogenic fermenta­ hydrolysis of lipids produces long chain fatty acids (LCFAs) and glycerol. tion of a solid or slurry-like waste, independently of its organic Glycerol can be converted to VFAs, but LCFAs could inhibit the meta­ composition, needs an alkaline pH to reach high VFA production yields. bolism of anaerobic bacteria, as they adhere to the cell walls, and they Solid or complex wastes usually require an alkaline pH to boost both, tend to decrease nutrient transportation [53]. Finally, the acidogenic hydrolysis and acidogenesis steps [68]. As an example, when primary or microorganisms have more difficulties to produce VFAs from glycerol WAS from a WWTP is used, the optimal pH range seems to be 8.0–12.0 than from carbohydrates or proteins such as glucose or peptone. Due to [88,93]. In this case, alkaline conditions enhance the VFA production their lower degradation rate, lipids tend to accumulate in the degraded since they stimulate the hydrolysis of the sludge by the ionization of the waste [63]. The highly reduced nature of carbon atoms in glycerol charged groups of the extracellular polymeric substances and the makes its utilization by microorganisms difficult under fermentative consequent release of fermentable carbohydrates and proteins [91,94]. conditions [80]. Nevertheless, the use of acclimatized cultures could be Moreover, an alkaline environment inhibits the growth of methanogens, a solution to increase the efficacy of acidogenic fermentation from such as Methanobacterium sp. and Methanobrevibacter sp., avoiding the lipidic waste [81]. consumption of the produced VFAs [91,94]. Conflicting results have Regarding the fermentation of protein-rich substrates, the hydrolysis been reported in studies using food waste as substrate. Dahiya et al. [95] of proteins could lead to the release of essential nutrients, but the performed several batch tests at pH 5.0, 6.0, 7.0, 8.0, 9.0, 10.0 and 11.0 degradation of some amino acids involves hydrogen consumption re­ and found that pH 11.0 led to the higher degree of acidification. In this actions [53]. Moreover, proteins are less easily biodegraded than car­ case, VFA production might be favored by a higher availability of hy­ bohydrates, as their structure is more complex than that of drolyzed compounds, similarly to the fermentation of sludge. On the carbohydrates. Thus, when using wastes with high protein content, contrary, Zhang et al. [96] carried out batch experiments at pH 5.0, 7.0, hydrolysis could be the rate-limiting step [82]. Additionally, proteins 9.0 11.0 and reported that the VFA concentration and production yield can contain different aminoacids, and their composition would also were maximized at pH 7. Moreover, Zhang et al. [96] quantified the 4 A. Vázquez-Fernández et al. Journal of Environmental Chemical Engineering 10 (2022) 107917 Table 1 VFA yield obtained by co-fermentation of substrates with different organic composition. Primary substrate Secondary Main component Main component of VFA yield of VFA yield of the Increment of the VFA yield Reference substrate of the primary the secondary the primary mixture (primary and obtained by co-fermentation in substrate substrate substrate secondary substrates) comparison to primary substrate fermentation* 1 Primary sludge Oleic acid Proteins Lipids 119 mg COD 176 mg COD g− 48% [86] from a WWTP g− 1 VS ** VS* * 1 Secondary sludge Oleic acid Proteins Lipids 41 mg COD 160 mg COD g− 288% [86] from a WWTP g− 1 VS ** VS* * 1 Secondary sludge Aged refuse Proteins Carbohydrates 83 mg COD 184 mg COD g− VSS 122% [61] from a WWTP g− 1 VSS − 1 Secondary sludge Rice Proteins Carbohydrates 101 mg COD 520 mg COD g VSS 413% [85] from a WWTP g− 1 VSS 1 Pretreated Potato peel Proteins Carbohydrates 132 mg COD 344 mg COD g− VS 160% [101] secondary waste g− 1 VS sludge from a WWTP 1 Primary sewage Organic Proteins Carbohydrates 250 mg COD 301 mg COD g− VS 20% [62] sludge from a waste g− 1 VS WWTP 1 Pretreated Food waste Proteins Carbohydrates and 132 mg COD 282 mg COD g− VS 113% [101] secondary proteins g− 1 VS sludge from a WWTP *The increment was calculated by dividing the difference between the VFA yield of the mixture and the VFA yield of the primary substrate, by the VFA yield of the primary substrate, expressed as %. * *VFA yields calculated by the authors from the reported data Table 2 Optimal pH reported to maximize VFA production yields for different substrates. Substrate Main component of the substrate Physical state pH range studied Optimal pH Reference Secondary sludge from a WWTP Proteins Slurry-like 7.0–12.0 11.0 for hydrolysis [93] 9.0 for acidification Primary sludge from a WWTP Proteins Slurry-like 3.0–11.0 10.0 [102] Slaughterhouse wastewater Proteins Liquid 5.5 and 10.0 10.0 [64] Gelatin-rich wastewater Proteins Liquid 4.0, 4.5, 5.0, 5.5, 6.0, 6.5 and 7.0 6.0 [98] 5.0 and 7.0 7.0 [99,100] Mushroom compost Carbohydrates Solid 4.0–12.0 10.0 [72] OFMSW Carbohydrates Slurry-like 5.5 and 10.0 10.0 [64] Maize silage Carbohydrates Slurry-like 5.0 and 11.0 11.0 [68] Food waste Carbohydrates Slurry-like 5.0, 6.0, 7.0, 8.0, 9.0, 10.0 and 11.0 10.0 [95] 5.0, 7.0, 9.0 and 11.0 7.0 [96] 4.0, 5.0, 6.0 6.0 [75] Cheese whey Carbohydrates Liquid 3.5–6.0 5.3–5.5 [65] 5.0 and 11.0 5.0 [68] Paper mill wastewater Carbohydrates Liquid 5.0–6.0 5.5–6.0 [65] 5.5 and 10.0 10.0 [64] Winery wastewater Carbohydrates Liquid 5.5 and 10.0 10.0 [64] Citrus wastewater Carbohydrates Liquid 4.0, 5.0, 6.0 and 7.0 7.0 [70] Crude glycerol Lipid derived product Liquid 5.5 and 10.0 10.0 at mesophilic conditions [64] 5.5 at thermophilic conditions Tuna waste Proteins and carbohydrates Solid 5.0–10.0 8.0 [71] Microalgae biomass Proteins, lipids and carbohydrates Slurry-like 5.0 and 11–0 11.0 [68] Olive mill solid waste Carbohydrates and lipids Slurry-like 5.0 and 9.0 9.0 [103] degree of solubilization of the substrate and found that pH 7.0 con­ at neutral pH in comparison to acidic or basic conditions. When using ducted to the maximum degree of solubilization for carbohydrates, paper mill wastewater, Bengtsson et al. [65] reported that neutral pH proteins and lipids in comparison to the rest of pH conditions tested. was more convenient, while Garcia-Aguirre et al. [64] found that alka­ These differences might arise from the different organic composition of line conditions result in higher VFA production. Despite being the same the food waste, which was not reported by Dahiya et al. [95]. type of waste, they might have a very different composition. Regarding liquid wastes, the optimal pH varies depending on its Garcia-Aguirre et al. [64] also found that alkaline pH leads to higher organic composition. Hydrolysis and acidification of lipids and proteins VFA production yields than lower pH for winery wastewater. need a different pH from simple carbohydrates to achieve high VFA From the aforementioned studies, it can be concluded that for liquid production yield [97]. Fermentation of monomer-rich substrates, which wastewaters that are mainly composed of carbohydrates, the optimal pH do not require such an extensive hydrolysis, seems to be more favorable for VFA production depends strongly on the complexity of the carbo­ at low pHs. Methanogens are active in a pH range between 6.5 and 8.2 hydrates forming the waste. As a general rule, alkaline pH would be and its activity is inhibited at higher or lower pHs [56]. However, needed when using wastewaters with complex carbohydrates and acidogens can grow in a wider range of pH than metanogens. In this way, neutral pHs are more favorable for liquid wastes with simpler carbo­ Table 2 shows that carbohydrate-rich liquid wastes, such as cheese whey hydrates that do not need intensive hydrolysis. [65,68] and citrus wastewater [70] lead to higher VFA production yield There are fewer studies on optimal pH conditions for protein-rich or 5 A. Vázquez-Fernández et al. Journal of Environmental Chemical Engineering 10 (2022) 107917 lipid-rich liquid substrates. Garcia-Aguirre et al. [64] found that the carbohydrate-rich substrates such as: food waste [106,108], OFMSW optimal pH when using crude glycerol as a substrate was 10.0 in mes­ [64,105] and cow manure mixed with maize silage [111]. Nevertheless, ophilic conditions and 5.5 in thermophilic conditions. In the case of several studies found higher VFA yields at thermophilic conditions than protein-rich substrates, one might find different results depending on the at mesophilic conditions when using protein-rich substrates such as: type of waste. Slaughterhouse wastewater was reported to yield more WAS [64,66,112,113] and meat and bone meal [64]. Based on the above VFAs at alkaline conditions [64], while gelatin-rich wastewater pro­ analysis, it can be concluded that mesophilic temperatures boost VFA duced better results at neutral pH [98–100]. Probably, these differences yields with carbohydrate-rich solid substrates rather than thermophilic are due to the different complexity of the proteins forming the waste. It conditions; and thermophilic temperatures enhance VFA production seems that the higher the complexity of the proteins, the higher the with protein-rich substrates in comparison with mesophilic conditions. optimal pH. The reason for the difference is that increasing the temperature from Recently, novel pH-stepwise control processes are being considered mesophilic to thermophilic range boosts the hydrolysis of the substrates as a promising alternative to enhance VFA production from a substrate by rising the key hydrolases activities [66], despite the key acid-forming mainly composed by complex proteins [73,93]. In pH-stepwise pro­ enzymes have been reported to have higher activities at mesophilic cesses, pH is maintained firstly at alkaline levels to promote the hy­ conditions rather than thermophilic conditions [67]. Proteins are char­ drolysis step, and then, is lowered to a pH level closer to neutrality to acterized by their low biodegradability because of their complex struc­ perform the acidogenesis. Wang et al. [73] demonstrated that a pH ture (in comparison to carbohydrates) so the hydrolysis is considered the stepwise control strategy that consisted in maintaining pH at 11.0 dur­ rate-limiting step in VFA synthesis from proteins [82]. Thus, ing the early stage and then keeping pH at 9.0 during the rest of the carbohydrate-rich solid substrates that do not need such extensive hy­ fermentation led to the higher VFA yields from primary sludge. pH 11.0 drolysis as proteinic substrates would have higher VFA yields at meso­ at the beginning increased the available soluble proteins and carbohy­ philic conditions than at thermophilic conditions. drates while inhibiting methanogenic activity and, subsequently, pH 9.0 Less research is reported regarding the effect of temperature on the increased the abundance and diversity of acidogenic bacteria while acidogenic fermentation of liquid substrates. Most of the studies of VFA keeping high levels of soluble substrates. synthesis are carried out in the mesophilic range [24,65,68,97,114]. During acidogenic fermentation, the accumulation of VFAs could Nevertheless, thermophilic conditions have been reported to be more lead to a severe drop of pH. At low pH levels, acids exist as undissociated optimal in terms of VFA production from several substrates such as molecules, which could pierce the cell membrane and reduce the mi­ gelatin-rich wastewater (protein-rich substrate) [98], slaughterhouse crobial activity by directing the energy generated to cell maintenance wastewater (protein-rich substrate) [64], paper mill wastewater (car­ [104]. Therefore, pH control and extraction of the produced acids would bohydrate-rich substrate) [64] and winery wastewater (carbohy­ serve to maintain a stable operation of the process. On the contrary, pH drate-rich substrate) [64]. Conversely, mesophilic temperatures are control would imply higher process costs due to the addition of acids and more convenient when employing glycerol as substrate (lipid-rich sub­ bases to adjust pH and the automatic control loop installation. In the strate) [64]. Further research about the influence of temperature on VFA design and scaling up of a VFA production process, it would be necessary production from liquid wastes is needed. to study if the increment of VFAs produced after implementing a pH Besides the substrate, the optimal temperature could also depend on control loop compensates the costs derived from pH control. the microbial population. Some bacterial consortia are more sensitive to temperature changes than others are [115]. Thus, inconsistent results 3.3. Temperature and organic composition of the waste between studies that use substrates with similar organic composition could be caused by the inoculum used in each case [116], specifically Temperature is another relevant operational parameter since it in­ when the acidogenesis is the rate-limiting step. In addition, synergic fluences the metabolic rates, enzymatic activities and microorganisms’ effects of the temperature and other process parameters on the VFA growth and decay rates [105]. For this reason, the optimal temperature production yield have been described, namely pH [64] and substrate for enhancing VFA yield depends on the microbial consortium compo­ composition [76]. sition and the organic composition of the waste. Several studies have carried out experiments on VFA production using different substrates at 3.4. Volumetric organic loading rate, hydraulic retention time and different temperature ranges, namely psychrophilic (4–20 ºC), meso­ organic composition of the waste philic (20–50 ºC), thermophilic (50–60 ºC) and hyperthermophilic (60–80 ºC) conditions [64,66,67,105–108]. The volumetric organic loading rate (ORLv), usually expressed as g Most of the authors agree on the mesophilic range being more COD⋅L− 1⋅d− 1, is a parameter that affects VFA production yield since it favorable in terms of VFA production yield in comparison to psychro­ represents the amount of substrate available per liter of reactor and per philic conditions [67,105,109,110]. Fernández-Domínguez et al. [105], day to be transformed into VFAs. However, to consider the effect of the for example, found that mesophilic conditions when using OFMSW as amount of inoculum added over the VFA production yield, a specific substrate led to higher VFA yields compared to psychrophilic tempera­ organic loading rate (OLRs) (expressed as g COD⋅g− 1 VSSbiomass⋅d− 1) tures. Similarly, if the substrate is WAS, increasing the temperature from should be used. psychrophilic to mesophilic ranges boosts the hydrolysis rate, making For a fixed working volume of reactor and a fixed substrate con­ carbohydrates and proteins more available and rising the activity of the centration in the influent, the OLRv is inversely proportional to the HRT. acid-forming enzymes, enlarging, therefore, VFA production yield [67, OLRv can be increased in two ways: by increasing the influent substrate 110]. Hydrolytic enzymes also show higher activities and the abundance concentration or by lowering the HRT. Since this review is dealing with of acidogenic bacteria was richer at mesophilic conditions compared to waste streams as substrate in acidogenic fermentation, its concentration psychrophilic conditions [110]. Due to all of this, there is a clear cannot easily be changed, so OLRv is normally regulated by tuning the agreement on the fact that mesophilic conditions are more favorable to HRT. Therefore, the impact of both OLRv and HRT is evaluated together VFA production rather than psychrophilic conditions. in this section, focusing on the effect of OLRv on the VFA production When comparing the effect of mesophilic temperatures with ther­ yield. mophilic and hyperthermophilic temperatures on VFA production yield In general terms, increasing the OLRv rises VFA production, as there from a solid or a slurry-like waste, the more convenient conditions are is more substrate available for the microorganisms. Furthermore, dependent on the organic composition of the waste. On one hand, some methanogen growth is slower than acidogen growth, so raising the OLRv studies found that mesophilic conditions were more favorable than is also a way to wash out the methanogens from the reactor [117,118]. thermophilic conditions for the acidogenic fermentation of different However, at very high OLRv, the reactor operation could become 6 A. Vázquez-Fernández et al. Journal of Environmental Chemical Engineering 10 (2022) 107917 unstable due to a sharp drop of pH caused by a high VFA production that component of organic matter is responsible for the formation of each can cease the activity of acidogenic microorganisms [119–121]. VFA. It was observed that butyric acid was the predominant compound The optimal OLRv is more dependent on the complexity of each one produced from glucose, acetic acid was the main product from peptone of the components of the waste rather than the proportion of the and propionic acid the main one when using glycerol. Additionally, different fractions (carbohydrates, proteins, lipids) on it. When working when a mixture of the three components was used, the VFA composition with complex substrates (such as WAS) or complex carbohydrates (like of the effluent was not the result of an additive effect of each component food waste), hydrolysis stage would be the rate-limiting step, so a larger since propionic acid proportion would be expected to be the 32% of the HRT of fermentation is needed. Thus, OLRv must be keep low when total VFAs (in a COD basis) and it was actually the 40% of the total VFAs working with rather complex proteins or carbohydrates to avoid the [63]. Once again, these studies suggest that the organic composition of increase in the broth viscosity produced by the accumulation of non- the waste is the main factor that influences the VFA production yield and hydrolyzed components, which would lead to a low VFA production the composition of the obtained VFAs, but other parameters may also due to mass transfer limitations [69,122]. Jankowska et al. [68] affect. Therefore, in the following sections, the interactional effect of pH, compared the VFA production yield obtained at different HRT and pH temperature and bacterial composition with the organic composition of from four different substrates, complex/simple carbohydrate-rich sub­ waste on the VFA composition is revised. Furthermore, more studies strates (maize silage/glucose), complex/simple protein-rich substrate using model compounds of carbohydrates, proteins, and lipids as sub­ (microalgae biomass/whey). They concluded that VFA production yield strates, but working in continuous or semicontinuous conditions are strongly depends on the complexity of each one of the components of the needed to evaluate the effect of organic composition of the waste on VFA waste and that an alkaline pH enhances VFA yield more than an increase composition in long term processes. in the HRT [68]. Alternately, several pretreatments of wastes described in the literature allow to apply larger OLRv by enhancing the hydrolysis 4.2. pH and organic composition of the waste step efficiency [123]. There is no doubt that the application of high OLRv would help to The effect of pH on VFA composition is by far the most studied wash out the methanogens, but if this ORLv is high enough, some among the different operational parameters. In this section, the joint acidogenic microorganisms would be also washed out the reactor. impact of pH and organic composition of the waste on the composition Therefore, the OLRv affects, at the end. all the microbial communities of the VFA rich stream produced is analyzed. Table 3 collect VFA dis­ present in the reactor [115,117] and, in consequence, the VFA compo­ tributions obtained in the fermentation of wastes with different organic sition distribution, which is discussed in Section 4. composition. According to the reviewed studies, there is agreement on the VFAs 4. Operational parameters affecting VFA distribution obtained from protein-rich substrates at different pH. pH lower than 5.0 leads to propionic acid while a pH between 5.0 and 11.0 leads to acetic In Section 3, we analyzed the influence of different operational pa­ acid [98,124,125]. Liu et al. [124] reported that a pH of 12.0 produced rameters together with the organic composition of the waste on the VFA butyric acid as a main component. The rest of the VFA components with production yield. However, the impact of these parameters on the pro­ lower presence vary among the different studies [98,124]. In any case, it duction of a particular VFA or a mixture of VFAs is even more difficult to can be affirmed that pH is an appropriate tool to change VFA compo­ determine. As we mentioned before, acidogenic fermentation is a com­ sition when using protein-rich substrates. plex process where numerous biological reactions take place. Moreover, Nevertheless, carbohydrate-rich substrates yielded very different when using MMC, the different microorganisms present in the reactor VFA compositions in the different studies shown in Table 3. On the one could follow different metabolic pathways and could also vary with hand, Atasoy et al. [114] performed experiments using glucose as sub­ applied operational conditions. Thus, the spectrum of obtained products strate (carbohydrate) and determined the VFA composition obtained from acidogenic fermentation by MMC could be very wide. Therefore, using different types of inoculums. The main VFA component was more knowledge is needed to understand how to drive the acidogenic butyric acid independently of the inoculum used for a wide range of pH fermentation process to obtain a specific VFA or a mixture of VFAs. conditions (pH between 5.0 and 10.0) [114]. On the other hand, pH has a strong effect when using substrates mainly composed of complex 4.1. Organic composition of the waste carbohydrates, namely paper mill wastewater and cheese whey [65]. In paper mill wastewater fermentation experiments, an increase of pH from Some authors studied the influence of the organic composition of the 4.9 to 6.0 changed the main VFA components from acetic acid (49%), waste on the VFA composition of the effluent form acidogenic fermen­ butyric acid (18%) and propionic acid (13%); to butyric acid (33%), tation without considering any other operational conditions. For propionic acid (23%) and acetic acid (22%). An increment of pH from example, Alibardi and Cossu [53] performed batch fermentation ex­ 5.3 to 6.0 in cheese whey acidogenic fermentation experiments, shifted periments using mixtures of four fractions of different organic wastes the main VFA components from acetic acid (51%), butyric acid (24%) and analyzed the produced VFAs. They only found a correlation between and propionic acid (19%); to propionic acid (41%), acetic acid (31%) butyric acid production and the chemical composition of the wastes (in and butyric acid (10%) [65]. terms of percentage of carbohydrates, lipids, and proteins). Carbohy­ Table 3 collects as well studies of VFA compositions obtained from drate content seemed to be the main factor influencing butyric acid fermentation of wastes that are formed of carbohydrates and proteins concentration. Nevertheless, the correlation was weak, since R2 value [51,108,126,127] and carbohydrates and lipids mixtures [97,103]. In was 0.809 [53]. Ma et al. [101] obtained similar results by carrying out these cases, the substrate heterogeneity makes it even more complicated fermentation batch experiments using mixtures of WAS and potato peel to link the organic composition and the pH with the final VFA compo­ or food waste in different ratios. In all the trials, acetic acid was the main sition obtained. Luo et al. [126] performed experiments of different pH product and when the carbohydrate content in the waste was higher of a mixture of WAS (protein-rich substrate) and wine vinasse (carbo­ than that of proteins, butyric acid and ethanol concentrations increased hydrate-rich substrate) (1:1, in COD proportions). The addition of wine while propionic and valeric acid decreased. They also correlated pro­ vinasse changed the main VFA component at different pH ranges: acetic pionic, butyric and valeric acid concentrations with lipid, starch, and acid (from pH 3.0–5.0), acetic and propionic acids (from pH 6.0–9.0) protein consumption but, again, R2 values were quite low (between 0.62 and acetic acid (pH 10.0) [126]. Food waste (carbohydrate-rich and and 0.81) [101]. Yin et al. [63] performed batch tests using glucose, protein-rich substrate) is one of the most studied substrates. Different peptone and glycerol as model compounds of simple carbohydrates, studies arrived at different VFA distributions at similar pH conditions proteins and lipids respectively, to elucidate more clearly which [51,108,127]. Feng et al. [51] discussed the different pathways that can 7 A. Vázquez-Fernández et al. Journal of Environmental Chemical Engineering 10 (2022) 107917 Table 3 Effect of pH on VFA distribution by using wastes with different organic composition. Substrate Main component of the Inoculum used pH Main products (in descending order) Reference substrate (% of the total VFA) Gelatin-rich wastewater Proteins Methanogenic anaerobic sludge 4.0 Propionic acid (32%) and acetic acid (15%) [98] 4.5 Propionic acid (27%) and acetic acid (18%) 5.0 Acetic acid (23%) and propionic acid (20%) 5.5 Acetic acid (25%), isobutyric acid (13%), butyric acid (13%), isovaleric acid (13%), propionic acid (12%) and valeric acid (12%) 6.0 Acetic acid (28%), butyric acid (16%), isobutyric acid (13%) and propionic acid (13%) 6.5 Acetic acid (32%) and butyric acid (21%) 7.0 Acetic acid (35%) and butyric acid (22%) Sewage sludge from a Proteins Pretreated sewage sludge (thermal 3.0 Propionic acid (56%) and acetic acid (44%)* [124] brewery pretreatment to inactivate methanogens) 5.0 Acetic acid (31%), butyric acid (24%) and valeric acid (20%)* 7.0 Acetic acid (26%), propionic acid (21%), butyric acid (16%) and valeric acid (15%)* 9.0 Acetic acid (44%) and propionic acid (19%)* 11.0 Acetic acid (44%) and propionic acid (19%)* 12.0 Butyric acid (37%) and acetic acid (31%)* Secondary sludge from a Proteins Slurry anaerobic biomass 7.0 Acetic acid (47%) [125] WWTP 10.0 Acetic acid (56%) Heat-alkaline pretreated Proteins Slurry anaerobic biomass 7.0 Acetic acid (57%) [125] secondary sludge 10.0 Acetic acid (65%) Glucose solution Carbohydrates Small granular anaerobic biomass 5.0 Butyric acid (58%) and acetic acid (24%)* [114] 8.0 Butyric acid (47%) and acetic acid (35%)* 10.0 Butyric acid (56%) and acetic acid (42%)* Large granular anaerobic biomass 5.0 Butyric acid (52%) and acetic acid (39%)* 8.0 Butyric acid (50%) and acetic acid (33%)* 10.0 Butyric acid (58%) and acetic acid (39%)* Slurry anaerobic biomass 5.0 Butyric acid (46%) and acetic acid (14%)* 8.0 Butyric acid (55%) and acetic acid (15%)* 10.0 Butyric acid (47%) and acetic acid (44%)* Paper mill wastewater Carbohydrates Acidogenic biomass from a reactor treating 4.9 Acetic acid (49%), butyric acid (18%) and propionic acid (13%) [65] paper mill wastewater 6.0 Butyric acid (33%), propionic acid (23%) and acetic acid (22%) Cheese whey Carbohydrates Acidogenic biomass from a reactor treating 5.3 Acetic acid (51%), butyric acid (24%) and propionic acid (19%) [65] paper mill wastewater 6.0 Propionic acid (41%), acetic acid (31%) and butyric acid (10%) Secondary sludge from a Proteins and No inoculum added (endogenous 3.0 Acetic acid (52%) and butyric acid (36%) * [126] WWTP + wine vinasse carbohydrates microorganisms from the substrate) 4.0 Acetic acid (41%), butyric acid (24%) and propionic acid (21%) * 5.0 Acetic acid (37%), propionic acid (31%) and butyric acid (19%) * 6.0 Propionic acid (40%) and acetic acid (38%) * 7.0 Propionic acid (45%) and acetic acid (38%) * 8.0 Propionic acid (42%) and acetic acid (40%) * 9.0 Acetic acid (43%) and propionic acid (40%) * 10.0 Acetic acid (38%) and propionic acid (33%) * Food waste Carbohydrates and Mesophilic anaerobic digested sludge 5.0 Acetic acid (60%) and butyric acid (31%) [108] proteins 6.0 Butyric acid (53%) and acetic acid (24%) 7.0 Butyric acid (43%) and acetic acid (34%) Carbohydrates and Sludge from anaerobic digester 3.2 Lactic acid (87%) [51] proteins 4.0 Lactic acid (81%) 4.2 Lactic acid (81%) 4.5 Lactic acid (57%) and acetic acid (25%) 4.7 Butyric acid (56%) and acetic acid (19%) 5.0 Butyric acid (40%) and acetic acid (40%) 6.0 Butyric acid (40%) and valeric acid (29%) Food waste + mature Carbohydrates and Biomass from anaerobic digester treating 6.0 Hexanoic acid (43–47%), butyric acid (21–25%) and acetic acid [127] compost proteins food waste (22–23%) 7.0 Acetic acid (36–38%), hexanoic acid (24–26%) and butyric acid (18–20%) Olive oil mil waste Carbohydrates and Anaerobic biomass acclimatized to 5.0 Acetic acid (60%) and propionic acid (23%) [103] lipids secondary sludge 9.0 Acetic acid (79%) Dairy wastewater Lipids and Anaerobic sludge treating synthetic dairy 4.0 Propionic acid (38%) and acetic acid (18%) [97] carbohydrates wastewater 4.5 Propionic acid (34%) and acetic acid (20%) 5.0 Propionic acid (28%) and acetic acid (26%) 5.5 Acetic acid (28%), propionic acid (18%) and butyric acid (13%) 6.0 Acetic acid (33%), butyric acid (14%) and propionic acid (13%) 6.5 Acetic acid (34%), butyric acid (14%) and propionic acid (12%) * The VFA compositions were obtained (and calculated in some cases) from graphically presented results. 8 A. Vázquez-Fernández et al. Journal of Environmental Chemical Engineering 10 (2022) 107917 take place at different pH conditions and would explain the VFA com­ 4.3. Temperature and organic composition of the waste positions obtained. Homolactic fermentation (following EMP and PPP pathways) takes place at pH 3.2–4.5 leading to lactate production. Less studies have been published about the effect of the temperature Heterolactic fermentation (following PK pathway) occurs at pH 3.2–5.0 on the VFA distribution. Each VFA can be produced by different strains, leading to lactate and ethanol production. Ethanol type fermentation and each of these species can have a different optimal growth temper­ (following EMP pathway) takes place at pH 4.5 and directs fermentation ature. Thus, the proportions of each VFA produced can vary with to ethanol, acetate and H2. Ethanol fermentation (following ED temperature. pathway) takes place at pH 4.4–6.0 and yields ethanol. Heterolactic From the results collected in Table 4, it can be gathered that VFA fermentation (following Bifidus pathway) occurs at pH 4.5–5.0 leading distribution for most of the substrates is not as dependent on tempera­ to acetate and lactate. Acetone-butanol-ethanol fermentation (following ture as it is on pH. The reported results show that the main VFA EMP pathway) takes place at pH 4.7–4.9 leading to acetone, butanol, component does not vary with the temperature used, from psychrophilic ethanol and H2. Butyrate fermentation (following EMP and PPP path­ to thermophilic ranges, for both protein-rich substrates [66,67,110] and ways) occurs at pH 5.0 and leads to butyrate, acetate and H2. Finally, carbohydrate-rich substrates [105,129]. The composition of the second mixed acid fermentation (following EMP and PPP pathways) takes place major and subsequent VFAs exhibited more variations with temperature at pH 6.0 and yields acetate, propionate, butyrate, valerate and H2 [51]. in some studies [110,129]. Nonetheless, several studies that used food Since several pathways can take place at the same pH conditions, the waste as substrate, reported different VFA compositions depending on differences between organic composition of the food waste used in the the temperature. Lim et al. [69] carried out food waste fermentation in a reported studies might explain the different VFA distributions obtained semicontinuous reactor at different temperatures and found that the [51,108,127]. main VFAs produced were propionic, acetic and valeric acids at 25 ºC; Moreover, the pH effect on the VFA composition also depends on the acetic and propionic acids at 35 ºC and acetic and hexanoic acids at microbial population present and, at the same time, pH influences the 45 ºC. Later, Jiang et al. [108] performed batch experiments of food microbial community structure. Thus, the pH could yield different VFA waste fermentation at different temperatures and pointed out that the composition depending on the culture history. As an example, Mohd- main VFAs produced were acetic and propionic acids at 35 ºC; propionic Zaki et al. [128] carried out glucose fermentation experiments using and acetic acids at 45 ºC and butyric acid at 55 ºC. two different pH regulation modes (progressive mode and reset mode) which led to non-identical VFA compositions at the same pH levels. 4.4. Bacterial composition in the anaerobic reactor and organic Progressive pH regulation led to a gradual change from butyric and composition of the waste acetic acids to acetic acid and ethanol as main components as pH increased, while resetting pH regulation caused a clearly defined change Regarding bacterial composition, numerous studies analyzed the from acetic and butyric acids to acetic acid and ethanol as main com­ bacteria present in the anaerobic reactors to link them to the production ponents when pH exceeded 6.5 [128]. of different VFAs. Despite the fact that some species of bacteria are known to be responsible for the production of specific types of VFAs Table 4 Effect of temperature on VFA distribution by using wastes with different organic composition. Substrate Main component of the Inoculum used Temperature Main products (in descending order) Reference substrate (ºC) (% of the total VFA) Ultrasonic-pretreated Proteins No inoculum added (endogenous 10, 20, 37, 55 Acetic acid (47–49%), isovaleric acid [67] secondary sludge from a microorganisms from the substrate) (16–23%) and propionic acid (9–15%) WWTP Secondary sludge from a Proteins No inoculum added (endogenous 15 Acetic acid (41%) and propionic acid [110] WWTP microorganisms from the substrate) (22%) 30 Acetic acid (49%), isovaleric acid (14%) and propionic acid (13%) Dewatered sludge Proteins No inoculum added (endogenous 35 Acetic acid (45%) and isovaleric acid [66] microorganisms from the substrate) (20%) 55 Acetic acid (51%) and isovaleric acid (22%) Palm mill oil effluent Carbohydrates Sludge from the treatment of palm oil 30 Acetic acid (37%) and propionic acid [129] mill effluent (26%) * 40 Acetic acid (43%) and propionic acid (25%) * 55 Acetic acid (58%) and propionic acid (14%) * OFMSW Carbohydrates No inoculum added (endogenous 20, 35, 45, 55, Acetic acid (28–33%), butyric acid [105] microorganisms from the substrate) 70 (25–29%) and propionic acid (22–26%) Food waste Carbohydrates and Anaerobic sludge 25 Propionic acid (44–46%), acetic acid [69] proteins (20–22%) and valeric acid (18–19%) 35 Acetic acid (30–33%) and propionic acid (25–28%) 45 Acetic acid (49–50%) and hexanoic acid (23–25%) Carbohydrates and Mesophilic anaerobic sludge 35 Acetic acid and (37%) and propionic acid [108] proteins (31%) 45 Propionic acid (38%) and acetic acid (33%) 55 Butyric acid (81%) * The VFA compositions were obtained from graphically presented results. 9 A. Vázquez-Fernández et al. Journal of Environmental Chemical Engineering 10 (2022) 107917 [17], the number of known acidogenic microorganisms is still limited one single hypothetical microbial population was capable of carry out [77]. all the most important metabolic pathways from glucose. It was the first Nevertheless, the bacterial communities present in a MMC strongly model capable of predicting the effect of pH on the product spectrum by depend on the organic composition of the substrate [130–132]. More­ considering the role of the different electron carriers (ferredoxin, NAD over, the bacterial composition could also change with pH [51,89,128, (H) and FAD(H2)) and a model for the transport of solutes across the cell 133] as well as other operational parameters such as temperature [134]. membrane. The model predicted high yields of butyric acid at low pH, Some authors analyzed the combined effect of the pH and the bacterial high yields of acetic acid and ethanol at high pH and acetic acid and community composition and structure on the VFA compositions ob­ propionic acid as secondary product at neutral pH [140]. The main tained [22,114]. Atasoy et al. [114] evaluated the VFA composition difference with the experimental study they took as reference is the obtained from glucose fermentation at different pHs when using three presence of acetic and butyric acids as main products at low pH, not only different inoculum types. They found that inoculum structure did not butyric acid [140,142]. Later, Regueira et al. [143] improved this model affect VFA composition while pH was determinant, as discussed before. by including electron bifurcation in the butyrate synthesis pathway and At the same time, the dominant microbial community affected VFA homoacetogenesis that consumed part of the H2 produced. After this production more than VFA composition [114]. Wang et al. [22] per­ modification, they succeed predicting both acetic and butyric acids at formed experiments of food waste fermentation at different pH condi­ low pH [143]. tions with two types of inoculums. pH did not affect VFA composition in Subsequently, Regueira et al. [141] built an energy-based metabolic the reactors working with anaerobic activated sludge, since acetic and model for predicting VFA formation from proteinic substrates. In that butyric acids where the main components in all pH conditions. However, study [141], they considered the same approach that when the inoculum was aerobic activated sludge, pH 4 and uncontrolled González-Cabaleiro et al. [140] held previously. In that case, it is again pH yielded acetic and propionic acids, while pH 5 and 6 led to butyric assumed that a virtual microorganism can perform all the pathways and and acetic acids [22]. that protein consumption can be limited in case it is not thermody­ Numerous studies carried out acidogenic fermentation experiments namically favorable. Moreover, the different amino acids could interact of different substrates and analyzed the VFA compositions obtained as among them limiting the degradation of others, mainly provoked by well as the bacterial communities at phylum, class, order, family, and NADH competition. Net NADH balance must be neutral since in absence genus level [68,77,89,91,114,132]. Nevertheless, the link of each VFA of oxygen, there is no electron acceptor. This balance can be affected by produced with the microorganisms responsible for its synthesis is still external pH, so this model is able to predict the preferred pathways that not clear. Further work is needed to understand the relationship of mi­ lead to different VFAs for the fermentation of a substrate with a defined crobial communities and VFA composition. amino acid composition at different pH [141]. Since wastes are made up of more than one component, Regueira 4.5. Prediction of VFA distribution by energy-based metabolic models et al. [32] proposed a model for fermentation of a mix with different ratios of carbohydrates and proteins by using glucose and gelatin as As explained above, the VFA composition of the effluents of an model substrates. The addition of glucose to casein fermentation adds a acidogenic fermentation process could depend on multiple factors, source of reductive power, changing the NADH balance and the path­ making it very difficult to predict what composition can be obtained ways that maximize ATP yield. In that case, protein consumption rate is depending on the experimental conditions applied. Against this back­ assumed to be lower than glucose consumption based on experimental ground, mathematical modelling of the fermentation processes could be results. One of the main limitations of this model is the little information a powerful tool to predict VFA production. The aim of this section in not available about the amino acid profile of the gelatin. If it is used as a to support the interpretations gathered in the previous sections for each design tool, the amino acid composition of the proteins used must be operational parameter and its correlation with organic composition. previously determined [32]. Here, the energy-based metabolic models are presented as the best In view of all the previously mentioned research, energy-based mathematical tool available up to date to predict the VFA composition. metabolic models seem to be the best method to mechanistically To the best of our knowledge, the energy-based metabolic models explain fermentation processes. Experimental data can be used to prove developed so far only consider simple molecules as substrate and pH as the predictable capacity of these models, but do not allow us to the variables that affect VFA composition. extrapolate to operational conditions different to those investigated. In thise sense, several types of models have been proposed to achieve Nevertheless, mechanistic models allow us to explore VFA distribution this objective. The first proposed models were based on the stoichiom­ under different operational conditions by changing those defined as etry of the fermentation reactions, considering it constant and not environmental conditions. Future work is needed to build models for considering the possible variations due to changes in operational con­ more complex substrates and introduce the hydrolysis step in the ditions [135]. From that point, several corrections have been applied to metabolic networks. consider operational conditions by using variable stoichiometry models [136,137]. However, these models do not have enough predictive ca­ 5. Example of application of bioproduced VFAs: how the pacity. They are very similar to ASM-family models that were created to composition of the VFAs determines the type of describe aerobic processes, which are usually controlled by kinetics polyhydroxyalkanoate (PHA) produced [138]. Nevertheless, anaerobic processes supply low energy, and their conversion is limited by the thermodynamic equilibrium [139]. There­ Polyhydroxyalkanoates (PHAs) are some of the multiple products fore, energy-metabolic modelling seems to be, nowadays, the best way that can be obtained from VFAs. PHAs can be defined as biodegradable to predict VFA composition in fermentation processes. polyesters, composed of hydroxyalkanoic acids, and synthesized by Energy-based metabolic models assume that in anaerobic fermenta­ bacteria [144,145]. There are more than 150 different hydroxyalkanoic tion processes, microorganisms would follow those pathways that return acids that can be integrated into the PHAs’ molecular structure. more net energy from the substrate and consequently lead to biomass Consequently, the properties of the resulting polymers depend on their growth. Thus, the VFAs linked to a higher ATP yield would be the composition [144]. PHAs are usually classified into two different groups dominant products of the fermentation [32]. Some studies developed depending on the number of carbon atoms of their monomers. Short models to predict fermentation product spectrum of glucose [140], chain length PHAs (scl-PHAs) are composed of monomers with 3–5 proteins [141] and co-fermentation of carbohydrates and proteins [32] carbon atoms while medium chain length PHAs (mcl-PHAs) contain by MMC. monomers with 6–14 carbon atoms [146]. scl-PHAs are characterized González-Cabaleiro et al. [140] elaborated a model considering that for having a high degree of crystallinity and high melting and low glass 10 A. Vázquez-Fernández et al. Journal of Environmental Chemical Engineering 10 (2022) 107917 transition temperatures, making them fragile and difficult-to-deform generations and the complex extraction of the polymer from the intra­ materials. Conversely, mcl-PHAs are less crystalline so they are elasto­ cellular compartments [154]. meric materials that can be used in high value-added applications [146]. Consequently, the current tendency in research is to develop pro­ Their glass transition temperature is below zero and their melting point cesses carried out by MMC processes. PHA production by MMC follows is lower than scl-PHAs, so it is easier to mold them [147]. Thus, PHAs natural selection principles since microorganisms with PHA storage with a wide range of properties can be produced and a great number of ability compete with those microorganisms that do not accumulate potential applications can be found for these polymers, as pointed out by PHAs. MMC based processes can be carried out in non-sterile conditions Prajapati et al. [148]. Nevertheless, PHAs have gained attention mainly and can use organic waste as a carbon source since MMC are more because they are biocompatible and fully biodegradable, and their me­ adaptable. Thus, these systems are considered more robust than pure chanical properties are comparable to petroleum-based plastics such as culture based processes, and simultaneously, more cost-efficient [146, polyethylene and polypropylene, as reported by Anjum et al. [149]. 151]. As an example, Crutchik et al. [150] estimated that the minimum Today, PHA production at an industrial scale is carried out by using PHA cost obtained from sewage sludge were 1.26 and 2.26 US$ kg− 1 pure cultures or genetically modified strains. The main disadvantage of PHA for large and small WWTPs, respectively. These values are quite PHAs production processes with pure cultures is that they usually similar to the fossil-fuel derived plastics cost (1.2 US$ kg− 1 plastic) and require highly pure substrates [146]. Therefore, the PHAs obtained are considerably lower to cost the PHA obtained by using pure cultures not commercially competitive against fossil-fuel-derived plastics since (between 2.4 and 5.5 US$ kg− 1 PHA) [150]. Furthermore, MMC based their production costs are between 2.0 and 4.6 times higher than the processes can produce a wide range of copolymers with a different conventional plastics costs [150]. Besides, pure cultures need large composition depending on the feedstock used [151]. Since MMC contain quantities of co-substrates to obtain polymers containing a relatively a high variety of bacteria, different pathways can be followed. Co­ low fraction of monomers different from 3-hydroxybutyrate (3HB) polymers based on 3HB synthesized by MMC can also contain monomers [151] possible to produce PHAs by chemical synthesis or by employing of 3-hydroxyvalerate (3HV), 3-hydroxyhexanoate (3HHx), 3-hydrox­ genetically modified plants, but these processes have been considered y-2-methylvalerate (3H2MV) and 3-hydroxy-2-methylbutyrate less interesting for the industry. (3H2MB) [155]. Therefore, in MMC based processes, one of the easiest On the one hand, PHAs can be chemically synthesized from ways to modify the final properties is by changing the carbon source substituted propiolactones, but this process will never be competitive used as substrate. In the literature, up to four natural PHA synthetic with bacterial fermentation since lactone monomers are very costly routes starting from different carbon sources have been described [152]. Furthermore, chemical synthesis results in polymers with lower (Fig. 2). Another ten engineered pathways have also been developed molecular weights in comparison to the ones obtained by microbial [156] but in this review, engineered pathways were not considered for synthesis [153]. On the other hand, PHA synthesis using genetically PHA production by MMC. modified plants is a process that still needs to deal with several limita­ Pathway I starts with the conversion of a sugar to acetyl-CoA by tions, namely control of monomer composition, plant transformation glycolysis and proceeds with the formation of acetoacetyl-CoA from the processes, expression of the transformed genes in the following condensation of two acetyl-CoA molecules by β-ketothiolase (PhaA), Fig. 2. Metabolic pathways for PHA synthesis. (adapted from Meng et al. [156]). 11 A. Vázquez-Fernández et al. Journal of Environmental Chemical Engineering 10 (2022) 107917 which is then transformed into 3-hydroxybutyryl-CoA by NADPH- and IV). On the one hand, VFAs are preferred with respect to other dependent reductase (PhaB). Finally, the polymerization of 3-hydroxy­ substrates, such as glycerol or carbohydrates, since the last two have the butyryl-CoA by PHA synthase (PhaC) leads to the formation of P(3HB) tendency to synthetize glycogen instead of PHAs [160]. Moreover, the monomers. This pathway is the most well-known and is typically used by synthesis starting from VFAs is energetically more favorable because Ralstonia eutrophia [157]. Concurrently, PHA depolymerase (PhaZ) their complete β-oxidation produces more energy in the form of ATP catalyzes PHA degradation to form 3-hydroxybutyrate, 3-hydrox­ molecules than the oxidation of a molar equivalent of glucose [161]. ybutyrate is transformed to acetoacetic acid by 3-hydroxybutyrate de­ However, when using bioproduced VFAs as substrate, the properties hydrogenase and acetoacetyl-CoA is regenerated from acetoacetic acid and monomer composition of the resultant PHAs would depend on the by acetoacetyl-CoA synthetase [153]. composition of the VFA-rich-stream [162–164] used as raw material. It Pathway II uses fatty acids as substrates, which are converted to acyl- has been demonstrated that when using VFAs with an even number of CoA, 3-ketoacyl-CoA, enoyl-CoA and S-3-hydroxyacyl-CoA by the carbons, polymers rich in 3HB monomers are formed. On the contrary, β-oxidation cycle. These intermediates are used to synthesize R-3- feeding VFAs with an odd number of carbons lead to 3HV monomers hydroxyacyl-CoA by the action of acyl-CoA oxidase and enoyl-CoA [165]. In this sense, the odd-to-even ratio is defined as the sum of hydratase, 3-ketoacyl-CoA reductase (FabG), (R)-enoyl-CoA hydratase/ odd-equivalent carboxylic acids, such as propionic or valeric acids, enoyl-CoA hydratase I (PhaJ) and epimerase, respectively. Polymeriza­ divided by the sum of even-equivalent carboxylic acids, such as acetic or tion of R-3-hydroxyacyl-CoA leads to mcl-PHAs by specific PhaZ [151, butyric acids [60] and it can be used as a parameter to evaluate the ratio 158]. between 3HB and 3HV monomeric units present in the final PHA Pathway III starts from the commonly acknowledged ‘unrelated’ product. Pure polyhydroxybutyrate (PHB) is brittle and stiff, but the carbon sources, which can be defined as carbon sources with a molecular incorporation of 3HV in the polymer (as in PHB-co-PHV) enhances structure completely different to the resultant PHA monomers [158]. elasticity and flexibility [166]. Thus, high odd-to-even ratios are desired Some examples of unrelated carbon sources are glucose, fructose, glyc­ in order to enhance polymer mechanical properties [60]. Therefore, erol, gluconate, ethanol, and acetate [159]. Through this pathway, also odd-equivalent acids must be the main components of the VFA stream named de novo fatty acid synthesis, carbon sources are firstly trans­ feed to the PHA accumulation reactor. Table 5 sums the composition and formed into acetyl-CoA, acetyl-CoA into malonyl-CoA, and malonyl-CoA properties of the PHAs obtained when using different VFAs or mixtures into malonyl-ACP. Malonyl-ACP is converted to R-3-hydroxyacyl-ACP, of VFAs in MMC based processes. Since the polymerization and crys­ which is then transformed into R-3-hydroxyacyl-CoA by 3-Hydroxyacy­ tallization have a great influence on the final properties of PHAs and l-ACP-CoA transferase (PhaG). Finally, R-3-hydroxyacyl-CoA polymeri­ they take place inside the cell cytoplasm, the properties of the PHAs zation gives rise to mcl-PHAs. Both Pathways II and III have been produced by MMC based processes are not necessarily the same as the described for most of the Pseudomonas spp. [157]. properties of PHAs produced by pure cultures [151]. Pathway IV is specific for butyric acid, which could be substrate in Furthermore, the cost of the carbon source for PHA biosynthesis can PHA synthesis without entering the β-oxidation cycle. Butyric acid is reach 50% of the final production cost [147], so the use of waste-derived transformed into S-3-hydroxybutyryl-CoA, which is then converted into VFAs would clearly reduce the price of the final product and make it acetoacetyl-CoA that would follow the same steps of Pathway I to pro­ competitive with fossil-fuel-derived polymers. Also, it would be possible duce P(3HB) monomers. This fourth pathway was observed in Rhizo­ to integrate PHA production and wastewater or organic waste treat­ bium (Cicer) sp. Strain CC 1192 [153]. ments [160]. In a MMC based process using waste or wastewater as Traditionally, the most widely used substrate in PHA synthesis with substrate, PHA synthesis takes place in four steps: pure cultures has been glucose (Pathway I) [146]. However, PHA pro­ duction by a MMC based process usually starts from VFAs (Pathway II Table 5 Thermal and mechanical properties of PHAs produced by MMC cultures. Tm, melting temperature; ΔHm, melting enthalpy; Tg, glass transition temperature; PDI, polydispersity index; Mw, weight average molecular weight. Substrate VFA composition PHA composition (% mol) PHA Thermal properties Molecular Reference (% mol) (HAc:HPr: (3HB:3H2MB:3HV:3H2MV:3HHx) content (% weight HBt:HV:other) gPHA g− 1 Tm Tm (2) Total Tg1 Tg2 PDI Mw VSS) (1) (⁰C) ΔHm (J (⁰C) (⁰C) (x 105) (⁰C) g− 1) Acetate 100:0:0:0:0 90:4:4:1:1 – 171 – 77 5 – 2.0 8.1 [155] Acetate 100:0:0:0:0 66:4:28:2:0 – 96/ 146/ 9 -6 – 1.4 5.5 [171] 109 161 Acetate 100:0:0:0:0 100:0:0:0:0 21 171 – 64 – – 1.3 32 [172] Acetate 100:0:0:0:0 96:0:4:0:0 52 – – – – – 2.2 0.9 [173] Acetate 100:0:0:0:0 NA 26 93 150 32 -6 – 2.3 2.2 [174] Propionate 0:100:0:0:0 12:6:63:14:6 – 89 – 18 -14 – 2.0 4.5 [155] Propionate 0:100:0:0:0 11:13:35:41:0 – 84 20 – -0.2 – 1.5 5.6 [171] Propionate 0:100:0:0:0 NA 30 92 38 104 -18 – 2.8 4.3 [174] Butyrate 0:0:100:0:0 83:5:7:2:2 – 137 150 50 3 – 1.7 9.0 [155] Valerate 0:0:0:100:0 12:5:78:4:1 – 98 – 2 -12 – 3.9 6.2 [155] Acetate + propionate 82:18:0:0:0 55:5:30:9:0 – 124 156 632 − 8 – 3.1 3.9 [171] Acetate + propionate 54:47:0:0:0 46:8:32:15:0 – 70 97 19 -2 – 1.7 5.0 [171] Acetate + propionate 22:78:0:0:0 20:9:39:32:0 – 119 155 10 -6 – 1.4 5.5 [171] Acetate + propionate 60:40:0:0:0 94:0:6:0:0 25 157 – 54 1 – 1.3 33 [172] Acetate + propionate 84:16:0:0:0 60.3:0:39.7:0:0 54 – – – – – 3.2 1.4 [173] Acetate + propionate 64:36:0:0:0 NA 32 82 24 93 -19 – 1.7 2.3 [174] VFA mix 47:18:22:13 61:0:39:0:0 77 113 138 – -16 – 2.3 2.1 [164] VFA mix 73:13:12:0.02 79:0:21:0:0 68 121 137 – -10 – 2.7 3.9 [164] VFA mix 74:7:16:3 85:0:15:0:0 56 134 147 – -1 – 2.3 6.5 [164] *NA: not available 12 A. Vázquez-Fernández et al. Journal of Environmental Chemical Engineering 10 (2022) 107917 1) Acidogenic fermentation of the waste material to produce a VFA-rich temperature and OLRv and HRT also play a role. In this sense, Table 6 stream. sums up the operational conditions that lead to a higher VFA production 2) Enrichment of aerobic activated sludge by feast-famine feeding yield depending on the organic composition of the waste. Likewise, the strategies that would select the PHA-accumulating populations. effect of pH, temperature and bacterial composition on VFA composition 3) Improvement of the accumulation of PHAs in the enriched biomass. was analyzed together with substrate composition. The effect of the 4) Extraction of the PHAs from the biomass. aforementioned factors on VFA composition might be interconnected and is still not clear. More research is needed to predict and control VFA Factors influencing PHA production in the enrichment and accu­ composition of the effluent of an acidogenic fermentation reactor. On mulation steps have been previously reviewed [160,167]. Also, extrac­ this matter, energy-based metabolic models are presented as the best tion methods for PHA recovery have been extensively evaluated [148, tool known to date to predict VFA composition from a substrate with a 168]. For an industrial application, it would be necessary to produce well-characterized composition under different pH conditions. Finally, PHAs with a fixed composition to maintain the quality and properties of the PHA synthesis process by MMC based processes was presented as an the bioplastics. It has been demonstrated that PHA composition can be example of one of the most interesting applications of the waste-based controlled by shifting the VFA composition in the feeding stream [155, bioproduced VFAs. 169,170]. Therefore, to obtain a PHA with a constant composition over Despite all the research performed in the last years in the VFA time, the VFA composition of the feeding stream must also be constant. fermentation platform, there is still limited knowledge in some areas Then, for developing a commercial PHA production from organic that should be studied deeply. Further work needs to cover the following wastes, more knowledge is needed in the control of VFA composition gaps: obtained in the acidogenesis fermentation. Furthermore, in both PHA enrichment and accumulation steps, the VFA concentration in the - Establishment of standard parameters to quantify both VFA pro­ VFA-rich stream used as substrate is also a key parameter. duction yield and VFA composition. In this review, studies of VFA fermentation of solid, slurry-like, and liquid wastes (mostly waste­ 6. Conclusions and research gaps to be solved waters) have been analyzed together. Nevertheless, the VFA pro­ duction yields are expressed differently, depending on the type of Volatile fatty acids are value added products and their synthesis from waste (solid and/or liquid waste) research field. Moreover, some organic waste or wastewaters by MMC based processes is a promising studies examine the operational parameters that maximize VFA technology that would avoid the use of fossil fuels. However, further production, quantifying it as the degree of acidification or the VFA knowledge about how to control VFA production yield and composition concentration obtained instead of the VFA production yield. Addi­ is needed to scale up bioprocesses based on the use of waste streams as tionally, VFA compositions are sometimes related to the sum of all feedstock. This review rounds up the main metabolic routes that lead to the VFAs obtained and on other occasions, related to the total soluble VFA synthesis by MMC. Next, the interactional effect of pH, temperature organic matter. The use of standard parameters would facilitate the and OLRv and HRT and the organic composition of the waste on VFA comparison between different studies. production yield is discussed. From the aforementioned analysis, it can - Further study of the VFA composition obtained from different sub­ be gathered that organic composition of waste, in terms of carbohy­ strates regarding their organic components rather than considering it drates, proteins and lipids, is the main factor that affects the VFA pro­ only as a concrete type of substrate. As emphasized in this review, duction yield, even though the operational parameters, like pH, organic composition of the substrate, in terms of carbohydrates, proteins and lipids, has a strong influence on both VFA production yield and the type of VFA produced. Additionally, attention should Table 6 be paid when reviewing literature since substrates with different Operational conditions that lead to high VFA production yields depending on the organic compositions and complexities have been grouped under the organic composition of the substrate. same substrate name, even when its composition, origin and sea­ Operational conditions that lead to high VFA production yields sonality is different. Also, the substrate consumption needs to be specifically analyzed in terms of carbohydrate, protein and lipid Parameter Main component of the substrate amounts apart from soluble COD amounts. Carbohydrates Proteins Lipids - Further analysis of the microbial communities to link the different pH Solid Alkaline pH Alkaline pH Alkaline pH species to the different VFAs obtained. There are still many un­ waste identified species among the acidogenic bacteria. Systematic studies Slurry- Complex Alkaline pH Alkaline pH of microbial communities in an acidogenic reactor will help to better like carbohydrates: waste alkaline pH know the bacteria responsible for the production of each VFA as well Simple as their metabolic interactions. carbohydrates: - Study of the specific organic loading rate (OLRs) as another param­ neutral pH eter that can affect both VFA production yield and VFA composition. Liquid Complex Complex Mesophilic waste carbohydrates: proteins: conditions: Unless in some studies of solid waste fermentation, where the alkaline pH alkaline pH alkaline pH parameter inoculum-to-substrate ratio is regarded, the amount of Simple Simple Thermophilic inoculum present in the reactor is rarely considered. We found little carbohydrates: proteins: conditions: data that allow us to analyze this parameter. On some occasions, neutral pH neutral pH neutral pH authors only detail the origin of the inoculum, but they do not specify Temperature Solid or Mesophilic Thermophilic ND slurry- conditions conditions the amount added or its concentration in the acidogenic fermenta­ like tion reactor. We consider that the amount of available substrate for a waste certain amount of biomass might affect the VFA yield or composi­ Liquid More research is More research Mesophilic tion. OLRs is a parameter that could serve to analyze this effect. waste needed is needed conditions OLRv Solid, Simple substrates: high OLRv to inhibit methanogenesis slurry- Complex substrates: maximum OLRv restricted by Declaration of Competing Interest like or hydrolysis liquid The authors declare that they have no known competing financial waste interests or personal relationships that could have appeared to influence 13 A. Vázquez-Fernández et al. 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