Volatile Fatty Acids Production by Acidogenic Fermentation of Wastewater: A Bibliometric Analysis

Abstract

This study presents a bibliometric analysis of the scientific literature on volatile fatty acids (VFA) production from wastewater fermentation published from 1981 to 21 June 2021. A total of 618 papers obtained from the Scopus database were analyzed using VOSviewer 1.6.16 software. According to the results, this topic has been capturing the attention of researchers over the years, but with different research approaches, including optimization of anaerobic digestion in two-stage reactors, biological removal of nutrients from wastewater, energy production in bioelectrochemical systems, and recovery of VFA as value-added intermediate products to be used as inputs in a variety of industries. In addition, the bibliometric networks obtained from the authors’ keyword frequency showed that wastewater treatment by using fermentation to obtain VFA as a value-added by-product is an emerging topic that undoubtedly requires further research and collaboration between scientific institutions. In this regard, different types of wastewater have been used as a substrate for acidogenic fermentation; however, and based on the results, the production of VFA from cassava processing wastewater is seen as one of the emerging issues of this field. Finally, evaluating the effect of operating conditions on the fermentation process, such as pH, hydraulic retention time, organic loading rate, temperature, inoculum and substrate concentration, independent of the final application of the VFA produced, is a relevant aspect for bioprocess optimization and implementation on a large scale.

1. Introduction

Recently, wastewater management has become increasingly one of the most critical problems faced by society. Its inadequate disposal generates a negative environmental impact on the water resource. Those disposals affect the different economic, social and ecological uses of water due to the decrease in dissolved oxygen and its impact on the species of the aquatic environment, the growth of microbial species and eutrophication that reduces the water mirror [1].

Wastewater can be treated through physicochemical and biological processes. Considering technical and economic aspects, physicochemical processes are preferably applied in the treatment of water with inorganic contaminants, non-biodegradable organic matter or compounds toxic to microorganisms, while biological processes are used when the main contaminants are biodegradable organic compounds and inorganic anions are present, such as nitrates, nitrites, sulfates, sulfides and phosphates [2]. To carry out biological treatment there are a variety of aerobic and anaerobic processes, depending on the presence or absence of oxygen as an electron acceptor, respectively.

Anaerobic digestion (AD), a process that involves a series of reactions (including hydrolysis, acidogenesis, acetogenesis and methanogenesis stages), was initially established to sludge stabilization, and later found an area of interest in wastewater treatment, especially in wastewaters with high organic matter content. However, today AD is considered, in addition to a technology for wastewater and waste treatment, a technology for resource recovery [3].

According to the United Nations World Water Development Report (2017), there is growing impetus for a paradigm shift in wastewater treatment as a component of a circular economy. In wastewater management, trends point to greater water reuse and resource recovery. In addition, alternative production chains involving the use of renewable resources are needed to reduce dependence on oil and associated environmental impacts [4,5].

Although methane is considered the main product of AD, studies confirm that some intermediate products have a greater added value and variety of applications in chemical and biological processes, as is the case with hydrogen and carboxylic acids [5] generated in the acidogenic stage of AD (also known as Acidogenic Fermentation—AF).

In AF, simple organic compounds (monosaccharides, long-chain fatty acids, glycerol and amino acids) generated by the hydrolysis of complex particulate material are metabolized inside the cells through fermentative metabolism, producing organic acids, mainly short-chain fatty acids (C1 to C5), commonly called Volatile Fatty Acids or VFA, such as acetic, lactic, butyric, propionic and formic acids. In addition, alcohols (ethanol, butanol, methanol, glycerol, isopropanol, propanol), ketones (acetone), carbon dioxide (CO₂), hydrogen (H₂), ammonia (NH₃, NH₄⁺), hydrogen sulfide and new bacterial cells are produced [6,7]. AF is carried out by a large and diverse group of acidogenic bacteria through a series of oxidation-reduction reactions, whose activity depends on changes in environmental and operational conditions [8].

To give an example of their applications, VFA have high potential as renewable carbon sources and are widely used for biological nutrient removal in the pharmaceutical, food and chemical industries, and as feedstock for products such as biogas, biodiesel, bioplastic and biohydrogen [9].

The production of VFA from AF of wastewater has been studied for several decades with different research approaches, which is reflected in a considerable volume of scientific publications. Approximately 3% of the indexed records are reviews on the subject, and most refer to a specific focus (biodegradable polymers and hydrogen production are two examples) [10,11,12]. Furthermore, and to the best of our knowledge, the available reviews do not make use of bibliometric analysis to analyze and follow up scientific publications on this topic, a tool that has been widely used for mapping a large volume of scientific studies.

Therefore, this work aims to identify and analyze the studies of the production of VFA from AF of wastewater by a bibliometric analysis of the world scientific literature published from 1981 to 21 June 2021. The work also includes the identifying, description and analysis of critical process variables that are susceptible to optimization and/or modeling to obtain a higher yield of VFA as a value-added product.

2. Materials and Methods

Data for the bibliometric analysis was obtained from the Scopus database on 21 June 2021. To address all articles related to VFA and wastewater fermentation, the following search equation with its respective Boolean operators was designed: “volatile fatty acid” AND wastewater AND fermentation. This search equation was used in the “article title, abstract and keywords” field (see Ramírez-Malule et al. [13] and Ramírez-Malule et al. [14] for further details of bibliometric analysis approach/methodology), and was adopted after performing the search with three preliminary equations, observing a greater number of records and verifying that key documents on the topic of interest previously reviewed were found within the results.

The information retrieved from the Scopus database was not limited in terms of type of document and corresponded to citation information, bibliographic information, abstract, keywords and funding details. Additionally, during the search the data was further refined (i.e., timespan) before errors such as duplicates and erroneous entries were eliminated. All this information was used for the analysis of bibliometric indicators, and for the elaboration and visualization of bibliometric networks based on co-authorship between countries and the author’s keyword co-occurrence by the VOSviewer 1.6.16 software [15]. One and nine were established as the minimum number of documents from a country and the minimum number of occurrences of a keyword for each type of analysis, respectively. The keywords were exported to Microsoft Excel® to adjust the thesaurus.

In addition, the titles and abstracts of each of the records were reviewed, and many of them (articles) were read in full.

Figure 1 shows a scheme of the methodology used.

3. Results and Discussion

3.1. Worldwide Scientific Production Related to VFA and Wastewater Fermentation

The search process yielded 618 publications, of which 85.6%, 9.7% and 2.9% were research articles, conference papers and reviews, respectively. The remaining 1.8% represented book chapters, conference reviews and short surveys.

Figure 2 shows the evolution in the number of papers related to VFA and wastewater fermentation published annually from 1981 to 21 June 2021, the journals that published them, and the countries that conducted the research.

Prior to 1997, the number of publications on the topic of interest in journals indexed in Scopus was low. Annually, the number of documents did not exceed four and the number of publications represented only 4.0% of the total worldwide scientific production identified for the period evaluated.

From 1997 onward, the overall production of scientific publications on this topic reflects a slight growth compared to the period 1981–1996; however, this has not been continuous, as short periods with marked decreases (e.g., 2001–2004, 2011–2013, 2014–2018) indicate.

Publications in scientific journals related to the topic of interest became more relevant in the scientific community as of 2001, increasing from 7 journals and 11 countries to 36 and 31, respectively, in 2020.

The number of publications increased in the last two decades due to various reasons. There has been a shift in awareness regarding the removal of nutrients from wastewater as an important measure to preserve the quality of water bodies receiving effluent from treatment systems [16,17]. In addition, the term “green biorefinery” was introduced in 1997 and major research and development contributions in the field of biorefinery systems were notable in Europe [18].

Likewise, resource maintenance and management were key policy areas within the sustainable development approved in Agenda 21 as a program of action for the 21st century. This required a search for novel solutions to reduce the rapid consumption of non-renewable fossil resources, demanding new research and development approaches that would redirect energy and material production towards biological raw materials as the main source [18]. For Ahmed et al. [19], the production of fuels and chemicals by biomass processing has become a worldwide practice to expand energy resources and reduce global warming, because, due to its characteristics, biomass is considered a renewable and carbon-neutral natural source.

Regarding the most cited articles on VFA and wastewater fermentation, the top 10 is available as Supplementary Material Table S1. This file shows the title of the article, the authors, the journal, the year of publication, the number of citations, the number of citations per year and some comments related to the research.

3.2. Most Productive Countries and Research Institutions Worldwide in the Scientific Literature on VFA and Wastewater Fermentation

Studies on VFA production and wastewater fermentation have been carried out in Asia (395 papers), Europe (245 papers), North America (98 papers), Oceania (37 papers), Africa (20 papers) and South America (15 papers). China is the leader with 202 papers, equivalent to 32.7% of the world’s scientific production for the period evaluated (Figure 3), followed by the United States (9.9%), India (7.1%) and Australia (5.3%).

Of the 52 countries reporting publications, the majority are European. Major research and development contributions in biorefinery systems were initially notable in Europe, with a high degree of contribution from Germany [20].

For its part, the U.S. President and Congress pushed for significant industrial developments for the first time in 2000 [20]. In the United States, in 2002, the Biomass Technical Advisory Committee published the work plan “Biomass technology in the United States”, in which research, development and construction of demonstration biorefineries were established as necessary, as reviewed by Kamm and Kamm [18]. China, which had the largest number of records, began publishing studies on this subject in 2001.

In the top 25 countries, Brazil is the only South American country publishing in this area of knowledge (ranking 22nd with 10 publications). Prior to 1997, publications were related to the optimization of the primary sludge fermentation process to maximize VFA production, essential for biological phosphorus removal. The most recent publications (after 2008) have focused on process optimization to produce value-added compounds. Brazil is no stranger to the global research interest in wastewater treatment, which in recent decades has been marked by the convergence of two areas: treatment and recycling, and reuse as a renewable source of energy and value-added products [21].

For the period studied, other Latin American countries such as Colombia and Chile are only in 34th and 43rd position with 4 and 1 publications, respectively. This shows the need to allocate more funding sources and establish effective policies that promote research and development to solve problems that can be addressed with technologies that have a more sustainable approach.

Regarding the topic of co-authorship, Figure 4 shows a collaboration network among different countries for scientific production related to VFA and wastewater fermentation. A total of 52 countries complied with the threshold of having at least one publication on the topic studied, but only 50 are linked in the network map. In this Figure, each node represents a country, the size of the circle represents the amount of publications and the width of the line linking them is proportional to the strength of the relationship between them [19,20]. China, in addition to being the most productive country, represented the place with the closest collaboration with other countries. This may be related to the country’s research capabilities and the availability of high-level research institutes, which becomes an attraction for foreign researchers to form partnerships.

Australia and the United States are also countries with a robust collaboration network.

If the analysis focuses on scientific production by research institutions, the predominance of Chinese institutions in the top 10 becomes evident, the exceptions being the Indian Institute of Chemical Technology (India) and the University of Queensland (Australia), which occupy the fourth and sixth positions, respectively (

Table 1. Top 10 most productive research institutions worldwide on the topic of VFA production from wastewater fermentation.

Position	Research Institution	Number of Publications
1	Tongji University	31
2	Harbin Institute of Technology	26
3	Ministry of Education China	25
4	Indian Institute of Chemical Technology	24
5	Chinese Academy of Sciences	19
6	The University of Queensland	18
7	Beijing University of Technology	16
8	The University of Hong Kong	14
9	Tsinghua University	14
10	University of Science and Technology of China	13
11	Jiangnan University	13

). It is worth noting that, although the United States has the second highest number of publications worldwide, none of its institutions are among the top 10, which is occupied by highly productive institutions.

3.3. Main Journals Publishing on VFA and Wastewater Fermentation

According to the documents reviewed, most of the publications have addressed the topic of interest from an environmental and technological perspective. Therefore, the journals with the highest number of publications are of environmental interest.

Figure 5a shows the top 10 scientific journals with the highest number of publications on VFA and wastewater fermentation, in which Bioresource Technology, Water Research, Water Science and Technology, International Journal of Hydrogen Energy and Water Environment Research stand out. These journals account for 40.3% of all the papers retrieved for the period evaluated (1981–21 June 2021). Figure 5b shows the evolution of the number of articles published per year in the five most important journals. This is consistent with the growth that these journals have experienced over the years due to increases in the frequency of publication.

The journal Bioresource Technology ranks first, with 102 publications since 1997. It is an international journal currently publishing on topics related to biomass, biowaste treatment, bioenergy, biotransformation and bioresource systems analysis, as well as technologies associated with conversion or production. The journals that occupy the second, third and fifth positions publish mainly on topics related to water cycle science and technology, water quality and management, wastewater and stormwater. The International Journal of Hydrogen Energy, which ranks fourth with publications reported since 2007, prioritizes aspects of hydrogen energy, such as production, storage, transmission, utilization, supporting technologies, environmental impact and economic and international aspects of hydrogen.

3.4. Main Research Approaches on VFA Production

3.4.1. Subject Areas of VFA Production

The studies were mainly related to the area of Environmental Sciences followed by the areas of Chemical Engineering, Energy, Biochemistry, Genetics and Molecular Biology, Engineering, and Immunology and Microbiology, where the number of publications corresponded to 71.0, 35.3, 30.7, 15.5, 11.8 and 11.2%, respectively (Figure 6).

It was to be expected that environmental sciences would take the first place because since 1981 the fermentation of wastewater for VFA production has had various research approaches in this area. The production of VFA to optimize AD for biogas production in two-stage reactors—for the biological removal of nutrients in wastewater—to be transformed into energy in bio-electrochemical systems and to be recovered as value-added intermediate products are some examples of those approaches.

However, most of the publications indexed in environmental sciences were also indexed in the areas of chemical engineering and energy. There is a strong connection between these areas, as the different environmental research approaches identified involve different chemical and biological transformations. The connection is mainly due to those investigations where the production of hydrogen, biogas, methane, PHA and the use of microbial fuel cells were part of the main objective.

3.4.2. Bibliometric Networks of VFA and Wastewater Fermentation Studies

The bibliometric network generated with the information exported from Scopus is shown in Figure 7. As a result, 1557 keywords were identified, of which 37 had a minimum number of nine occurrences; that is, each of these terms appears at least nine times (frequency). Five clusters were formed, consisting of 37 nodes.

The network analysis allows us to identify different thematic groups related to the field of knowledge of interest. In addition to the keywords used in the search, the terms with the highest frequency were biohydrogen, anaerobic digestion, anaerobic fermentation, acidogenic fermentation, waste activated sludge and enhanced biological phosphorus removal, representing the largest nodes in the network (Figure 7). Moreover, Figure 8 exhibits a word cloud of the abstracts of the articles used in this study, which supports the findings presented in Figure 7.

Cluster No. 1 (red color) is formed of the biohydrogen, anaerobic digestion, dark fermentation, anaerobic process, biogas, methane, mixed culture, cheese whey and thermophilic nodes (Figure 7). This cluster represents research oriented towards AD and dark fermentation processes for the transformation of organic matter into by-products such as biogas/methane and hydrogen, respectively.

AD is a process that was applied almost exclusively to the stabilization of sludge from treatment plants; however, in the 1980s AD found an important area of interest in the treatment of wastewater, especially wastewaters with high carbonaceous organic matter content such as those generated in agri-food industries [3]. This is consistent with the dates of the oldest publications indexed in Scopus used in this bibliometric analysis.

The aim of those investigations were mainly to decrease the pollutant load of the wastewater to optimize biogas production and/or to evaluate the efficiency of different reactor configurations [22,23]. Athanasopoulos et al. [24] evaluated at pilot scale the applicability of an upflow fixed-bed anaerobic reactor for the treatment of sulfite-rich wastewater from the raisin finishing process, obtaining chemical oxygen demand (COD) removals between 90 and 95%. The authors observed that, with small organic loads, VFA concentrations in the digester were less than 0.5 g/L and the biogas had 63% v/v CH₄.

Additionally, hydrogen production from petroleum compounds or fossil fuels involves the release of greenhouse gases (e.g., CO₂, CH₄) into the environment, which has made it necessary to investigate a renewable and environmentally friendly route for its production. Dark fermentation is considered one of the most appropriate methods for hydrogen production [25], which involves anaerobic microorganisms that grow in the dark and use carbohydrate-rich substrates (wastewater, solid waste, biomass).

Wu and Lin [26] evaluated the hydrogen production potential of molasses wastewater, with waste activated sludge from a municipal wastewater treatment plant (WWTP) as inoculum and a fermentation time of 104 h, to optimize the substrate concentration (10 to 160 g COD/L) and pH (4 to 8). The authors found that hydrogen production depended on the two parameters evaluated (substrate concentration and pH). In this study, hydrogen contents in the digestate gas of 49–58% and 20–27% (v/v) were observed for concentrations of 10–60 and 80–160 g COD/L, respectively, indicating some inhibition at higher substrate concentrations. In addition, hydrogen production was accompanied by VFA and solvents, among which butyric acid was present in the highest proportions, and the fraction (butyric acid) increased gradually from 7.8 to 17 g COD/L (41–62.5%) when the substrate concentration increased from 10 to 160 g COD/L. A substrate concentration of 40 g COD/L and a pH close to 6.0–7.0 were considered optimal for molasses wastewater fermentation.

Cluster No. 2 (green color) consists of the nodes of anaerobic fermentation, activated sludge withdrawal, denitrification, treatment plant sludge, acidification, hydrolysis, carbon source, food waste and short-chain fatty acids. This cluster represents research focused on the first stages of AD (hydrolysis and acidogenesis) to improve the production of VFA as a carbon source for the optimization of methanogenesis, the increase of AD stability [27] and the removal of nitrogen from wastewater or sludge fermentation liquid from a WWTP.

According to Dinopoulou et al. [27], the separation of the acidogenic and methanogenic phases represented advantages over conventional AD:

Increased reactor stability by avoiding methane overload because of adequate control of the acidogenic phase.

Maintenance of appropriate densities of acid and methane forming bacteria in separate reactors to maximize acidification and methanogenesis rates under optimal operating conditions.

The acidification reactor acts as a buffer system with wastewater of varying composition and helps to remove toxic compounds from methanogenic archaea.

The acidification reactor provides a constant substrate for methanogenic archaea, which adapt slowly to varying substrate content and composition.

Undoubtedly, the two-stage AD process is favorable because the conditions in the two reactors can be optimized separately for acidogenic and methanogenic microorganisms. However, the need for an additional large reactor for wastewater acidification can make the two-stage process more costly than the single-stage process [28].

Two-stage AD systems have been widely used for solid waste treatment [26,28,29], mainly with food waste as substrate [26,29]. Wang et al. [30] investigated a hybrid two-stage batch mode system for food waste digestion. The authors used two reactors for the acidification stage (inoculated with sludge from a WWTP anaerobic digester) and a methanogenic reactor (UASB type) (inoculated with sludge adapted to synthetic wastewater containing VFA). With each acidification reactor, one cycle (A and B) was carried out at 35 °C and times of 16 and 10 days, respectively. The results showed that the two-phase hybrid system is effective and efficient for the conversion of biodegradable food waste into biogas. The authors reported treatment efficiencies of 77–79% total organic carbon removal, 57–60% volatile solids removal and 79–80% total of COD reduction. Approximately, 99% of the total biogas yield came from the methanogenic phase with a high CH₄ content (68–70%).

On the other hand, WWTPs face the problem of the scarcity of carbon sources for denitrification, using as a carbon source the VFA obtained from the AF of the sludge (primary sludge and waste activated sludge). However, the AF of activated sludge withdrawal can present drawbacks related to the separation of VFA from nitrogen and phosphate, and low production efficiency, for which pretreatment and alkaline fermentation are effective alternative solutions [31].

Cluster No. 3 (blue color), formed by the nodes of fermentation, enhanced biological phosphorus removal, biological nutrient removal, activated sludge, pH, pre-fermentation, modeling and sequential batch reactor, includes research focused on the improvement of conventional treatment processes (e.g., activated sludge, UASB) to optimize the biological removal of nutrients (mainly phosphorus).

Enhanced biological phosphorus removal in the activated sludge process is a mechanism where the organisms present in the activated process remove a greater mass of phosphorus from the wastewater than they need for their basic metabolic purposes. For this removal to take place in the activated sludge process, the mixture must be subjected to anaerobic conditions (free of dissolved oxygen and nitrate/nitrite) and the influent from the anaerobic zone must have a certain concentration of simple carbonaceous substrates, with VFA such as acetate and propionate being the preferred substrates [32].

The production of VFA can take place in a separate fermenter (pre-fermentation) or directly in the anaerobic reactor of the treatment process [33]. Comeau et al. [34] demonstrated at the laboratory scale its feasibility and determined the maximum phosphorus removal potential of wastewater from a phosphorus-rich cheese industry (60 to 80 mg P/L), which was being treated in a plant composed of an anaerobic equalization tank, followed by a UASB reactor and aerated lagoons. In the laboratory, they used the effluent from the equalization tank and the UASB reactor in a sequential batch reactor. The authors observed that nutrient removal in the UASB reactor effluent was difficult due to the low VFA concentration, while it improved with the equalization tank effluent (higher VFA concentration), generating an effluent with a concentration of 5 to 10 mg P/L.

Several types of research have been directed to the kinetic and mathematical modeling of pre-fermenters or of the acidogenic stage [31,33,34] with the final aim of favoring the activity of phosphate accumulating organisms.

Cluster No. 4 (yellow color) consists of the nodes of acidogenic fermentation, microbial community, polyhydroxyalkanoates, resource recovery, sludge fermentation and primary sludge. This cluster represents the research where AF emerges as an interesting process in which, in addition to treating organic matter, favoring the biological removal of nutrients and mitigating the environmental impact, it produces chemical compounds that are currently generated by the petrochemical industry and have diverse applications in chemical and biological processes [35,36].

In recent years, there has been increasing research interest in the recovery of VFA from solid waste [37] and wastewater [38,39]. Research has aimed to evaluate and optimize operational parameters such as pH, temperature, organic loading rate, substrate concentration, hydraulic retention time and inoculum [7,38,40,41], through laboratory-scale experiments in continuous [38,42] and batch [43] reactors. VFA production presents additional challenges because the products are soluble and there is no automatic phase separation [44]. Therefore, the production of medium-chain acids, such as caproic acid, perhaps represents greater advantages than VFA because, being less soluble, their separation can be easier [45].

Other work has focused on the production of polyhydroxyalkanoates (PHA), which are bio-based and biodegradable polymers that are usually produced from expensive sugars or VFA; these biopolymers are considered an alternative for the replacement of traditional plastics (petroleum polymers) [45,46]. Elain et al. [46] evaluated two wastewaters rich in vegetable protein (legume processing water) and reduced sugars (fruit processing water) as a growth medium for PHA synthesis with a single marine bacterial species (Halomonas i4786). The authors demonstrated that agro-industry by-products offer an effective alternative to an expensive conventional culture medium (marine broth) to produce PHA. In that study, biomass was produced from the media studied to obtain PHA productivities of 1.6 and 1.8 g/L in 72 h for legume and fruit processing water, respectively. The results showed that these alternative media produce relative PHA contents (in relation to the dry mass of the cells) above 50% and reach almost 80% in the case of fruit processing water. For their part, Karaca et al. [47] investigated the optimization of PHA production from VFA produced in the fermentation of dairy wastewater and food waste, using activated sludge acclimated with domestic wastewater and VFA supplements under different conditions (feed–starvation regime and anaerobic–aerobic–anoxic operation). However, the authors demonstrated that the PHA accumulation potential of acclimatized activated sludge was mainly dependent on the VFA components available for polymer storage, rather than on the acclimatization conditions. In this case, the researchers obtained maximum PHA contents of 67.3 and 4.5% using VFA obtained from the acidification of solid food waste and dairy wastewater, respectively.

Finally, cluster No. 5 (purple color) includes as nodes two of the key terms used to obtain the information (volatile fatty acids and wastewater) and three keywords directly related to them (acetate, chemical oxygen demand and dairy industry wastewater). However, this cluster is small and does not clearly define a research approach related to the topic under study.

Figure 9 shows the areas with the most and least development, as well as the thematic areas where future scientific projects are most needed. The recovery of resources such as VFA (see the yellow nodes in Figure 9) from various substrates is an unconsolidated/emergent topic which requires further laboratory- and pilot-scale research to provide inputs for projecting this sustainable technology on a larger scale. It is perceived that the production of compounds from other substrates also rich in biodegradable organic matter (see item 3.4.3), to be used as a carbon source in subsequent processes and the evaluation of the microbial community involved in the AF process, are also emerging topics.

Therefore, this bibliometric analysis delves into the review of scientific literature related to the influence of the main operational parameters on the production of VFA from the AF of wastewater, since it is an indispensable aspect for understanding the phenomena involved in the process.

3.4.3. Operational Parameters of VFA Production by Wastewater Fermentation

Wastewater (real and synthetic) derived from agricultural or food sources, or more complex effluents such as municipal or industrial wastewater, have been used for VFA production through AF in laboratory and pilot scale experiments. Some examples include dairy wastewater [48,49,50,51], cheese whey [38,52], slaughterhouse wastewater [37], paper mill wastewater [37,38], winery wastewater [37], olive oil mill wastewater [53], saline cooked mussel processing wastewater [54], synthetic soft drink wastewater [55], wood mill wastewater [56], swine wastewater [28,57,58], sucrose-rich wastewater [59,60], municipal wastewater [61] and cassava processing wastewater [39,40].

The concentration, composition and efficiency of VFA production from the AF process depends on wastewater characteristics, reactor configuration and operational parameters such as pH, hydraulic retention time, temperature, organic loading rate and inoculum, among others [7,23,47,48]. Operational conditions largely determine which thermodynamically viable reactions and microorganisms can grow most efficiently [37,49], allowing researchers to exert some control over the product spectrum. This section describes the operating parameters that are commonly evaluated in studies related to the production of VFA through wastewater fermentation.

• Effect of pH on VFA production by AF

The pH in the reactor is a key factor in producing carboxylic acids as most hydrolytic and fermentative bacteria cannot survive in extremely acidic or alkaline environments [62]. pH value affects the growth of organisms, which influences the product spectrum, mainly of acetic, propionic and butyric acids, in this process [7,50].

The optimum pH to produce VFA can range from 5.25 to 11.0; however, the specific value will depend mainly on the type and characteristics of the substrate [62] and the species and enzymes involved in the process [44]. Gallert and Winter [63] reported that hydrolysis of proteins and lipids requires a neutral or slightly alkaline pH, while for carbohydrates a slightly acidic pH is desirable. An increase in the degree of hydrolysis is reflected in a higher concentration of total VFA. Niz et al. [39], who used wastewater from cassava processing, concluded that neither sludge adaptation nor heat treatment to eliminate methanogenic archaea activity was as relevant as pH in improving VFA production.

Table 2. Optimal pH for obtaining carboxylic acids from different types of wastewater.

Type of Wastewater	Substrate Concentration (mgCOD /L)	Optimum pH	Total Acids (mg/L)	Acid Composition	Degree of Acidification	Reference
Meat extract	2920	7.0	NA	Acetic acid (39%) Propionic acid (35%)	40%	[27]
Cheese whey	4590	5.25–5.5	2270	Acetic acid (43%) Butyric acid (42%) Propionic acid (15%)	83%	[38]
Pulp and paper	7740	5.5–6.0	3960	Butyric acid (78%) Acetic acid (9%) Propionic acid (6%) Others (7%)	76%	[38]
Cassava processing	6040 to 7600	5.50–6.09	5080 to 5760	Butyric acid (87%)	84.10%	[39]
Cassava processing	8865	5.9	4000	Butyric acid (42%) Acetic acid (35%) Propionic acid (20%)	63%	[40]
Cassava processing	50,000 (glucose)	6.5	40,000 (lactic acid)	Lactic acid Acetic acid Propionic acid	NA Note: the paper was oriented only to the production of lactic acid.	[42]
Dairy wastewater	111,450 ± 1937	8	20,585	Acetic acid (17.5%) Butyric acid (61.1%) Propionic acid (9.3%) Valeric acid (12.0%)	NA	[64]
Cheese processing	20,000	5.0	3688	Propionic acid (24%) Acetic acid (21%) Valeric acid (20%) Butyric acid (17%)	93%	[65]
Cassava Processing	1000–25,440	5.5–6.0	200 to 4520	Lactic Acid (82%) Butyric Acid (6%) Acetic Acid (5%) Small concentrations of tartaric, succinic, propionic, iso-butyric and iso-valeric acids were detected.	NA	[66]
Cassava vinasse	36,000	8.0	24,000	Butyric acid (40%) Acetic acid (36.5%) Propionic acid (20%)	71%	[67]
Starch	19,400	8.0	8600	Acetic acid (68.3%) Propionic acid (15.5%) Butyric acid (13.8%)	68%	[67]
Agro-industrial (oil mill, cheese whey and cow manure slurry—55:40:5 v/v/v)	95,000	6.5	13,430	Butyric acid Propionic acid Acetic acid Ethanol Lactic acid	NA	[68]
Synthetic wastewater (with glucose) DQO:N:P—300:5:1	4000	6.0	2139.11 as mgHAc/L	Acetic acid (49.88%) Butyric acid Propionic acid	56.64%	[69]

NA: Information not available.

lists several works showing the optimum pH for the production of carboxylic acids from different types of wastewater.

From the studies reviewed, it is perceived that, when wastewater is used as substrate, the optimum pH can vary between 5 and 8 (see Table 2). However, its specific value must be determined for each case since it depends on the substrate characteristics (e.g., substrate concentration) and possibly on other operational parameters (e.g., hydraulic retention time). The optimum pH range is generally very tight. Bengtsson et al. [38] identified an optimum pH for cheese whey wastewater of 5.25–5.5 and for pulp and paper industry effluent of 5.5–6.0, with acidification degrees of 0.83 and 0.76, respectively. For wastewater from cassava processing, VFA production generally happens under acidic conditions, with optimum values ranging from 5.0 to 6.5.

A considerable amount of research has been conducted on the optimal pH range to produce carboxylic acids from fermentation processes. The results are often diverse due to differences in the type of substrate, inoculum and other operating conditions adopted. In addition, the aim of the studies must be taken into account because, while a significant number of works are oriented towards identifying the pH necessary to optimize the total production of carboxylic acids, others seek the optimization of a specific acid. Da Silva et al. [42] used wastewater from cassava processing to optimize lactic acid production, while the work of Dareioti et al. [68], despite having evaluated VFA production, took optimize hydrogen production as its main objective.

The pH value influences the composition of the carboxylic acids. Bengtsson et al. [38] observed—for cheese whey wastewater—that the acid composition was stable between pH 3.5 and 5.25. When the pH value increased from 5.25 to 6, the amount of acetate and butyrate decreased and the amount of propionate was favored (51:19:24 to 31:41:10 acetic:propionic:butyric). In the case of pulp and paper industry effluent, butyrate and propionate increased with pH in the range of 4.9–6.0, varying the VFA composition from 42:13:18 to 22:23:33 in % acetic:propionic:butyric. Dareioti et al. [68] observed an increase in propionic acid concentration from 0.09 to 4.02 g/L when the pH varied from 4.5 to 7.5. Wu and Lin [26], who evaluated hydrogen production from molasses wastewater, observed that a pH between 4 and 5 favored acetic acid production (50.6–55.4%), while a pH between 6 and 8 increased butyric acid production (47.2–58.7%). According to Guo et al. [70] metabolic pathways involving the production of acetic and butyric acid are favored at pH ranging from 4.5 to 6.0, while a neutral or higher pH favors ethanol and propionic acid production.

• Effect of Hydraulic Retention Time on VFA production by AF

The hydraulic retention time (HRT) represents the average time that the substrate remains in the reactor. It can vary between 0.5 to 10 days depending mainly on the characteristics and type of substrate as this considerably influences the rate of hydrolysis of particulate organic matter [62]. The HRT should be sufficient to allow the solubilization of complex organic matter and favor subsequent AF. A very long HRT, on the other hand, will reduce the amount of substrate available per day, making it easier for methanogenic archaea to develop at an appropriate pH [71].

Dinopoulou et al. [27] observed a tendency for the degree of acidification to vary proportionally with HRT. However, the authors state that there is a critical time that may vary slightly with substrate concentration and under which the degree of acidification drops sharply. Of the parameters evaluated (HRT, substrate concentration and organic loading rate), the authors identified HRT as the most statistically important variable for predicting the degree of acidification and indicated the need for long HRT, even for less concentrated substrates. Fang and Yu [50] used dairy wastewater and observed that VFA production nearly doubled when HRT increased from 4 to 12 h, but when it varied from 16 to 24 h the production only increased by 4 to 5%. For the authors, this behavior may be due to the complexity of the wastewater (composition and concentration) since more than 70% of the glucose, starch or other easily biodegradable carbohydrates could be acidified in less than 12 h.

Ben et al. [72] evaluated the possibility of producing bioplastics from wood mill wastewater. The AF tests were performed in a continuous stirred tank reactor (CSTR) at pH 5.5, 30 °C and HRT of 1 and 1.5 d, observing that the concentration of VFA in the effluent was similar for the two HRT tested, with acidification degrees of 37 and 42%, respectively. However, the VFA composition was different, with an increase in the concentration of long-chain VFA at higher HRT evaluated.

Khan et al. [73] investigated VFA production from low-strength wastewater at different HRT and organic loading rates in a continuous anaerobic membrane bioreactor using glucose as carbon source, no inhibition of methanogenesis, pH of 7 ± 0.1, constant influent COD of 550 mg/L and organic loading rate of 68.75 mg COD/(L.h). The authors evaluated HRT of 48, 24, 18, 12, 8 and 6 h. The results showed that the overall VFA yield in the reactor (mg VFA/mg COD in the feed) changed from 48 to 24 h, registering a rapid increase in the yield (13.39 ± 1.21 to 32.88 ± 2.56%). The influent carbon content increased at the shortest HRT and VFA yield increased to 48.20 ± 1.21% at 8 h. However, a further decrease in HRT (6 h) caused a drop in the overall VFA yield to 42.32 ± 2.32%, indicating that this was an insufficient contact time between the microorganisms and the substrate.

Likewise, Lv et al. [69] varied the HRT between 12 and 2 h for VFA production from synthetic wastewater with glucose as sole carbon source at pH 6, 35 ± 1 °C and 4000 mg COD/L in a CSTR. The results showed that, when the HRT gradually decreased from 12 to 6 h, the VFA concentration progressively increased and peaked at HRT of 6 h with value about 2540 mg HAc/L and an acidification degree of 68.28%. Consistently, when HRT was reduced to 4 h, the VFA concentration decreased abruptly and achieved the minimum of 1892.90 mg HAc/L.

Most studies on AF of wastewater were conducted in batch bioreactors [7,39,40]. Therefore, there is limited information in the scientific literature evaluating the effect of HRT on the production of carboxylic acids, mainly if wastewater from cassava processing is treated. However, some studies that did not assess the impact of HRT reported the fermentation time in which the maximum concentration of organic acids was observed. Da Silva et al. [42] aimed to evaluate the effect of initial pH on the lactic acid production from cassava processing wastewater in 48 h experiments. The authors indicated a rapid decrease in pH to 4.5 within 6 h after starting fermentation, but after 24 h pH remained stable between 4.0 and 4.5 until the end of the process. In this case, the pH value can be considered an indirect indicator of the amount of organic acids present in the medium.

Controlling HRT can favor specific microbial populations. Because other operating parameters (for example, substrate concentration, pH and temperature) also affect mixed cultures, the HRT will affect not only the total concentration of organic acids but also their composition, especially when using pure cultures [44].

Demirel and Yenigun [48] evaluated the production and distribution of VFA from dairy wastewater at the laboratory scale in a continuous-flow, complete-mix acidogenic reactor coupled to a conventional gravity settling tank and a continuous recycling system. The authors varied HRT between 24 and 12 h, and up to an organic loading rate of 9.3 kg COD/(m³.d), without pH control and at 35 ± 1 °C. The results showed a gradual increase in VFA production, proportional to the organic loading rate, with decreasing HRT. The highest degree of acidification and VFA production rate was 56% and 3.1 g/(dm³.d), respectively, at 12 h of HRT. The VFA produced were acetic, propionic, butyric and valeric, with a higher production of propionic acid for the optimum HRT of the evaluated range. However, in the experiments performed by Lv et al. [69], the VFA were mainly composed of acetate, butyrate and propionate. In addition, HRT reduction favored butyrate production and formate retainment.

• Effect of Organic Loading Rate on VFA production by AF

Organic loading rate (OLR) refers to the amount of substrate fed to the bioreactor per day per unit volume in terms of total solids (TS), volatile solids (VS) or COD [71]. The ORL combines the effect of substrate concentration and HRT, and determines the feed–microorganism ratio [44].

Theoretically, increasing the ORL can generate an increase in the concentration of carboxylic acids; however, studies show that this is not always the case and that the effect of this variable on acid production also depends on the type and characteristics of the substrate [27]. Dinopoulou et al. [27] conducted experiments with meat extract wastewater and constant HRT. They observed a tendency for the degree of acidification to vary inversely with OLR and initial substrate concentration. However, the authors reported that changes in the degree of acidification were not significant in most of the experiments as it remained almost constant for COD loads of 9.4 and 72 g/(L.d).

Azbar et al. [74], in experiments aimed at hydrogen production from the fermentation of cheese whey wastewater under thermophilic conditions (55 °C) and pH 5.5 ± 0.05, evaluated the effect of different ORL (47, 35 and 21 g COD/(L.d)) in a CSTR and constant HRT of 1 d. The authors observed in the effluent a concentration of VFA ranging from 118 to 27,012 mg/L and mainly composed of acetic acid, iso butyric acid, butyric acid, propionic acid, formate and lactate. The most abundant VFA in all OLR tested was lactate, as lactose was converted to lactic acid by mixed culture instead of acetic acid being formed. The second most abundant VFA in the experiments was formate.

Other studies mention the influence of ORL on VFA composition and point out that low values (5 g ST/(L.d)) favor the production of propionic and butyric acids, while high values (16 g ST/(L.d)) promote the production of acetic and valeric acids [75]. Additionally, the VFA composition may depend on how the ORL is adjusted. According to Arslan et al. [44], in the range of 5 to 240 g COD/(L.d) when a high ORL is achieved by increasing the substrate concentration, the carboxylate concentration increases and the product type tends to be reduced. In contrast, when a high ORL is achieved by decreasing the HRT, hydrolysis may be less effective, resulting in a lower concentration of total VFA and/or shifts towards more acetate, because only fast-growing bacteria such as acetate or lactate-producing bacteria are retained in the reactor.

Operating a reactor with a high ORL (11 to 16 g ST/(L.d)) must consider the presence of inhibitory substances in the substrate as it can affect not only methanogenic archaea but also hydrolytic and fermentative bacteria [71].

Khan et al. [73] evaluated ORL of 43.75, 68.75 and 89.38 mg COD/(L.h) adjusting the input COD (350, 550 and 715 mg COD fed, respectively) in wastewater with glucose as carbon source, HRT of 8 h, pH of 7 ± 0.1 and temperature of 22 ± 1 °C. The maximum acetic and propanoic acid concentrations were obtained (1.1845 ± 0.0165 and 0.5160 ± 0.0141 mmol/L, respectively) at 550 mg COD, the concentration at which the highest VFA production yield was also observed. The isobutyric acid concentration was highest (0.3580 ± 0.0407 mmol/L) at 715 mg COD fed, indicating butyric-type fermentation at a higher ORL.

• Effect of Temperature on VFA production by AF

Temperature influences microbial growth and metabolism, and most fermentative bacteria cannot survive in extreme temperatures. Each type of microorganism has an optimal temperature range; therefore, a change in bioreactor temperature can alter the structure of the microbial consortium involved in AF [71].

Reviewing the scientific literature reveals a wide range of outcomes. Dinopoulou et al. [27] evaluated the effect of temperature on the percentage of acidification using meat extract wastewater as a substrate and observed an increase with increasing temperature in the range studied (25–40 °C). For their part, Hasan et al. [40] evaluated the effect of alkalinity and temperature in the same range on VFA production from wastewater of cassava processing and observed a negative effect of temperature on VFA production, indicating higher yields at a lower temperature (30 °C).

Infantes et al. [76] concluded that pH, temperature and concentration of non-dissociated acids (formic, acetic, propionic, lactic, butyric acid) influence the fermentation process (oriented to hydrogen production), as they had a significant effect on cell growth and substrate consumption. The authors observed less biomass growth as temperature increased. At pH of 6, the biomass increased up to 1.4 and 0.9 g/L when the temperature was 26 and 40 °C, respectively, showing a similar behavior at pH 4 and 5, although at these pH values the increase in biomass growth was negligible. Regarding substrate consumption, the effect was more important at pH 4 and temperature values above 33 °C, where glucose was not completely consumed, and biomass growth decreased due to the undissociated acid concentrations reached (50 to 70 mM).

In addition, the concentrations of undissociated acids affected fermentation exponentially, mainly at high temperatures (40 °C), because the higher the temperature the higher the permeability of the cell membrane, and the undissociated acids can pass through it more easily.

According to the review by Lee et al. [62], the effect of temperature on VFA composition appears minor when compared to pH, challenging the widespread idea that microbial composition changes with temperature. The authors raise the concern of whether similar microbial species are present at different temperatures or whether there are different microbial species that produce similar types of VFA, being relevant to examine the microbial community involved in AF at different temperatures. Khan et al. [77] also mentioned that the type of VFA produced does not alter much when the temperature is changed during VFA production.

Kim et al. [28] investigated the influence of HRT and temperature on VFA production in thermophilic acidogenesis of swine wastewater. The authors verified through ANOVA of the response surface methodology that VFA produced by acidogenic bacteria were more affected by HRT than by temperature. However, from the qualitative analysis of the microbial consortium, they observed that the changes were due more to temperature variation than to HRT. The authors deduced that VFA production was affected by the physicochemical properties of the acidogenic microorganisms (specific growth rate or contact time with the substrate), rather than by the change in microbial diversity.

Additionally, Yang et al. [58] conducted experiments to optimize VFA production in a laboratory-scale CSTR from swine wastewater under mesophilic conditions. Initially, the authors evaluated different concentrations of ammonia nitrogen, finding that a concentration lower than 1.2 g/L did not significantly affect the biochemical acidogenic potential of the swine wastewater. Additionally, the authors used a response surface as a statistical methodology, simultaneously varying HRT (1 to 3 d) and temperature (25 to 35 °C), and keeping the substrate concentration at 80,000 mg COD/L for all trials. The results showed optimal physiological conditions for acetic and butyric acid production of 2.4 d of HRT at 34 °C and 2.1 d of HRT at 35 °C, respectively.

• Effect of Inoculum on VFA production by AF

The biomass of acidogenic reactors and anaerobic digesters is rich in acid-producing organisms and methane-producing organisms, requiring pretreatment to prevent methanogenic archaeal activity if this control is not done by managing some operational parameter, e.g., HRT [44]. The most common pretreatment methods are thermal [39,40,59], acid/alkali conditioning and chemical inhibitors [60,61,73]. Pretreatments may not be as cost-effective or technically feasible at the industrial level, but they do allow for increased production of organic acids from a given substrate, if there is a risk that these are consumed for methane production [44].

Amorim et al. [41] evaluated the effect of methanogenesis inhibition on the production of carboxylic acids and hydrogen from cassava processing wastewater and indicated that chemical (with acetylene) and thermal treatment effectively inhibited methanogenesis, avoiding methane production. However, the maximum hydrogen production potential (~563 mL) upon application of the heat treatment was more than twice that of the acetylene treatment (~257 mL). Moreover, butyrate was the main carboxylic acid produced (~3 g/L). Furthermore, Niz et al. [39] investigated, in batch reactors, the effect of inoculum adapted and not adapted to cassava wastewater, collected from the upflow anaerobic reactor of a beverage industry, with and without methanogenic archaea inhibition techniques. The study aimed to improve the production of VFA by fermentation of cassava processing wastewater. The authors observed that in non-adapted sludge an acidification of 84.1 and 66.1% was obtained for thermally inhibited and non-inhibited conditions, respectively, 10% and 45% higher than the acidification degrees obtained in the same conditions with adapted sludge.

Amorim et al. [41] evaluated three types of inoculums obtained from anaerobic sludge: municipal WWTP sludge, bovine rumen and WWTP sludge from the textile industry. The authors observed that the best inoculum was bovine rumen, which produced the highest amount of butyric acid at the lowest substrate concentrations (10 and 20 g O₂/L); however, they observed a decrease after 20 days of operation, as acetic acid became available. This behavior was like that observed by Hasan et al. [40], but after 32 h of operation and using sludge from an anaerobic digester treating swine manure as inoculum. The consumption of hydrogen and subsequent decrease in the partial pressure thermodynamically favored the conversion of butyric acid to acetic acid [41].

• Effect of Substrate Concentration on VFA production by AF

Although the product concentration increases as the substrate concentration in the reactor rises, the increase in VFA concentration is limited when the feed level exceeds 40 g COD/L due to overload or inhibition [44]. However, some studies show that inhibition can occur at lower concentrations. Dinopoulou et al. [27] conducted experiments with influent COD concentrations of 3.15 and 12 g/L and similar HRT and showed that the degree of acidification achieved during the first series (3.15 g/L) was significantly higher than that obtained during the second (12 g/L), evidencing a negative effect at higher initial substrate concentrations.

Da Silva et al. [42] evaluated the effect of initial pH and five glucose concentrations (10, 15.8, 30, 44.2 and 50 g/L) on lactic acid production from cassava processing wastewater. In the experiments with higher initial glucose concentrations, the authors observed a stabilization of fermentation 24 h after the start of the process. Nevertheless, in the experiments with lower glucose concentration, there was a rapid decrease in the first 12 h and it remained stable until the end of the experiment. Statistical analysis showed that only substrate concentration had a significant effect on lactic acid production at the 95% confidence level. High substrate concentrations (50 g/L) favor lactic acid production.

Wu and Lin [26] evaluated hydrogen production from molasses wastewater with concentrations ranging from 10 to 160 g COD/L, at 35 °C, pH 6 and 104 h of fermentation, but during the experiments VFA production was monitored. The results showed butyric acid, acetic acid and ethanol as the dominant liquid fermentation products. The butyric acid fraction gradually increased from 7831 to 17,000 mg COD/L (41.0–62.5%) when substrate concentrations increased from 10 to 160 g COD/L.

The initial substrate concentration can also influence the composition of VFA in the reactor. The number of electrons available per cell and the NADH/NAD⁺ ratio in the reactor increase as the substrate concentration rises, and an excess of electrons and NADH favors reactions in which NADH can be consumed, leading to the formation of more reduced compounds such as ethanol, n-butyrate or n-caproate instead of acetate and propionate [44].

Yu and Fang [78] performed experiments to evaluate the acidogenesis of synthetic dairy wastewater in batch reactors at pH 5.5 and 55 °C, with feed solutions containing 2, 4, 8, 12, 20 and 30 g COD/L. For all concentrations tested, the authors identified acetic, propionic and butyric acids as the main VFA. The concentrations of propionic acid and its time to peak increased with increasing substrate concentration. However, for acetic and butyric acid concentrations a different behavior was perceived. The concentrations of the acids increased rapidly at the beginning, peaked at some point during the experiment, and then decreased in reactors with initial concentrations equal to or greater than 8 g COD/L. A decrease in the degree of acidification from 49.6% at 2 g COD/L to 30.4% at 30 g COD/L was observed in this study, allowing the authors to conclude the significant influence of substrate concentration on AF.

Despite the large amount of research that has been oriented towards evaluating the effect of operational parameters on the production of VFA from wastewater, there is still much diversity among the results. This confirms the need for further research and improved understanding of the phenomena involved in the process, before its application at pilot and real scale, mainly when less explored substrates such as wastewater from cassava processing are used. In addition, it is worth noting that a complete physicochemical characterization, mainly of the organic content, can alleviate efforts to transform wastewater into value-added by-products.

Finally, since several recent studies of VFA production by AF of wastewater from cassava processing were found during the analysis of the information retrieved in this bibliometric analysis, it may be considered an emerging topic in this field.

3.5. Future Perspectives and Challenges

In the 21st century, providing clean, accessible and reliable sources of energy and chemicals has become a priority from both socioeconomic and environmental perspectives [19]. To manage this problem, it has been shown that low-cost renewable resources such as solid waste and wastewater from various sources (e.g., industrial, agricultural, etc.) can be an ideal source of raw material for the production of valuable compounds such as VFA. These are produced synthetically from petrochemical derivatives, a production method supplying about 90% of this market [5,79].

Undoubtedly, technologies normally responsible for eliminating pollutants from wastewater (e.g., cheese whey, swine, dairy, cassava processing, etc.), which have a high biodegradable organic load, should be oriented towards the generation of products to be incorporated into a circular economy. A massive change in current practices is not expected in the short term, but more research focused on improving these technologies with a focus on recovery rather than compound removal is a priority.

It is in this context that AF should be considered an alternative and advanced strategy which consists of stopping methanogenesis in the conventional AD process to favor the production of VFA. These are direct precursors of energy or higher value-added products when subjected to downstream processes, playing an important role in the future market of sustainable energy and green chemistry technologies, and representing an important process if the resource–waste–resource cycle is to be completed [80].

However, major obstacles have been identified in this issue, such as lack of control of metabolic activities, the persistence of methanogenic microorganisms in the biological production of VFA, maximizing the production of VFA in appropriate proportions to facilitate their subsequent use, limitations for the extraction of produced acids, developing integrated systems, to detect the genetic expression of microorganisms and regulation of key enzymes in order to increase the specificity and yield of specific products [9,81,82].

Limited understanding is perceived of the mechanisms involved during metabolic changes, i.e., changes in the product range, which mainly depend on operational parameters such as HRT, pH, temperature, nature and concentration of the substrate, nutrient composition and availability, and partial pressure [35,36,62]. Optimizing the biological process—that is, finding the perfect combination of operational parameters to maximize the product—can be achieved by combining experimental studies with modeling tools. According to Lorenzo-Llanes et al. [83], an understanding and description of the acidogenic stage of AD would improve with the use of process simulation tools, allowing predictions and optimization in less time and at lower cost, compared to laboratory- or real-scale work.

Concerning the extraction of the produced acids, it is interesting to evaluate the biological process of chain elongation to favor the transformation of short- to medium-chain carboxylic acids, since their chemical properties would make them easier to separate from the fermentation broth. Furthermore, according to de Sousa e Silva et al. [5] the added value of short-chain carboxylic acids (400–2500 US$/ton) is lower than the market price of medium-chain carboxylic acids (2000–2500 US$/ton).

Although the optimal set of operational parameters to produce VFA and their separation from the fermentation broth remains a challenge, their high energy potential and competitive cost make them a strong contender for AD technology [84].

Few pilot-scale studies have been conducted using wastewater as a substrate for VFA production. The pilot scale is an intermediate stage in which the operating conditions (e.g., type of regime, agitation speeds, etc.) of the processes are as stable as possible in order to establish the most optimal operating parameters, as well as to determine the economic feasibility of the project. In addition, it is necessary to evaluate the reactor configuration (batch, continuous, semi-continuous, etc.) that best suits the desired application.

Finally, bioaugmentation strategies should be considered to identify bacterial strains that participate in a specific metabolic pathway and produce an acid of interest, isolate them, promote their growth and introduce them into the acidogenic reactor. This in order to favor the predominance of a particular microorganism within the reactor.

Therefore, due to the economic and environmental advantages of this field of knowledge, an accelerated development of research in this area is expected in the near future.

4. Conclusions

The bibliometric networks showed five clusters, which identified thematic groups where VFA production through wastewater fermentation is a key aspect. Methane production through AD, hydrogen production in dark fermentation, AD in two-phase reactors, biological nutrient removal, generation of value-added chemicals and energy production in bio-electrochemical systems were the main research focuses identified.

Additionally, in the context of resource recovery from wastewater, VFA production is an emerging issue and requires further research, mainly when less explored substrates such as cassava processing wastewater are used.

The technological challenges in this field of knowledge should be mainly oriented towards always trying to maximize VFA concentration, avoid product inhibition and direct the fermentation process towards the production of a specific VFA depending on the desired application; all this in order to reduce costs associated with the concentration and purification of VFA.