Exploring Potentials for Bioresource and Bioenergy Recovery from Vinasse, the “New” Protagonist in Brazilian Sugarcane Biorefineries

Abstract

Vinasse management in biorefineries bears a burden for sugarcane industries. Despite its high potassium-related fertilizer potential, a series of negative environmental impacts is expected to occur in long-term soil applications of vinasse through fertirrigation. Conversely, a high biodegradable organic content characterizes vinasse as a potential substrate for bioresource and bioenergy recovery from numerous (bio)technological perspectives. This review presents the alternative approaches proposed for sugarcane vinasse management in Brazil, with special attention dedicated to the role of anaerobic digestion as the core conversion step. The suitability of applying phase separation, i.e., the separation of fermentation from methanogenesis in sequential reactors, is discussed in detail. Laboratory and full-scale experiences were considered to discuss the energetic potential of sugarcane vinasse through biogas generation. With a national installed capacity of up to 1603 MW, energy from vinasse could replace half of the coal-derived electricity in Brazil. Meanwhile, investing in vinasse fermentation to obtain soluble organic metabolites could provide more than 10 g L⁻¹ of (iso)butyrate. This is the first review addressing the potential use of sugarcane vinasse in anaerobic biorefineries that discusses applications far beyond conventional biogas production, and encourages the rational use of vinasse as a raw material for bioprocesses, either in short- or long-term scenarios.

1. Residual Stream Management in Sugarcane Biorefineries

Sugarcane-based sugar and ethanol plants are representative examples of the biorefinering concept based on the potential production of a wide range of value-added products from sugarcane, primarily sugar and ethanol [1]. The bagasse resulting from cane crushing is burnt in cogeneration plants for the production of both electricity and steam, which directly supply various processes and operations of the plants (Figure 1a). Steam is fully consumed in specific steps of sugarcane processing, such as juice evaporation (sugar production) and wine distillation (ethanol production). Meanwhile, only a fraction of the produced electricity is consumed within the boundaries of the biorefinery, and the remaining (surplus) amount is exported to the grid [2,3]. This characterizes electricity as a third major product of sugarcane biorefineries. Junqueira et al. [4] indicated a potential surplus electricity amount of 186 kWh per ton of cane (TC) in optimized first-generation (1G) biorefineries, which are characterized by a series of technological improvements compared to conventional industrial schemes, such as recovering trash from the fields, using molecular sieves instead of azeotropic distillation, and efficient heat integration within the whole plant to reduce steam consumption.

With respect to residual stream management from sugarcane biorefineries, two different scenarios can be observed when considering solid and liquid fractions. Relatively well-defined strategies are established for the solid residues, while some uncertain approaches are still observed in the case of wastewaters. Two primary destinations are virtually defined for solid residues from sugarcane processing: [i] the thermoelectric generation from bagasse in the short- to medium-term, as aforementioned, and [ii] the integration of second-generation (2G) ethanol production in the long-term (Figure 1b) [5,6,7,8]. 2G processes aim to increase the ethanol productivity in biorefineries by converting lignocellulosic materials via biochemical or thermochemical processes. The first ones are based on the fermentation of sugars obtained after pretreating lignocellulosics (Figure 1b), while the latter use the gasification of the residues [3,7,9]. Recent studies also propose the biodigestion of lignocellulosics for biogas production [8,10,11,12,13] as an alternative route for enhanced bioenergy generation. In addition to bagasse and trash, filter cake is another important solid residue from biorefineries. This nutrient-rich material is generated specifically in the treatment of juice (before the production of sugar and ethanol), and also presents a defined destination (together with trash) as a soil conditioning agent [14,15].

Conversely, the management of residual liquid streams in biorefineries, especially considering vinasse, may still be considered a burden for the sugar and ethanol industry. Vinasse is characterized as a high-strength wastewater (HSW) resulting from distillation in ethanol plants, and it presents a high content of biodegradable organic matter, macro- and micronutrients, and solids [16,17,18]. Fertirrigation is virtually the only management approach for vinasse in sugarcane biorefineries and is characterized by the direct land disposal of the effluent into sugarcane fields for recycling nutrients and water in crops [3,18,19]. Although fertirrigation leads to significant reductions in terms of mineral fertilizer use, notably potassium and phosphorus, many negative environmental impacts should be observed in continuous long-term vinasse applications, such as soil salinization, soil/water acidification, and an increase in greenhouse gas (GHG) emissions [17,18,20,21,22]. Drawbacks in terms of wastewater management in sugarcane biorefineries may become even more relevant in the future, because new sources of residual streams from 2G processes will be made available, which include pentose liquor (and eventually deacetylation liquors from trash processing [8]) and 2G-vinasse (Figure 1b). The pentose liquor is generated during the pretreatment of lignocellulosic residues, while 2G-vinasse results from 2G-ethanol distillation. Although little is known in terms of the compositional aspects of these HSW, high concentrations of toxic compounds may be found in the pentose liquor, such as furfural and 5-hydroxymetilfurfural, while 2G-vinasse is poor in terms of nutrients [3,23].

When focusing on 1G sugarcane distilleries, numerous treatment approaches have been proposed in an effort to reduce the polluting load of vinasse. These include conventional coagulation–flocculation [24,25,26] and adsorption processes [27], advanced electrochemical and oxidative processes [25,26,28,29,30], and conventional aerobic [31,32] and anaerobic processes [33,34,35,36,37,38,39,40]. Although these approaches are environmentally relevant, from the biorefinery perspective, the treatment processes should add value to residual fractions instead of only being a step to reduce environmental impacts [41,42,43]. In this context, the high biodegradable organic content (e.g., organic acids, residual ethanol, carbohydrates, and glycerol) usually found in sugarcane vinasse, i.e., biochemical oxygen demand (BOD) values ranging from 11.3–29.7 g L⁻¹ [32,44,45,46,47], is a potential substrate for a series of biotechnological applications, including the recovery of energy through optimized anaerobic processes [3,48,49,50,51,52,53] and the production of value-added products, such as enzymes and biosurfactants [54,55]. The high nutrient content found in vinasse may also be properly exploited in alternative bioprocesses, such as the growth of microalgae and protein-rich microorganisms [56,57,58].

Anaerobic digestion, or simply biodigestion, is the most attractive technological approach for managing sugarcane vinasse within the context of biorefineries because the environmental suitability of the effluent (resulting from a decreasing polluting load) may be coupled to the production of a wide range of bioresources and/or biogas. Biogas production characterizes the primary approach for the exploitation of HSW through biodigestion, as observed in experimental (lab and pilot scale) schemes [33,35,36,37,38,40,59,60] and theoretical estimations [48,49,50,61,62,63,64,65,66] proposed for sugarcane vinasse. In particular, using phase separation as an operating strategy for anaerobic systems, in which fermentation (first phase) is separated from methanogenesis (second phase), considerably extends the range of options for the recovery of bioresources from organic waste [41,42,43,67,68]. The fermentative (or acidogenic) step can be exploited to produce numerous metabolites other than methane-rich biogas, such as biohydrogen (bioH₂), organic acids, solvents, and biopolymers. An even more efficient use of the primary raw material, i.e., sugarcane, can be observed for this case, with a view to adopting the cascade approach [41,69]. This concept is based on the application of sequential processes to directly and/or indirectly (i.e., through the production of bioproducts, such as sugar and ethanol) recover energy from a central energy flow, which represents the energetic potential of the raw material.

This review aims to discuss the potentials of sugarcane vinasse as a substrate for bioresource and bioenergy recovery from different (bio)technological routes, using the concept of biorefinery as the reference. The compositional aspects of sugarcane vinasse are first presented, especially considering the biodegradable organic fraction and nutrient content. Different applications for vinasse are then considered, such as using it as a carbon and nutrient source for growing microorganisms and producing organomineral fertilizers. Special attention is given to the role of biodigestion as the central process for efficiently exploiting vinasse, while considering the state of the art of conventional biodigestion schemes and proposing layouts with phase separation for diversifying the recovery of resources in sugarcane biorefineries. Focus is given to the Brazilian case, which is characterized by the second largest world ethanol production, in which vinasse mostly results from the conversion of blends of sugarcane juice and molasses in biorefineries, due to the integration of distilleries and sugar plants. This review describes the fate of sugarcane vinasse based on the biorefinering concept, i.e., proposing the maximum exploitability of this energy-rich substrate.

2. Sugarcane Vinasse: Biodegradable Organic Fraction and Nutrients

The composition of vinasses from various feedstocks is well characterized in the reference literature, as observed in studies published throughout the last decades, which include España-Gamboa et al. [16], Fuess and Garcia [17], Wilkie et al. [23], Christofoletti et al. [70], and Sheehan and Greenfield [71]. Therefore, this section focuses specifically on the characterization of the biodegradable organic fraction and nutrient content of sugarcane vinasse, because the technological application of this HSW largely depends on these aspects. Table S1 shows the compositional characteristics obtained for sugarcane vinasses in various studies, in which both organic and inorganic fractions are described in detail. The high biodegradable content found in sugarcane vinasse is observed based on specific parameters, such as the BOD/COD ratio (values usually above 0.40), the concentration of total carbohydrates (up to 16.3 g L⁻¹), and the concentration of organic acids—primarily lactic (up to 12,697 mg L⁻¹), iso-butyric (1547–4597 mg L⁻¹), and malic (2639–10,460 mg L⁻¹) acids (Table S1).

The biodegradable organic fraction found in vinasse results from low molecular weight organic compounds that are separated from ethanol in the distillation process [72,73]. These compounds result from three major sources: residual concentrations of non-fermented carbohydrates, residual concentrations of non-recovered ethanol, and metabolites from parallel conversion (fermentative) pathways carried out by yeasts and bacteria. The proportion of organic components in vinasse mainly depends on the conditions used in the fermentation step, such as the concentration of reducing sugars, pH, and microbial control. The presence of organic metabolites in sugarcane vinasse usually accounts for a large fraction of the biodegradable organic content of the effluent (Table S1), so that the presence of fatty acids, mainly lactic, results directly from the contamination of fermentation vessels by bacteria. Glycerol also comprises a highly relevant metabolite found in vinasses and is characterized as the main byproduct released by yeasts subjected to stress conditions [73,74,75]. Approximately 2–9% of the total reducing sugars available to yeasts are converted into glycerol, leading to concentrations in the range of 1.4–5.9 g L⁻¹ [43,72,75,76,77,78,79,80,81]. Comparatively, the production of acetic acid in parallel metabolic pathways should be minimal, accounting for only 0.1–0.7% of the available reducing sugars [74]. In turn, residual ethanol concentrations vary considerably in sugarcane vinasse, with the observation of low (250 mg L⁻¹ [82]) and very high (3.8 g L⁻¹ [72], 5.5 g L⁻¹ [77]) values. Ethanol concentrations in vinasses resulting from spirit production can reach even higher values (15.8 g L⁻¹ [78]), because both the head (55–65% v/v of ethanol) and the tail (<38% v/v of ethanol) of the distilled product are discarded together with vinasse [83]. The terms “head” and “tail” refer to the liquid fractions recovered respectively at the beginning and at the end of the distillation step, representing 5–10% (head) and 10–15% (tail) of the total distilled volume [83]. The head usually contains relatively high concentrations of methanol and aldehydes, while the ethanol fraction in the tail is below the desirable content (43–45% v/v) found in the spirit. Hence, only the “heart”, i.e., the amount of liquid collected between the head and the tail, effectively corresponds to the spirit (75–80% of the total distilled volume) [83].

With respect to the nutrient content, vinasses resulting from sugarcane processing are characterized primarily by high concentrations of potassium and sulfate (Table S1). Potassium comprises one of the most abundant nutrients in vegetable tissues [84] and is incorporated into the ethanol production chain when sugarcane is crushed to extract the juice. In turn, high levels of sulfate result from using sulfur compounds in specific steps of sugar and ethanol production. In particular, high levels of sulfuric acid are dosed during the fermentation to both prevent the bacterial contamination and the flocculation of yeast through pH control [85,86], which directly favors the buildup of sulfate in vinasse [48,79,87]. The presence of other compounds, such as nitrogen and phosphorus, may also be explained by their incorporation from plant tissues, as well as by using chemicals in sugarcane harvesting and processing [17,85].

3. (Bio)Technological Applications (Other Than Biodigestion) for Sugarcane Vinasse

The agricultural use of sugarcane vinasse in fertirrigation has historically limited the study of alternative approaches for its management. In practical aspects, fertirrigation is still considered the most suitable techno-economic approach (by decision-makers in the sector) for vinasse in the Brazilian sugar and ethanol industry. In fact, fertirrigation has virtually been the only management approach for sugarcane vinasse in Brazil since the mid-1970s [3], which has led to numerous studies (targeting mainly a local audience) on the effects of vinasse for soil and sugarcane crops. These studies include Fuess and Garcia [17], Fuess et al. [18], Fuess et al. [88], Brito et al. [89], Brito et al. [90], Camilotti et al. [91], Gunkel et al. [92], Lyra et al. [93], Miranda et al. [94], Moore et al. [95], Ribeiro et al. [96], Rolim et al. [97], Silva et al. [98], Tasso Jr. et al. [99], Uyeda et al. [100], and Zolin et al. [101]. Despite the scientific relevance of these studies, this scenario reinforces the importance of developing options that enable more efficient exploitation of this HSW as a raw material in diversified processes.

The reference literature indicates relatively few biotechnological alternatives in which sugarcane vinasse was used as the raw material, as compiled in Table S2, which highlights the use of its biodegradable organic and nutrient contents as substrate in bioprocesses dedicated to the cultivation of microorganisms and/or the production of biochemicals. In this case, some examples based on using sugarcane vinasse obtained directly from molasses fermentation were also considered. The growth of microalgae on sugarcane vinasse was studied by Ramirez et al. [57], Bonini [76], Kadioğlu and Algur [102], Marques et al. [103], Santos et al. [104], Candido and Lombardi [105], and Soto et al. [106], while Nitayavardhana and Khanal [56], Pires et al. [107], Ricci et al. [108], and Santos et al. [109] used vinasse as a nutrient source in the cultures of protein-rich fungal biomass (Table S2). Most of these studies have two primary motivations: the reduction in the polluting load of vinasse and the minimization of production costs of the target products, such as single-cell protein [108] by using low-cost substrates. Despite the successful application of sugarcane vinasse as a nutrient source, diluting the effluent is a common characteristic in most of the considered studies [56,57,103,104,107]. This strategy may lead to a major drawback in full-scale applications, due to the high specific vinasse generation rate (12–14 m³ per m³ of ethanol) [17,19,64], which directly requires huge processing units. Alternatively, vinasse clarification using proper coagulants can eliminate dilution requirements while maintaining adequate nutrient concentrations in the cultivation broth [110].

With respect to the recovery of biochemicals, Aguiar et al. [54] and Kahraman and Gurdal [111] assessed the role of vinasse as a nutrient source to produce high-value enzymes (laccase and peroxidases) from fungi, while Oliveira and Garcia-Cruz [55] and Colin et al. [112] studied the recovery of natural emulsifiers (Table S2). Bastos et al. [113] also assessed the potential to recover citric acid from bagasse by using vinasse as a moistening agent. Similarly to studies focusing on biomass production, vinasse dilution is characterized as an imperative factor for achieving successful results in such cases [111,112,114]. Moreover, using pure cultures, as well as processes based on microbial biomass production, usually requires the sterilization of wastewater streams, which also limits full-scale applications. Vinasse naturally carries yeast and bacteria from the fermentation vessels. It is important to stress that studies on the recovery of biochemicals from sugarcane vinasse are relatively scarce compared to exploiting vinasses from different feedstocks, as observed from surveys in the specific literature. Vinasses resulting from the processing of corn, wheat, grains, grape and rice (also known as thin stillages for the cases of corn, wheat, and grains) have already been assessed as raw material to produce a wide range of bioproducts, such as: acid protease [115], polysaccharides (10,000–200,000 Da) [116], eicosapentaenoic acid [117], lipids (single-cell oils) [118], bacterial cellulose [119,120], 1,3-propanediol [121], carbon-based solid acid catalyst [122], lactic acid coupled to animal feed [123], cellulosic fibers for 2G-ethanol production [124], and chitosan [125]. This discrepancy in terms of diversified studies also results from the massive land disposal of sugarcane vinasse via fertirrigation, which characterizes a limiting factor for enhancing the exploitation of sugarcane vinasse using alternative processes.

In particular, recycling vinasse in fermentation to produce fuel ethanol and spirits has been demonstrated to be an attractive approach for the recovery of biochemicals in sugarcane biorefineries, as proposed by Navarro et al. [73], Menezes et al. [114], Pereira [126], and Madaleno et al. [127] (Table S2). Using optimized operating conditions (which include recycling yeasts), Navarro et al. [73] obtained high ethanol concentrations (>70 g L⁻¹) throughout 14 cycles meant to reduce the inputs of nutrients, fresh water, and sulfuric acid in fermentation vessels. Moreover, vinasse recycling characterized a bioconcentration step, which could reduce the energy consumption in further concentration steps using conventional evaporators [73]. Regarding different feedstocks, recycling thin stillages in corn- and cassava-to-ethanol industries has been widely studied [128,129,130,131], and results also demonstrated the technical feasibility of this approach. Bialas et al. [128] highlighted that up to 75% of the fresh water used in the fermentation step could be replaced by stillage (subjected only to simple steps of cooling down and centrifugation prior to recycling), while the energy consumption using stillage concentrations in multi-evaporator processes could be considerably reduced [131]. Therefore, among the technological approaches for the enhanced recovery of biochemicals in sugarcane biorefineries, vinasse (fresh or biodigested) recycling for enhanced ethanol production could be a wise choice, based primarily on a more efficient energy extraction from the biodegradable organic fraction of vinasse.

Additional alternative approaches to sugarcane vinasse management reported in the literature include the production of organomineral fertilizers [132,133], the use in nutritive solutions for hydroponics [134], the generation of energy from burning [135,136], and the use as a biostimulating agent for the biodegradation of hydrocarbons and oily residues [137,138] (Table S2). A recent highly innovative application was described by Tamashiro et al. [139], in which vinasse replaced water in concrete manufacturing, together with recycled concrete aggregates as substitutes to sand. The production of organomineral fertilizer may also be considered an attractive option for vinasse management in full-scale biorefineries, which works primarily by facilitating the transportation and field applications of vinasse, as well as by improving some physical-chemical properties of the vinasse due to incorporating solid residues [140]. Adding different residues, namely grape-marc, olive-pressed cake, and cotton gin trash to beet molasses vinasses improved the nutrient release in previous reports [141,142,143], and therefore a similar behavior should be observed for sugarcane vinasse. However, when considering biorefineries, unlike the scheme proposed by Gurgel et al. [132] (Table S2), in which raw vinasse is concentrated and blended with other residual streams from biorefineries, the biodigestion of vinasse (prior to the concentration step) could improve the energetics of the process, as proposed by Nandy et al. [133] in the Indian ethanol industry. Concentration is a highly energy-consuming step, which may seriously affect the economic feasibility of full-scale systems [47,144]. Moreover, condensed liquids recovered from raw vinasses are still characterized by high acidity [145] due to the volatilization of organic acids, which requires additional treatment steps to enable proper reuse, either as process water or for return to the sugarcane fields. Peiter et al. [146] pointed out the sequential application of anaerobic digestion and reverse osmosis as the most attractive approach to manage vinasse on an exergy basis; however, the economic performance of evaporators outperformed that from reverse osmosis.

The high energy inputs associated with the concentration step were also considered a major drawback in the system proposed by Cortez and Pérez [136], which was characterized by the combustion of concentrated vinasse blended with fuel oil (Table S2). Additional drawbacks reported by these authors included negative effects from increasing vinasse concentrations over burning oil, as a direct result of the emulsification of the hydrocarbon, as well as the fusion of ashes within the bed combustor. Alternatively, Akram et al. [135] co-fired raw sugarcane vinasse with coal, and also reported operating problems due to the agglomeration of ashes within the combustor (Table S2). In addition to the unsuccessful experiences with vinasse burning, using complementary fossil energy sources associates negative environmental impacts with this approach, such as the depletion of fossil resources and the release of GHG. The potential recovery of potassium-rich ashes from combustors, which present high agricultural value (similar to that of organomineral fertilizers), represents one of the few benefits of this approach [47,133].

In summary, the large-scale application of most of the technological alternatives evaluated for the management of sugarcane vinasse still demands process improvements while considering important limitations, such as diluting the vinasse and nutritional supplementation to optimize the recovery of bioresources. Moreover, little is reported on the physical-chemical characterization of vinasse after (bio)processing, which further limits understanding of the potential of these approaches in full-scale plants. Among the listed alternatives, recycling vinasse as a complementary carbon and nutrient source for the fermentation step and organomineral fertilizer production may be characterized as important alternatives, as minor modifications would be required in the operating plants. However, the recovery of bioresources or bioenergy from vinasse in sugarcane biorefineries could be maximized by using biodigestion, particularly with phase separation.

4. Biodigestion as the Core Technology for Processing Sugarcane Vinasse

4.1. Fundamentals of Anaerobic Processes

The reference literature historically compiles a series of critical studies on the anaerobic digestion of wastewater, which range from fundamental and general aspects [147,148,149,150,151,152] to specific studies dealing with different types of high-rate systems [153,154,155,156] and using biodigestion for vinasses [23,157]. Biodigestion is a natural biological process in which the organic matter is converted, when there is a lack of free oxygen, by diverse groups of microorganisms that are syntrophically associated. The primary product of the process is biogas, a gaseous blend mainly consisting of methane (CH₄) and carbon dioxide (CO₂). Using biodigestion as an approach to treat organic residues has been reported for several decades. However, a significant development in the process was observed only from the 1970s onwards, when the search for alternative energy sources stimulated a significant technological improvement of anaerobic systems. The development of high-rate reactors was sine qua non for such progress [149,153,155]. High-rate reactors triggered significant improvements in the anaerobic conversion of HSW by decoupling the solid retention time (SRT; or sludge age) from the hydraulic retention time (HRT) in order to provide adequate contact periods for microorganisms, particularly methanogens, to degrade organic compounds [149,155,158].

Biodigestion may be divided into four main steps, according to the biochemical conversion of the organic compounds—hydrolysis, acidogenesis, acetogenesis, and methanogenesis—wherein at least five microbial groups participate in the process. Figure 2 depicts a simplified diagram of the anaerobic process, in which hydrolytic and fermentative bacteria initially convert complex substrates, i.e., polymers, such as carbohydrates and proteins, into short-chain compounds, such as volatile fatty acids (VFAs), alcohols, CO₂, and hydrogen (H₂). Acetogenic bacteria further convert VFAs (other than acetic acid) and alcohols into acetic acid, CO₂, and H₂, which correspond to the primary substrates utilized by methanogenic archaea (methanogens). Hydrogenotrophic methanogens grow autotrophically on CO₂ and H₂ to produce CH₄, while acetate is used as the carbon source by acetoclastic methanogens. Table 1 shows some relevant reactions carried out by the different microbial groups in the anaerobic conversion process.

Many factors directly affect the biodigestion process, which are strictly related to operating parameters that include pH, alkalinity, organic loading rate (OLR, i.e., the amount of substrate supplied to the microorganisms), nutrient supplementation, and temperature [159,160,161]. The presence of toxic compounds (e.g., phenolics naturally found in wastewaters or ammonia/sulfide released during substrate conversion) and the characteristics of the organic substrate, i.e., biodegradability and recalcitrance, also play important roles in the anaerobic process. Particularly for sugarcane vinasse, two primary factors must be considered: pH/alkalinity and temperature conditions. The high biodegradable organic fraction of sugarcane vinasse, especially in terms of carbohydrates (Table S1) and glycerol, promptly stimulates the activity of acidogenic bacteria, which present high growth rates (with a minimum doubling time of about 30 min [151]). Consequently, the rapid acidification of the wastewater may seriously affect the global anaerobic process [162], because methanogens (with a minimum doubling time of 2–3 days for acetoclastic methanogens [151]) are susceptible to the accumulation of VFAs [159]. Providing adequate alkalinity levels is imperative to prevent unbalancing conditions between acidogenic bacteria and methanogens.

Regarding the temperature, the application of thermophilic conditions (50–55 °C) may be considered the most suitable option for biodigestion systems used for sugarcane vinasse, based on the following relevant advantages: [i] vinasse is collected from distillation columns at temperatures as high as 90 °C [3,163], wherein cooling systems may be excluded as vinasse naturally reaches the required temperature conditions in intermediate transportation steps; and [ii] thermophilic systems support the application of higher OLR and present equivalent efficiencies compared to mesophilic systems, which leads to the installation of more compact reactors [23,47].

Figure 2. Simplified diagram of the anaerobic bioconversion of organic matter into methane, including the main involved microbial populations. Bold lines indicate the process without the interference of sulfate reduction; dashed lines indicate possible biochemical pathways associated with the reduction of sulfate into sulfide. Note: The diagram is based on McCarty and Smith [150] and Chenicharo [160].

Figure 2. Simplified diagram of the anaerobic bioconversion of organic matter into methane, including the main involved microbial populations. Bold lines indicate the process without the interference of sulfate reduction; dashed lines indicate possible biochemical pathways associated with the reduction of sulfate into sulfide. Note: The diagram is based on McCarty and Smith [150] and Chenicharo [160].

Finally, the high concentrations of sulfate observed in sugarcane vinasse are also a matter of concern when using biodigestion [79,81]. Applying sulfate-rich wastewaters to anaerobic systems stimulates the sulfidogenic pathway (Figure 2), in which sulfate is reduced primarily into sulfide (H₂S) by a specific group of microorganisms, namely, the sulfate-reducing bacteria (SRB) [164,165]. SRB are characterized as an extremely versatile microbial group insomuch as they outcompete methanogens when using common substrates, such as acetate and H₂ [159]. This characteristic limits the extraction of energy from organic matter by CH₄ production. Moreover, the high toxicity of H₂S may also affect the methanogenic activity in anaerobic systems, either by permeating microbial cells as the non-ionized form (H₂S) or by the precipitation of essential metals as the ionized forms (HS⁻ and S²⁻) [159]. Kiyuna et al. [87] studied the influence of the COD/sulfate ratio on the biodigestion of sugarcane vinasse, and indicated that methane production is negatively affected by high sulfate concentrations. Conversely, these authors reported that the COD removal was not affected by sulfidogenesis, and also highlighted that the conversion of acetate improved when higher sulfate concentrations were applied. These observations also result from the versatility of SRB, which may act as acetogenic bacteria by partially oxidizing VFAs to acetate (Table 1) [154,166], and thus kinetically favor the activity of methanogens.

Table 1. Biochemical reactions performed by different microbial populations in anaerobic environments and the respective Gibbs free energies.

Microbial Group	Reaction	ΔG°′ (kJ mol−1)	Equation
Fermentative bacteria	Glucose + 2H2O → 2Acetate + 4H2 + 2CO2	−206.0	(1) 1
	Glucose → Butyrate + 2H2 + 2CO2	−254.0	(2) 1
	Glucose + 2H2 → 2Propionate + 2H2O	−279.4	(3) 1
	Glucose → 2Lactate + 2H+	−198.0	(4) 2
	Glucose → 2Ethanol + 2CO2	−164.8	(5) 3
	Glucose + H2O → Ethanol + Acetate + 2H2 + 2CO2	−205.2	(6) 3
	Acetate + 2Lactate → H2 + 1.5Butyrate + 2CO2 + H2O	−156.6	(7) 4
	3Lactate → 2Propionate + Acetate + CO2 + H2O	−170.0	(8) 5
Acetogenic bacteria	Propionate + 2H2O → Acetate + 3H2 + CO2	+76.2	(9) 3
	Butyrate + 2H2O → 2Acetate + 2H2	+48.4	(10) 3
	Ethanol + H2O → Acetate + 2H2	+9.6	(11) 3
	Lactate + 2H2O → Acetate + 3HCO3− + 2H2	−4.2	(12) 3
	Glycerol + 2H2O → Acetate + HCO3− + 2H+ + 2H2	−73.2	(13) 6
Homoacetogenic bacteria	4H2 + 2CO2 → Acetate + 2H2O	−104.0	(14) 3
Acetoclastic methanogens	Acetate → CH4 + H2O	−31.0	(15) 3
Hydrogenotrophic methanogens	4H2 + CO2 → CH4 + 2H2O	−135.0	(16) 3
Sulfate-reducing bacteria	SO42− + Acetate → HS− + 2HCO3−	−47.3	(17) 7
	SO42− + 2Butyrate → HS− + H+ + 4Acetate	−55.5	(18) 7
	SO42− + 2Ethanol → HS− + 2H2O + H+ + 2Acetate	−132.7	(19) 7
	3SO42− + 4Propionate → 3HS− + 4HCO3− + H+ + 4Acetate	−150.6	(20) 7
	SO42− + 4H2 + H+ → HS− + 4H2O	−152.2	(21) 7
	SO42− + 2Lactate → HS− + 2HCO3− + H+ + 2Acetate	−160.1	(22) 7
	3SO42− + 2Ethanol → 3HS− + 4HCO3− + H+ + 2H2O	−227.3	(23) 7
	Glycerol + 1.25SO42− → 0.5Acetate + 1.5H2CO3 +0.5HCO3− + 1.25HS− + 0.75OH− + H2O	−424.5	(24) 8

¹ Mosey [151]. ² Anzola-Rojas et al. [167]. ³ Saady [168]. ⁴ Matsumoto and Nishimura [169]. ⁵ Seeliger et al. [170]. ⁶ Thauer et al. [171]. ⁷ Zhou and Xing [172]. ⁸ Bertolino et al. [173].

Table 1. Biochemical reactions performed by different microbial populations in anaerobic environments and the respective Gibbs free energies.

Microbial Group	Reaction	ΔG°′ (kJ mol−1)	Equation
Fermentative bacteria	Glucose + 2H2O → 2Acetate + 4H2 + 2CO2	−206.0	(1) 1
	Glucose → Butyrate + 2H2 + 2CO2	−254.0	(2) 1
	Glucose + 2H2 → 2Propionate + 2H2O	−279.4	(3) 1
	Glucose → 2Lactate + 2H+	−198.0	(4) 2
	Glucose → 2Ethanol + 2CO2	−164.8	(5) 3
	Glucose + H2O → Ethanol + Acetate + 2H2 + 2CO2	−205.2	(6) 3
	Acetate + 2Lactate → H2 + 1.5Butyrate + 2CO2 + H2O	−156.6	(7) 4
	3Lactate → 2Propionate + Acetate + CO2 + H2O	−170.0	(8) 5
Acetogenic bacteria	Propionate + 2H2O → Acetate + 3H2 + CO2	+76.2	(9) 3
	Butyrate + 2H2O → 2Acetate + 2H2	+48.4	(10) 3
	Ethanol + H2O → Acetate + 2H2	+9.6	(11) 3
	Lactate + 2H2O → Acetate + 3HCO3− + 2H2	−4.2	(12) 3
	Glycerol + 2H2O → Acetate + HCO3− + 2H+ + 2H2	−73.2	(13) 6
Homoacetogenic bacteria	4H2 + 2CO2 → Acetate + 2H2O	−104.0	(14) 3
Acetoclastic methanogens	Acetate → CH4 + H2O	−31.0	(15) 3
Hydrogenotrophic methanogens	4H2 + CO2 → CH4 + 2H2O	−135.0	(16) 3
Sulfate-reducing bacteria	SO42− + Acetate → HS− + 2HCO3−	−47.3	(17) 7
	SO42− + 2Butyrate → HS− + H+ + 4Acetate	−55.5	(18) 7
	SO42− + 2Ethanol → HS− + 2H2O + H+ + 2Acetate	−132.7	(19) 7
	3SO42− + 4Propionate → 3HS− + 4HCO3− + H+ + 4Acetate	−150.6	(20) 7
	SO42− + 4H2 + H+ → HS− + 4H2O	−152.2	(21) 7
	SO42− + 2Lactate → HS− + 2HCO3− + H+ + 2Acetate	−160.1	(22) 7
	3SO42− + 2Ethanol → 3HS− + 4HCO3− + H+ + 2H2O	−227.3	(23) 7
	Glycerol + 1.25SO42− → 0.5Acetate + 1.5H2CO3 +0.5HCO3− + 1.25HS− + 0.75OH− + H2O	−424.5	(24) 8

¹ Mosey [151]. ² Anzola-Rojas et al. [167]. ³ Saady [168]. ⁴ Matsumoto and Nishimura [169]. ⁵ Seeliger et al. [170]. ⁶ Thauer et al. [171]. ⁷ Zhou and Xing [172]. ⁸ Bertolino et al. [173].

4.2. Biodigestion of Vinasse in Brazil: Background and Current State of the Research

The anaerobic processing of organic residues and wastewaters has two primary targets: the bioenergy recovery from the methane-rich biogas (biogas-CH₄) produced by methanogens and the minimization of the polluting load of residues. In this context, various reactor configurations and operating conditions were proposed for the vinasses of various raw materials [23] in an effort to optimize the energy extraction mainly from single-phase systems, i.e., in which all the steps of the anaerobic process (Table 1) are carried out in a single reactor. However, studies may still be considered incipient when using biodigestion in the processing of sugarcane vinasse in Brazil, especially in terms of full-scale experiences. Initial studies were reported in the early 1980–1990s [34,37,174,175], and then a gap of approximately 20–25 years was observed until complementary relevant studies were made available [33,35,36,38,39,59,60,176,177,178]. The relatively recent growing interest in vinasse biodigestion was motivated by the search for alternative bioenergy sources and the need for reducing negative environmental impacts from fertirrigation. The ever-increasing concern for the environment has begun to critically assess any and all aspects of biofuel production chains, and the environmental burden of fertirrigation has gained considerable prominence in this context.

The low adherence of distilleries to the biodigestion of vinasse directly resulted from unsuccessful experiences after implementing full-scale biodigestion plants in biorefineries in the past, such as the METHAX Dedini^® system, which resulted from a consortium (Codistil-Dedini/PEM/Paques) in the early 1980s [179]. The METHAX Dedini^® system included a sludge-blanket reactor coupled to a biogas purification plant for producing biomethane (bioCH₄) to be used primarily as an automotive fuel. Despite the robustness of this system, its relative black box character, as well as the lack of fundamental knowledge in terms of plant operation and specificities of vinasse bioconversion seriously affected the acceptance of this process as a reliable technological approach for managing vinasse in Brazilian sugarcane biorefineries. The susceptibility to fast acidification and the requirements for long HRT [3,48,162] represent some of the specific points to be carefully considered. Moreover, prohibiting the sale of electricity imposed on biorefineries until the mid-1990s and the proven techno-economic (but not environmental) feasibility of fertirrigation were also preponderant factors that discouraged studies on the biodigestion and the energy exploitation of biogas from vinasse in Brazil [3].

The characteristics of some highly relevant (according to the suitability to industrial scale application) studies on sugarcane vinasse biodigestion from the Brazilian sugar and ethanol industry are listed in Table S3, which especially consider the achievements of each proposed system. Studies based on the processing of vinasses resulting only from sugarcane molasses, e.g., Acharya et al. [180], España-Gamboa et al. [181], Fernández et al. [182] and Kumar et al. [183], were not considered for comparison purposes, because the direct use of molasses for ethanol production is not carried out in Brazil. Blending molasses with juice or using pure sugarcane juice in fermentation (i.e., annexed and autonomous distilleries, respectively) generates considerably less concentrated vinasses than using molasses as the only substrate [17,23,88], and requires different operating strategies for optimizing the biodigestion systems. One of the main differences refers to the HRT, because lower values can be more easily implemented in the case of less concentrated vinasses. Initial studies on the biodigestion of sugarcane vinasse in Brazil showed promising results with the application of pilot systems, as presented in Craveiro et al. [34], Souza et al. [37] and Costa et al. [174] (Table S3). Despite the temporal gap in terms of relevant studies, recent reports [33,35,36,38,39,40,176,177,178] (most listed in Table S3) have shown significant improvements regarding the biodigestion of sugarcane vinasse, i.e., obtaining satisfactory COD removals (>70%) by associating high OLR (25–30 kg COD m⁻³ day⁻¹) with low HRT (<24 h). Nevertheless, these results were mostly obtained in bench-scale systems. These promising results may be attributed to two primary factors, namely, using thermophilic temperature conditions and phase separation for the systems.

Regarding future steps, the scale-up of new reactor configurations, such as the anaerobic structured-bed reactor (ASTBR) [28,36] and the optimization of using alkalizing compounds, is characterized as an essential step for encouraging the implementation of full-scale biodigestion plants in sugarcane biorefineries. Using fixed-film systems with high bed porosity (such as the ASTBR) tends to be more suitable than sludge-blanket reactors in the case of sugarcane vinasse, because specific compositional aspects (e.g., the relatively high contents of solids and salts) hinder an efficient granulation [155]. In terms of the system scale-up, the literature currently indicates the long-term operation (>20 years) of only one full-scale reactor (5200 m³) in the Brazilian sugar and ethanol industry [179,184], which is used for processing approximately 25% of the vinasse (37 m³ h⁻¹) generated in a large scale annexed biorefinery (milling capacity of 10 million tons of sugarcane -MTC- per harvest). Although the design of this reactor was based on the system proposed by Souza et al. [37], which successfully reported the operation of a pilot-scale (75 m³) thermophilic reactor for over 250 days at high OLR (26.5 kg COD m⁻³ day⁻¹; Table S3), the implemented full-scale system operates far below the potential (OLR = 5 kg COD m⁻³ day⁻¹) [179]. Recent interest on the application of vinasse biodigestion (frequently associated with the co-digestion of filter cake, which depends on low rate reactors) is on course in Brazil; however, the results of the experiences will be observed in the coming years. In any case, the scale-up of efficient anaerobic systems is imperative for identifying real-life operating drawbacks, as well as for precisely assessing the energetic potential of the biogas from sugarcane vinasse. In fact, all the data concerning the assessment of the energy potential of vinasse (see Section 4.3) still derive from estimates, meaning that the potentials and limitations of biogas as a source of bioenergy in biorefineries will only be fully understood by pilot- and full-scale experiences [48].

Finally, the alkalinization of full-scale systems directly affects the economic feasibility of the biodigestion plants, as was recently described elsewhere [50,163]. Sodium bicarbonate (NaHCO₃) is often used as the alkalizing agent in studies reporting the biodigestion of sugarcane vinasse [35,36,59,60]. However, even low doses of NaHCO₃ (0.28–0.36 g NaHCO₃ g⁻¹COD) [163] lead to highly unfavorable economic scenarios. Biogas production costs in biodigestion systems using NaHCO₃ as the alkalizing compound may be up to 70-fold higher compared to using sodium hydroxide (NaOH) [50]. In this context, although alternative alkalizing approaches have been proposed, such as using urea [65,162,185], using NaOH may be the most feasible option [163]. Low doses of NaOH (4 g NaOH kg⁻¹ COD) may provide adequate conditions for the bioconversion of the organic matter in high-rate reactors, as referenced by the results obtained by Souza et al. [37] (Table S3). Using effluent recirculation for alkalinity recycling is also characterized as a feasible option, as attested by a few pilot- and full-scale experiences [37,133] and economic assessments [50].

4.3. Predicting the Energetic Potential of Sugarcane Vinasse

The lack of full-scale experiences regarding the biodigeston of sugarcane vinasse in Brazil limits the assessment of its energetic potential in terms of practical results, as aforementioned. When considering the only case reported in the literature for the Brazilian industry [179], biogas is used for yeast drying in a spray dryer (8 tons of dried yeasts per day). However, approaches based on theoretical calculations and scenario prediction have historically presented relevant results for assessing the energy recovery capacity of vinasse-derived biogas in Brazilian biorefineries [46,48,50,61,62,63,64,65,66,184,186,187,188,189,190,191], and have even surpassed the number of published experimental approaches. The use of scenarization-based studies both complements the lack of experimental/field data and encourages the implementation of full-scale plants to enhance the energy balance of distilleries. Table S4 shows data on the estimation of the energetic potential of sugarcane vinasse from different studies, with a focus on the generation of electricity by applying biogas-CH₄ in different prime movers.

The energetic potential estimated for sugarcane vinasses from different studies ranged from 0.0001 to 2.61 MJ L⁻¹ of ethanol (Table S4). The huge data variability directly results from the different assumptions considered in each study, primarily the specific vinasse production (8.14–15 L vinasse L⁻¹ of ethanol), the type (internal combustion engines, gas and steam turbines) and conversion efficiency (23–43%; which depends on the plant size, the implementation of heat recovery, and also on the type) of the prime movers (Table S4). The type of feedstock used in ethanol production (juice or blends of juice and molasses), the organic content of sugarcane vinasse (COD = 21–45.8 g L⁻¹, in addition to one case considering the co-processing of vinasse and filter cake [192]), and the lower heating value (LHV) of the biogas (or methane; at 20.02–39.62 MJ Nm⁻³) (Table S4) also influenced the obtained values. Nevertheless, the results indicate that the energy from biogas can offset the consumption of fossil fuels in distilleries, which corresponds to an average of 0.47 ± 0.20 MJ L⁻¹ of ethanol, based on data available elsewhere [193,194,195,196,197,198,199,200]. A global analysis of the energetic potential data suggests that the values obtained from simple energy balances (>1.0 MJ L⁻¹ of ethanol [48,61,62,65,184]) may be overestimated, because such studies do not usually include detailed characteristics of biorefineries and prime movers (Table S4).

Regarding the application of the energy recovered from biogas–CH₄, the studies propose numerous options. Fuess et al. [50] and Moraes et al. [63] indicated that the electricity produced from biogas could supply populations of approximately 130–140 thousand inhabitants during the sugarcane harvesting period, for biorefineries processing 4 and 2 MTC, respectively, per harvest. The operation of biodigestion-power plants during the inter-harvest could further supply 60 thousand inhabitants [50]. The aforementioned studies also highlighted that the energy from biogas could significantly improve the generation of surplus electricity in biorefineries, because more than 50% of the electricity exported to the grid could be obtained from biodigestion-power plants. Salomon et al. [64] reported that the generation of electricity from biogas in engines could lead to more favorable economic scenarios compared to the application of microturbines, although the most profitable option was characterized by co-firing biogas and bagasse in boilers. These authors also highlighted the high profitability of using biogas as fuel in spray dryers for yeast drying (further description on economic aspects is provided in Section 4.3.1). In global terms, i.e., considering the conversion of the total volume of sugarcane vinasse generated in Brazil into biogas–CH₄, the installed capacity reached values in the range of 819–1603 MW [46,49,50,63,184], depending on the assumptions used in each study. Although directing the entire amount of vinasse to biodigestion may not represent a factual scenario (because implementing biodigestion plants may not be economically attractive in small-scale sugarcane processing plants), these values correspond to 7.0–13.7% of the installed capacity of electricity generation from bagasse (11,712 MW [201]). Even more favorable scenarios could be obtained using the consumption of fossil sources as a reference, as up to 49.2% and 50.0% of the electric power of fuel oil and coal (3256 and 3203 MW [201]) could be respectively replaced by the energy from biogas.

Improvements in terms of energy extraction by applying two-phase biodigestion to the processing of sugarcane vinasse may also be highlighted from data shown in Table S4, because the electric power of biogas–CH₄ (7.0–10.8 MW [50]) reached levels between 10–115% higher compared to single-phase systems (5.02–6.37 MW [63,64]). The operation of biodigestion-power plants during the inter-harvesting period (off season) also greatly improved electricity production from vinasse, and led to electric power values of 12.3 MW and 15.5–16.3 MW for single- and two-phase biodigestion schemes, respectively. The use of gas turbines coupled with steam turbines (combined cycle) was considered in this case [50]. The unavailability of sugarcane vinasse during the off season (once ethanol production is interrupted) is one of the main bottlenecks hindering efficient high rate biogas production in vinasse-fed plants. Defining strategies to achieve year-round operation, as proposed elsewhere [50,52,53], represents one of the main topics to be addressed in new vinasse-based research within the context of biodigestion. In particular, Fuess et al. [50] also reported estimates concerning the recovery of thermal energy from biogas in engines and gas turbines with values of 6.8–8.3 MW and 3.4–4.2 MW for the harvesting and inter-harvesting periods, respectively. Although these values (8.17–9.94 kWh TC⁻¹ [50]) are considerably lower than the consumption of steam in distilleries (300 kWh TC⁻¹), the association between electricity production and thermal energy recovery could lead to marked improvements in terms of biogas conversion, with values as high as 86.7% when using gas turbines as the prime movers [50]. In practical terms, the thermal energy recovered from biogas could be used for maintaining the temperature of the reactors [3], mainly in thermophilic systems.

4.3.1. Economics of Vinasse-Derived Bioenergy

Estimates on the energy production from sugarcane vinasse are frequently associated with economic assessments used to both measure profitability levels and identify key factors impacting process profitability (sensitivity analyses). Common tools used to assess the economic performance of anaerobic systems include the net present value (NPV), the internal rate of return (IRR), and the discounted payback period [49,50,63] when considering greenfield and brownfield scenarios. In the case of vinasse biodigestion, greenfield investments refer to the installation of an entirely new sugarcane biorefinery (i.e., including steps from sugarcane processing to biogas production and conversion), while brownfield ones address the implementation of anaerobic-power plants in operating biorefineries (i.e., the investments refer only to the infrastructure demanded by vinasse processing and biogas use). Consequently, potential revenues include sugar (in the case of annexed plants), ethanol, and biogas-derived energy in the first case, while only the biogas-derived energy is a marketable product in the latter. A direct comparison among different studies is hindered due to the use of different assumptions in each case, including technical (e.g., biorefinery size, quantitative and compositional characteristics of vinasse, type of anaerobic system, type and efficiency of the prime movers, etc.) and economic (e.g., minimum acceptable rate of return—MARR, project lifetime, tax rates, etc.) aspects. Nevertheless, results indicate that the production and use of biogas from vinasse can be economically feasible in both greenfield and brownfield approaches, provided that the following conditions are achieved: NPV > 0, IRR > MARR and payback period < project lifetime.

Considering brownfield scenarios, Salomon et al. [64] reported on the economic feasibility of using vinasse-derived biogas to produce electricity in reciprocating combustion engines or in cogeneration systems (coupled with the commercialization of surplus bagasse in the latter case), as well as to generate heat directed to yeast drying (in all cases: IRR > MARR = 15%). Electricity generation in micro gas turbines was demonstrated to be unfeasible in this case (IRR = 7.06%). In another brownfield approach, Fuess and Zaiat [49] also observed the economic feasibility (IRR > 12% vs. MARR = 8.76%) of three different biogas uses in annexed biorefineries, namely, the enhanced electricity generation in combined cycle power plants, the cogeneration of electricity in boilers, and the production of bioCH₄ for grid injection. The authors demonstrated that achieving process profitability in autonomous plants (IRR < 12%) would directly depend on the type of biodigestion plant, i.e., single- or two-phase. The use of phase separation would negatively impact process profitability due to the higher investment costs required to build facilities processing higher vinasse flow rates (compared to the annexed plants). The profitability of electricity production from biogas in engines was associated with a wide range of electricity prices (from USD 59.83 to USD 76.93) by Nogueira et al. [189], with IRR values directly dependent on the operating period considered for the power plant.

With respect to greenfield investments, the results usually show profitable scenarios regardless of the biogas use, because of the high market values of ethanol and sugar (which represent the main biorefinery products in this type of approach). Moraes et al. [63] observed that the use of biogas for electricity production (using either boilers or internal combustion engines) or for diesel replacement could provide some extra revenue in autonomous ethanol plants. However, IRR values (13–14%) were relatively low compared to the IRR (12%). In this case, annexed biorefineries were demonstrated to be almost economically unfeasible due to higher investment costs. In contrast, Fuess et al. [50] showed that electricity production from biogas in annexed plants could be highly profitable (IRR of ca. 20% vs. MARR = 12%) by using different prime movers (internal combustion engines, gas turbines, or the combined cycle). The operation of the biorefinery as an autonomous plant processing energy cane during the inter-harvest directly increased revenues with electricity. The authors also demonstrated the significant negative impacts of dosing alkalinizing agents (NaHCO₃) on the economic performance of the entire biorefinery. Implementing a flex ethanol facility processing sugarcane (harvest) and maize (inter-harvest) also was demonstrated to be an attractive strategy to improve the economic performance of sugarcane biorefineries [192]. While relatively low IRR values (ca. 15%) were estimated for electricity production, bioCH₄ production and use (grid injection or diesel replacement with IRR values of ca. 25% and 40%, respectively) would be the most profitable option in this case (considering MARR = 12%). Additional details on the production of bioCH₄ from vinasse can be found in Section 4.6.

The complementary use of sensitivity analyses indicates the main factors impacting the profitability of bioenergy production from vinasse, which is highly useful to identify investment opportunities and provide guidelines for policy makers within the biofuel industry context. Overall, investment costs and the product (electricity or bioCH₄) prices impact the profitability to a higher extent than other factors, such as the operating costs [49,66,189]. In practical aspects, these results indicate that both the agents controlling tax rates (governments) and the market demands directly impact the economics of biogas production and use.

4.4. Phase Separation: Enhancing Resource Recovery from Vinasse

The use of biodigestion with phase separation aims to optimally exploit the potentials of different microbial groups acting in the bioconversion of organic matter, primarily hydrolytic and fermentative bacteria in the acidogenic phase (Equations (1)–(8); Table 1) and methanogenic archaea (Equations (15) and (16); Table 1) in the subsequent methanogenic phase. Despite breaking some syntrophic relationships among the microbial populations (Figure 2) [202], phase separation brings numerous advantages, including [i] optimization of potentially limiting anaerobic conversion steps, such as the hydrolysis of complex materials in the acidogenic phase and the consumption of acids by acetogens and methanogens; [ii] enhancement of operating stability by preventing the exposure of methanogens to the accumulation of VFAs, which also dramatically reduces the requirements of alkalizing compounds; [iii] enhancement of both the substrate biodegradability and energy extraction from organic residues; and [iv] reduction in the start-up period coupled with increasing processing capacities, i.e., the application of high OLR for methanogenic reactors [35,202,203,204,205,206,207]. In particular, these characteristics suggest that drawbacks resulting from the susceptibility of vinasse to rapid acidification [162] could be successfully offset in two-phase biodigestion schemes. Moreover, phase separation may also characterize a strategy to prevent the accumulation of VFAs in thermophilic systems as the activity of acidogenic bacteria is enhanced at higher temperatures [206].

The reference literature is still poor in terms of using two-phase biodigestion of sugarcane vinasses from Brazilian distilleries, with a few recent studies available [35,36,177], all detailed in Table S3, in addition to Ramos et al. [39]. However, the results reported in these cases, particularly by Ferraz Jr. et al. [35] and Fuess et al. [36], indicated the technical feasibility of vinasse bioconversion in two-phase systems, as both the organic matter removal and energy extraction through methane could be markedly enhanced when applying high OLR and relatively low HRT (Table S3). Complementary studies based on estimates and simulations of biodigestion systems applied to vinasse further confirmed the economic and environmental feasibility of scaling-up two-phase schemes in sugarcane biorefineries [49,50,163]. Fuess et al. [163] pointed out that the costs of producing biogas and electricity obtained in two-phase biodigestion systems can reach equivalent or lower values compared to single-phase layouts. In this case, the higher capital and operating costs intrinsic to two-phase schemes were demonstrated to be offset by the savings resulting from much lower consumption of chemicals. Fuess et al. [50] later associated lower environmental impacts with two-phase biodigestion systems applied to sugarcane vinasse, provided that an optimized use of chemicals in the reactors is observed.

The benefits of phase separation in anaerobic processes extend far beyond enhanced bioenergy recovery through CH₄ production, as the acidogenic step may be optimized for the recovery of a series of bioproducts, such as biohydrogen (bioH₂), organic metabolites (in particular, short-chain organic acids and solvents) and biopolymers. Figure 3 depicts different routes for processing vinasse through two-phase biodigestion in sugarcane biorefineries, in which the methanogenic step may be potentially coupled with different stages of the global process. In terms of practical results, the production of bioH₂ from sugarcane vinasse has been extensively studied in Brazilian research groups in recent years [44,77,79,208,209,210,211,212,213,214,215,216,217,218,219,220,221,222], while studies on the additional pathways considered in Figure 3, i.e., enhanced production of metabolites and biopolymers, are still relatively scarce or non-existent.

Efforts in using H₂ as a bioenergy carrier consider two primary factors: its excellent fuel properties, such as high energy density (120 MJ kg⁻¹ [223]), rapid burning speed, and low minimum ignition point [224]; and the generation of water as the only “residue” from burning [203,225]. Moreover, the biological production of H₂ could offset the energetic and environmental drawbacks inherent to conventional electro- and thermochemical production processes, which are characterized by the intense use of fossil resources [67,226]. Investigations on bioH₂ production, which comprises the gas stream evolved from the acidogenic phase together with CO₂ (Figure 3), i.e., biogas–H₂ (Equations (1), (2), (6), and (7); Table 1), may still be considered fundamental in an attempt to identify adequate conditions to achieve continuous and stable long-term production profiles. Table S5 shows characteristics of the studies on bioH₂ production from sugarcane vinasse in Brazil, while also considering the main aspects of each case. BioH₂ production may be carried out according to different pathways, including photosynthetic, photo-fermentative and exclusively fermentative (dark fermentation) processes [41,225]. Dark fermentation corresponds to the acidogenic phase of the biodigestion process (Equations (1)–(8), Table 1; Figure 2), in which adequate conditions of substrate availability and HRT are provided to stimulate the activity of fermentative bacteria rather than methanogens. In turn, bioH₂ production from photosynthetic processes may be carried out either directly (i.e., using the metabolism of algae, protists, and photosynthetic bacteria to convert water into chemical energy) or indirectly (i.e., growing purple non-sulfur bacteria on VFAs resulting from acidogenesis; Equations (25) and (26)) [41,67,213,225,227].

2Acetate + 4H₂O+ Light → 8H₂ + 4CO₂

(25)

Butyrate + 3H₂O+ Light → 10H₂ + 4CO₂

(26)

Studies on the application of dark fermentation to sugarcane vinasse are more numerous than light-based processes (Table S5), and present a few common characteristics, such as the preference for thermophilic temperature conditions (55 °C) and biofilm systems (fluidized- and fixed-film reactors). High temperatures thermodynamically favor the production of bioH₂ [209,224], and the reduced solubility of gases in such conditions also enhances the release of bioH₂ to the gas phase. In practical aspects, using fixed-bed reactors in the acidogenic phase for processing sugarcane vinasse has led to important achievements, such as defining optimized OLR [77,209] and suitable support material [211], as well as implementing operating strategies to maintain long-term bioH₂ production rates [44] (Table S5). The strict dependence of fermentation on the pH has also been demonstrated with the favoring of lactate accumulation (pH < 5.0), the production of bioH₂ and butyrate with lactate consumption (pH = 5.0–5.5), or the production of the latter coupled with sulfate reduction (pH > 6.0) [77,79]. The fermentative microbial groups respond differently to different pH levels, which directly impact the distribution of soluble metabolites. Moderate-to-high acidic conditions (pH < 5.0) are suitable for the growth of a limited number of microbial groups (among them, lactate-producing bacteria [77]) and are, therefore, highly unsuitable for most microbes (such as those consuming lactate to produce butyrate and other organic acids). Meanwhile, a more diversified metabolite distribution can be expected at higher pH values (>6.0), which may include the participation of propionate-forming bacteria [43] in association with butyrate- and acetate-producing groups and non-fermentative bacteria, e.g., sulfate-reducing groups. Overall, suitable pH conditions are maintained through dosing chemicals (neutralizing and/or alkalinizing agents) in reactors, which is similar to conventional methanogenic reactors. In the particular case of sugarcane vinasse, dosing NaOH is the main pH control approach used [44,77,79,209,210,211,215].

Fluidized-bed systems also show a great potential for the production of bioH₂ from sugarcane vinasse, and usually present the highest volumetric production rates (Table S5), which is most likely due to the enhanced gas–liquid separation triggered by the high recirculation rates. However, some limitations may affect the scale-up of these systems, which primarily include the need to dilute vinasse to prevent negative effects from organic overloads and the high energy consumption associated with the fluidization of the bed (Table S5). The characterization of microbial communities involved in bioH₂ production from sugarcane vinasse was also investigated in recent studies [209,210,211,212,213,219,220,221,222,228] in an effort to associate optimal operating conditions with specific microbial groups, such as those belonging to the Thermoanaerobacterium genus. Despite the marked progress, some aspects still require careful attention for an efficient bioH₂ production from sugarcane vinasse, and these include [i] the definition of suitable reactor configurations, especially considering the improvement of fixed-bed reactors through the application of ASTBR systems; [ii] the adequate control of biomass concentrations within the reactors; and [iii] the adequate control of the pH of the systems coupled with the rational use of chemicals. In turn, light-based processes have further limitations, such as large areas requirements (primarily to offset the negative effects of the high color of vinasse) and external inputs of luminous energy [41,67,205]. Nevertheless, practical results indicate that the application of photo-fermentative processes to acidified effluents may enhance bioH₂ yields by at least 50% of the values usually reported for dark fermentation systems [41], removing organic compounds from the wastewater.

Although the aforementioned aspects still hinder an efficient recovery and exploitation of bioH₂ at larger scales, Fuess et al. [50] simulated different alternatives for the use of bioH₂ in sugarcane distilleries in an attempt to enhance the bioenergy extraction from vinasse, which included biogas–H₂ (i.e., bioH₂ + CO₂) purification and sale of the bioH₂ as a value-added product and biogas–H₂ blending with biogas–CH₄ for biohythane production and biogas–H₂ purification, followed by the injection of bioH₂ into the methanogenic reactor for enhanced biogas–CH₄ production. The production of biohythane aims to improve the fuel properties of biogas–CH₄ [203,229,230]. The term biohythane derives from the term hythane^®, which is characterized by adding hydrogen (typically 10–25%, v/v) to methane-based fuels to improve specific properties of the gas, such as the flammability limit, flame speed, and temperature ignition [203,230,231,232]. In turn, the injection of bioH₂ into the methanogenic reactor aims to upgrade the biogas–CH₄ (i.e., increase the CH₄ content) by stimulating the activity of hydrogenotrophic archaea (Equation (16), as seen in Table 1 [233,234,235]. The production of biohythane was characterized as the best option, and led to energetic gains compared to the use of biogas–CH₄ without requiring the previous purification of the biogas–H₂ stream [50]. Injecting purified biogas-H₂ into the methanogenic reactors could also lead to similar energetic gains compared to using biohythane. However, the purification step would require additional capital and operating costs [50]. Using phase separation exclusively to increase the vinasse biodegradability in the subsequent methanogenic step also proved to be an attractive alternative to such an extent that the limitations in terms of bioH₂ recovery did not hinder the operation of an acidogenic reactor in full-scale biodigestion plants [50,163]. Figure 4 depicts the potential technological approaches to enhance the bioenergy recovery from bioH₂ in biorefineries, which may also include using fuel cells for the production of electricity and thermal energy.

4.5. Alternative Pathways for Bioresource Recovery from Vinasse via Acidogenesis

The optimization of acidogenic systems for recovering bioproducts from the liquid phase, i.e., organic acids and solvents (Figure 3), usually impacts the recovery of bioH₂, based on the application of specific operating parameters to select target microbial populations and further enhance the production of soluble metabolites [43,236,237,238]. In other words, enhanced production of bioH₂ may not always be directly associated with the enhanced production of a given soluble metabolite. Solventogenic pathways should be observed at low pH values (4.0–4.5)—which are unfavorable for bioH₂-producing groups [77]—as a response of the microbial metabolism to the accumulation of VFAs [239], while the proportion of organic acids also depends directly on the type of substrate and operating conditions applied to the systems [43,205,214,240,241]. Organic acids may be directly used in food and chemical synthesis industries [205,240]. In turn, the production of solvents from residues, such as ethanol and butanol, directly increases the energy balance in biorefineries [241].

In particular, results from studies assessing the production of bioH₂ suggest the potential for recovering butyric acid from sugarcane vinasse (Table S5), although the optimization of the hydrogenogenic activity should focus on the acetic type fermentation (Equation (1), Table 1), in which the hydrogen yield is maximized. Butyric acid production from direct sugar fermentation (Equation (2), Table 1) and from the co-fermentation of acetate and lactate (Equation (7), Table 1) frequently prevails in the acidogenesis of vinasse [43,77,79,215,228,238]. Butyric acid concentrations in fluidized-bed reactors reached values often exceeding 2000 mg L⁻¹ [214,217,220,221,222], while values within the range of 1400–6000 mg L⁻¹ were observed in fixed-bed (both APBR and ASTBR) systems [44,77,79,209,210], depending on both the inoculation and operating conditions. Butyric acid may be used as raw material for the production of butanol, either via catalytic [242] or biochemical [243] routes. Butanol is a promising biofuel, and shows important advantages compared to ethanol. In addition to a 30% higher energy density, butanol may be blended with gasoline and diesel at higher proportions, due to the hydrophobicity of its hydrocarbon chain, and it also performs better for the cold start of engines [244,245,246]. The esterification of butyric acid in association with butanol produces butyl butyrate, an alternative jet fuel [205,242], while the production of esters for food flavoring is also a widespread application of butyric acid [205,247]. The production of butyric acid from biochemical routes is more advantageous than the conventional process (butyraldehyde oxidation), because the starting material for this process (propylene) is obtained from crude oil [205].

In contrast to bioH₂ production (characterized by numerous experiments dealing with continuous reactors; Table S5), the few recent studies focusing on the recovery of soluble metabolites from sugarcane vinasse are at an earlier stage of research, and are still based on experiments carried out in batch conditions. Moraes et al. [82] reported butyric and iso-butyric acid concentrations, as high as 10.60 and 10.34 g L⁻¹, respectively, when fermenting blends of sucrose and sugarcane vinasse using microbial inocula subjected to different pretreatment methods (thermal and acidic-thermal). In this case, vinasse was used primarily as a nutrient (rather than carbon) source. Sydney et al. [248] also exploited sugarcane vinasse as a nutrient source for the recovery of organic acids; however, the potential for the concomitant bioH₂ production was also considered. In this case, different supplementary carbon sources were assessed, including sucrose, sugarcane juice, and sugarcane molasses. High butyric acid concentrations were also reported by these authors, with values ranging from 6.3–8.0 g L⁻¹, 4.3–6.4 g L⁻¹ and 4.8–7.0 g L⁻¹, respectively for juice, molasses, and sucrose as carbon sources. Fuess et al. [249] further observed butyric and iso-butyric acid concentrations within the ranges of 6.0–6.4 g L⁻¹ and 5.8–6.4 g L⁻¹, respectively, using vinasse as the only carbon source and the same types of inoculum pretreatment reported elsewhere [82]. A simple energetic assessment carried out by Fuess et al. [249] demonstrated that enhanced butyric acid production could represent an energy recovery potential of 30.9–33.4 MW, which exceeded the numbers observed for highly efficient biohydrogen (<1.0 MW) and methane-producing (25.5 MW) biodigestion plants. Sánchez et al. [43] and Eng et al. [238] presented the systematic assessment of environmental factors impacting VFA production from sugarcane vinasse. Sánchez et al. [43] observed maximum acidification (regardless of the VFA types) at alkaline (pH = 8.8–10.0) and mesophilic (40 °C) conditions. Interestingly, acetate was the main metabolite found in the mesophilic range (30–40 °C), while butyric-type fermentation prevailed at higher temperature (55 °C). The authors suggested that investing in butyrate recovery from vinasse (in concentrations exceeding 3500–4000 mg L⁻¹) represents an economically more attractive option than maximizing the production of medium chain VFAs (e.g., caproic and valeric acids), which eventually demands exhaustive operating efforts.

Butyric acid, in association with other organic compounds, may also be used as a precursor in the production of polymers in biological processes (Figure 3). The production of biopolymers from wastewaters aims to reduce the production costs of bioplastics (up to 15-fold higher than conventional plastics [250]) in an effort to replace oil-derived polymers [251,252,253,254]. These compounds present mechanic and thermoplastic properties similar to conventional polymers, with an additional advantage characterized by their biodegradable character [68,205,255,256,257,258]. Polyhydroxyalkanoates (PHA) are the main group of biopolymers studied and are characterized as linear aliphatic polyesters accumulated as cytoplasmic inclusions in microbial cells subjected to conditions of essential nutrient shortages [251,252,256,259]. The production of poly-β-hydroxybutyrate (PHB) and poly-β-hydroxyvalerate (PHV), which are the most common PHA stored as internal carbon and energy reserve by microorganisms [251], strictly depends on the type of organic acids found in the substrate. Butyric and acetic acids are the precursors for the synthesis of PHB, while PHV is produced primarily from propionic and valeric acids [251,252].

Sugarcane vinasse could be used either in natura or acidified as a substrate for the production of biopolymers (Figure 3). However, a critical assessment of both types of vinasses suggests that better results could be obtained by using acidified streams. Butyric acid concentrations in raw vinasse are considerably lower than in acidified vinasse, with values being usually less than 1000 mg L⁻¹ (Table S1). Similar patterns are observed for acetic and propionic acids (Table S1), which are also characterized as important precursors in the production of PHA. In fact, the production of biopolymers from carbohydrate-rich and/or VFA-poor effluents, such as raw vinasse, is less attractive than using acidified streams, because the supply of sugars to stressed cells favors the accumulation of glycogen as the energy storage instead of PHA [260]. In this context, the application of acidified sugarcane vinasse to the production of biopolymers could most likely result in high proportions of PHB, based on the suitability of vinasse for the production of butyric acid via acidogenic processes [82,248,249]. In turn, the production of PHV from vinasse could be coupled with acidogenic systems operated at high OLR, based on the enhanced production of propionic acid at such conditions, i.e., 4036–5026 mg L⁻¹ (OLR > 360 kg COD m⁻³ day⁻¹ [221]), 3280–4378 mg L⁻¹ (OLR > 90 kg COD m⁻³ day⁻¹ [222]), and >5000 mg L⁻¹ (OLR = 360 kg COD m⁻³ day⁻¹ [214]).

The production of PHA from sugarcane vinasse in the Brazilian sugar and ethanol industry is still poorly exploited, and only two experimental reports could be found up to the publication of this review. Using mixed microbial cultures and fermented vinasse, Oliveira et al. [261] observed maximum PHA storage yields (0.60 g COD-PHA g⁻¹COD) when applying an OLR of 4.5 kg COD m⁻³ d⁻¹ in a sequencing batch reactor after selecting a PHA-accumulating consortium from activated sludge-derived inoculum. PHB (86–77%mol) was the prevailing PHA, as previously speculated for the case of fermented vinasse, while PHV (14–23%mol) accounted for the remaining fraction of the produced PHA. Silverio et al. [262] used concentrated vinasse (7°Brix) as the carbon and mineral source to produce PHB assessing two bacterial strains, namely, Halomonas sp. HG03 and Ralstonia eutropha L359PCJ in continuous stirred-reactor tanks (CSTR). In the case of R. eutropha L359PCJ, which grows on glycerol, vinasse, together with a concentrated glycerol solution, were used as the carbon sources, while in the case of Halomonas sp. HG03, vinasse was used mainly as a nutrient source, such that the carbon supply derived from a concentrated sucrose solution. A higher PHB proportion (66.3%) was found when cultivating R. eutropha L359PCJ, which was associated with a PHB yield of 0.32 g PHB g⁻¹ of glycerol. Halomonas sp. HG03 yielded 0.16 g PHB g⁻¹ of sucrose, with a PHB content of 42.2%.

The integration of PHB production into Brazilian sugarcane biorefineries was also proposed by Nonato et al. [255]; however, they considered a completely different approach based on using sugar as the carbon source. These authors proposed a PHB production model by using facilities, materials, and surplus energy from biorefineries, to significantly reduce PHB production costs. The application of 3.85 tons h⁻¹ of sugar would lead to a PHB production of 1.28 tons h⁻¹ (10,137.6 tons year⁻¹) and require 4147 kW of electricity and 50.56 tons h⁻¹ of steam during the harvesting and inter-harvesting periods (330 days). The diversion of sugar for PHB production would not affect sugar stocks, nor would it significantly affect sugar prices, although approximately 17% of the total sugar produced in the mill would supply the PHB plant. Further assessments of biopolymer production in the context of sugarcane biorefineries were proposed by Amulya et al. [251] and Albuquerque et al. [260], while considering the application of acidified molasses and acidified sugarcane (molasses) vinasse as the substrates, respectively. Although these cases do not refer to the Brazilian sugar and ethanol industry, important results were obtained, especially considering the findings obtained by Amulya et al. [251]. These authors reported the successful production of PHA by Bacillus tequilensis with different proportions of PHB and PHV, which depended on the applied OLR (0.66–2.64 kg COD m⁻³ day⁻¹). PHA accumulation within the cells reached up to 40% of the dry cell weight, with an associated COD removal of up to 73%.

Despite the promising results from using sugarcane vinasse to produce organic metabolites and biopolymers, the recovery of target products via purification processes still negatively affects the economic feasibility of the global process that requires further massive research to optimize full-scale applications. The costs incurred with the recovery of organic acids from fermented substrates typically vary between 30–40% of the total production costs [263], while this value reaches approximately 30% in the recovery of biopolymers [250,253]. However, considering the context of biorefineries, the purification step of both organic acids and biopolymers could be economically and environmentally improved by using solvents, specifically fusel oil generated as byproducts from the fermentation step and recovered through distillation [255]. Bekatorou et al. [263] assessed the application of numerous solvents in the recovery of organic acids from fermented media, and reported recovery efficiencies for the iso-butyric and butyric acids of up to 78.2% and 75.0%, respectively, when using fusel oil. Regarding the environmental performance, the gains could result from the replacement of chlorine-based compounds, which are usually used in the purification of polymers [255].

Apart from focusing on the production and recovery of bioH₂ and organic value-added products, the potential to stimulate the establishment of SRB populations in vinasse-fed acidogenic reactors has recently (re)gained attention as an efficient approach to boost methane production in two-phase biodigestion. Sulfate constitutes one of the main compounds with the potential to negatively impact methanogenesis, as detailed in Section 4.1. Some characteristics of mesophilic (35 °C) vinasse-fed acidogenic-sulfidogenic reactors were first addressed in the early 1990s [264,265], with preliminary results (at relatively high HRT, i.e., 28.8 and 22 h) indicating the superior performance (enhanced sulfate removal coupled with acetate accumulation) of fixed-bed reactors compared to suspended cell growth ones (CSTR). A sharp decrease in the sulfidogenic activity at pH values below 6.2 was also observed in these studies. Operating conditions were not restricted enough to prevent the occurrence of methanogenic activity occurring mainly in the fixed-film reactor [264]. This indicates the importance of selecting adequate inocula to simultaneously reach three metabolic demands, which include [i] the removal of sulfate, [ii] the fermentation of easily degradable substrates, such as glycerol and carbohydrates (with both processes ideally stimulating the acetogenic activity), and [iii] the inhibition of methanogens.

After a gap of ca. 30 years, this topic was re-introduced after the observation of sulfidogenesis during the thermophilic fermentation of sugarcane vinasse when dosing NaHCO₃ to ASTBR systems [77,79]. A sulfate removal efficiency exceeding 80% at harsh operating conditions (HRT = 6 h and OLR = 100 kg COD m⁻³ day⁻¹) was observed, with results indicating the need to maintain adequate pH values (>6.0) as the only environmental requirement of sulfate-reducing groups. Meanwhile, the harsh operating conditions usually applied in vinasse fermentation (Table S5) directly inhibit methanogens. Within this context, the production of fermentative inocula from the natural fermentation of vinasse, as consistently carried out elsewhere [44,77,79,209,210,211,215], may be the key aspect to achieve such a “metabolic success”, because specific SRB (genera Desulfotomaculum and Desulfohalotomaculum [238,266]) have the ability to sporulate and then resist high VFA accumulation, which is in contrast to methanogens. The definition of optimal operating conditions in both continuous sulfidogenic-fermentative and methanogenic reactors is still required to better understand the effective energy gains in the biomethanation of sulfate-free (and eventually acetate-rich) fermented vinasses. Nevertheless, batch experiments showed the potential to maintain methane proportions as high as 80–90% in biogas (compared to less than 70% in the processing of raw, and therefore sulfate-rich, vinasse), which led to a 14% increase in the lower heating value (LHV) of the biogas–CH₄ [81]. From an energy exploitation perspective, this is more representative than investing on bioH₂ production, whose contribution to the global energy potential of biodigestion tends to be lower than 5% [35,36].

4.6. Additional Opportunities for Sugarcane Vinasse Exploitation via Biodigestion

Using biodigestion to process sugarcane vinasse offers additional opportunities for recovering bioresources in biorefineries, regardless of applying two-phase schemes. Regarding the direct recovery of bioenergy through biogas, in addition to electricity and thermal energy production, the biogas–CH₄ stream evolved from methanogenic systems can replace diesel in engines as a vehicular fuel and can be injected into the natural gas grid (Figure 4) [3,46,49,63,192,267,268,269,270]. “Raw” biogas–CH₄, i.e., still containing CO₂ but free of H₂S, could be directly used to replace diesel, but only partially. The lower calorific power of CO₂-containing biogas (which results from the non-fuel character of CO₂) demands the use of diesel as a supplementary fuel source, and the amount of diesel required will depend on the CH₄ content in biogas [63]. Moraes et al. [63] estimated that the use of biogas-CH₄ containing 60% CH₄ could replace only 40% of the diesel used in the fleet of sugarcane biorefineries processing 2 MTC per harvest. Hence, a more efficient use (on both energetic and environmental terms) of biogas–CH₄ for such purposes, i.e., diesel and natural gas replacement, depends on a purification step. In practical terms, the upgrade of biogas–CH₄ to bioCH₄ is required to obtain a final CH₄ content of at least 90% and trace levels of H₂S (<10 mg m⁻³), as defined by the Brazilian National Agency of Petroleum, Natural Gas, and Biofuels (ANP) [271].

Table S6 shows estimates on the potential production and application of bioCH₄ in Brazilian sugarcane biorefineries. Souza et al. [268] indicated that the bioCH₄ obtained from vinasse biodigestion in a large scale sugarcane biorefinery (8 MTC per season) could potentially supply up to 788 buses per day, with a daily autonomy of 400 km. Moraes et al. [63] reported that fossil fuel replacement could be the most economically attractive alternative for biogas in biorefineries when compared to the generation of electricity in engines and the cogeneration of electricity and steam in boilers. The total displacement of diesel, gasoline, and ethanol could reach 8000, 8900, and 11,500 m³, respectively, in annexed or autonomous distilleries over a harvesting period of 167 days. No biogas upgrade was considered in this case. The results presented by Junqueira et al. [267] and Fuess and Zaiat [49] also indicate that the production of bioCH₄ from biogas–CH₄ in distilleries could lead to more favorable economic performances compared to the direct electricity generation in prime movers when considering greenfield and brownfield scenarios. Comparatively, the installation of prime movers is more expensive than the implementation of technological packages dedicated to biogas purification and distribution [49,270]. The replacement of diesel in agricultural operations and trucks directly reduces the production costs (both economic and environmental) of sugarcane. However, relatively significant infrastructure modifications are still necessary to manage eventually high bioCH₄ production levels, such as implementing gas grids nearby sugarcane facilities, and vice-versa. Conversely, the infrastructure required for managing electricity production is found in most sugarcane plants, in view of the sharing of equipment used in thermoelectricity (derived from bagasse) distribution.

The purification of biogas–CH₄ for bioCH₄ production also offers the opportunity for sulfur recovery in biorefineries (Figure 3) [3,192,272], depending on the technology used in the H₂S removal step. The desulfurization of biogas–CH₄ may be carried out according to various physical-chemical (e.g., in situ precipitation with iron salts; chemical adsorption in packed-bed columns, which may also use iron compounds or activated carbon as adsorbents; membrane separation; absorption using water or organic solvents) or biological (e.g., biofiltration; microaeration for H₂S oxidation; microalgae-based) processes [192,273,274]. Despite the technological consolidation of physical-chemical processes [274], the application of biological pathways for the upgrade of biogas–CH₄ offers important advantages, especially in terms of full-scale plants. Reaching equivalent removal efficiencies with reduced operating costs, eliminating chemical inputs and secondary residual streams (e.g., chemical sludge from H₂S precipitation with metallic salts), and potentially recovering bioresources, such as elemental sulfur and microalgae [192,273,275,276] exemplify the list of advantages. Among the upgrading options, the recovery of elemental sulfur in biorefineries could be obtained through the application of microaeration for the partial oxidation of H₂S (Equation (27)). In practical aspects, air (or pure oxygen) can be supplied either directly into the influent stream of the methanogenic reactors (liquid phase) or into the headspace of the systems. This technology is based on the activity of specific bacterial groups that grow lithoautotrophically on H₂S under limiting conditions of oxygen availability [273]. Consequently, sulfur reaches an intermediate oxidation state instead of being transformed into sulfate.

H₂S + 0.5O₂ → S + H₂O

(27)

Experimental results from the monitoring of pilot-scale methanogenic reactors applied to sugarcane vinasse indicated H₂S concentrations in the biogas–CH₄ in the range of 0.5–2.2% v/v (5000–22,000 ppmv) [276,277]. Assuming the value of 256 g mol⁻¹ for the molar mass of elemental sulfur [278], a H₂S concentration of 2.2% v/v in biogas (98.22 mol H₂S Nm⁻³ of biogas) could result in a theoretical elemental sulfur production of 25.14 kg S Nm⁻³ of biogas. Concerning economic aspects, gross earnings of 2.26 USD Nm⁻³ of biogas could be obtained by selling elemental sulfur, using a market price of 90.00 USD ton⁻¹S as a reference [279]. Using a very simple comparative approach (once prices are quoted in different periods), this value is at least 90% higher than the production costs estimated for bioCH₄, i.e., 0.95 USD Nm⁻³ [64], 0.30–0.31 USD Nm⁻³ [267], and 0.08–0.10 USD Nm⁻³ [49].

Elemental sulfur could also be blended with the biodigested vinasse and other residual streams from processing sugarcane to form an organomineral fertilizer (Figure 3), when considering some modifications in the system proposed by Gurgel et al. [132]. Junqueira et al. [267] also proposed the production of an organomineral fertilizer from biodigested sugarcane vinasse, which included a concentration step by evaporation (25°Brix) followed by the addition of ammonia up to a desired nitrogen–potassium ratio. In this system, the condensates recovered from the concentration step could be recycled as process water in different processes of the biorefinery, such as preparing yeast prior to fermentation. In particular, these authors reported the opportunity to incorporate relevant economic and environmental gains to the global sugarcane processing chain, primarily by reducing the inputs of diesel and mineral fertilizers, whose production chains also require high fossil fuel levels. Barrera et al. [272] highlighted that the sulfur recovered from biogass–CH₄ has a great potential for replacing the mineral sulfur supply provided by the inputs of ammonium sulfate in the cultivation of sugarcane vinasse.

5. Outlook: Implementing Biodigestion in Sugarcane Biorefineries

There are many opportunities for exploiting the recovery of bioresources from sugarcane vinasse based on the direct or indirect bioenergy generation from biogas and value-added metabolites obtained from the controlled bioconversion of organic matter. Regarding a preliminary (short-term) approach, the application of biodigestion as a robust technology for processing vinasse should be first disseminated in the Brazilian sugar and ethanol industry. Therefore, the production of biogas–CH₄ and its direct conversion into energy (instead of investing in the exploitation of fermentation products) should characterize the most immediate target of biodigestion in sugarcane biorefineries. Many favorable aspects could be observed, including [i] the technological maturity of large scale prime movers, such as engines and gas turbines, which combine high conversion efficiencies with operating robustness [280]; [ii] the suitability for recovering both electricity and thermal energy from biogas [280]; and [iii] the already existing infrastructure in biorefineries for exporting electricity to the grid. This approach requires a relatively simple infrastructure and has potential to promptly supply electricity demands, which are still much higher than the demands for bioCH₄ [270] or soluble products (from fermentation) in the Brazilian case.

The increasing relevance of bioenergy projects also favors the prompt implementation of full-scale biodigestion-power plants for generating electricity from biogas in biorefineries. In particular, the first full-scale project for electricity generation from biogas in the sucro-alcohol industry was approved in a public auction in Brazil just a few years ago (2016) for a thermoelectric plant with a 20.8 MW-capacity. The Brazilian Electricity Regulatory Agency (ANEEL) uses public auctions to purchase renewable energy from different sources in an attempt to encourage the production of bioenergy, decentralize the production of electricity exported to the grid, and diversify the Brazilian energy matrix [48]. Historically, projects related to using biogas have been absent from public auctions of renewable energy in Brazil, due to the higher profitability provided by other energy sources, such as wind, biomass, and bagasse [3]. However, financial incentives from the government, such as subsides, funding programs to reduce investment costs, and more restrictive environmental legislations, are still imperative to enable the implementation of full-scale biodigestion plants in biorefineries [3,49,184]. Although the lack of a national biogas program also affects the processing of wastewaters through biodigestion [184], the price paid by the electricity produced from the biogas in the aforementioned project (R$ 251.00 MWh⁻¹ or USD 46.83 MWh⁻¹) was the highest value compared to other biofuels, such as sugarcane bagasse (R$ 181.25–245.20 MWh⁻¹ or USD 33.82–45.75 MWh⁻¹) and wood chips (R$ 199.00–243.21 MWh⁻¹ or USD 37.13–45.37 MWh⁻¹), which characterizes the bioenergy recovery from sugarcane vinasse as a promising approach.

The further diversification of alternatives for the exploitation of sugarcane vinasse will largely depend on the profile of each biorefinery in order to consider different aspects, which include: [i] the type of destination for the solid residues, i.e., bagasse, filter cake, and trash; [ii] the technological maturity of the alternative bioprocesses; and [iii] the location of the biorefinery relative to the natural gas grid. The cogeneration of energy from solid residues may favor adopting approaches for vinasse management that do not aim to directly recover energy from biogas, because electricity and steam obtained from bagasse tend to completely supply the requirements of the plants. However, this option does not eliminate the possibility of inserting biogas to boost the energy balance of the biorefineries, as detailed in Section 4.3. bioCH₄ production from biogas also characterizes a technology that could be promptly coupled with the implementation of vinasse biodigestion in biorefineries to focus on the replacement of diesel in transport operations. Although exporting bioCH₄ to the natural gas grid is an attractive option [49,267,270], the project may only be economically feasible in cases where the biorefinery is close to the grid. Distribution costs for long distances may be prohibitive because constructing networks for bioCH₄ distribution is costly [267].

Regardless of the predicted use for biogas, the operation of an independent acidogenic reactor characterizes a suitable option for full-scale biodigestion plants in biorefineries. Regarding short-term applications, the acidification of vinasse may be used exclusively for enhancing both the substrate biodegradability and bioenergy recovery through CH₄, as supported by experimental results [35,36]. The non-recovery of bioH₂ or other types of bioproducts from acidogenesis does not affect the economic feasibility of phase separation, as described by Fuess et al. [163,270]. In terms of medium- to long-term applications, the implemented acidogenic reactors could be modified to meet the requirements of the desired production process such that the extent of the modifications depends on the target bioproduct. For instance, the enhanced production of organic metabolites is likely to mainly require changes in operational parameters and chemical dosing, rather than installation aspects. In turn, additional reactors could be required when considering the production of biopolymers from the acidified vinasse, which also require the installation of aeration systems. Before any definitions, techno-economic assessments and scenarization-based approaches will be imperative to identify market opportunities. In contrast to the case of bioenergy production from biogas (Section 4.3.1), the literature is poor in terms of detailing techno-economic aspects of vinasse fermentation dedicated to the production and recovery of soluble metabolites. Nevertheless, it should be highlighted that the methanogenic step will fit into different stages of the global process, regardless of the type of bioresource recovered from the acidogenic phase (Figure 3).

Considering a broader analysis, it is worth noting that biodigestion has a great potential to be used as the core conversion step of the raw materials in biorefineries [270,281], instead of only processing residual streams (Figure 5). When focusing on the sugar and ethanol industry, important advantages could be observed by directly applying biodigestion to the processing of energy crops, i.e., sugarcane in this case:

• Ethanol plants are capital intensive and require several unit processes (e.g., fermentation, centrifugation, and distillation) that demand high levels of energy, while biodigestion requires much less investment and energy inputs [46,269]. Fuess et al. [50] estimated that the installation costs of biodigestion systems coupled with power plants for recovering bioenergy from vinasse would be less than 10% of the total investment with large scale sugarcane biorefineries (with milling capacities of 4 MTC and 1.7 MTC in the harvesting and inter-harvesting periods, respectively). This particular analysis already considers the installation of two-phase anaerobic plants. Similarly, Junqueira et al. [267] also indicated that the biodigestion of vinasse coupled with the recovery of bioenergy accounts for a minimal fraction of the total investment in sugarcane biorefineries, and ranges from 3.4–7.5% depending on the technological package considered for exploiting the biogas–CH₄ (cogeneration of electricity and steam in boilers, electricity generation in engines, bioCH₄ production for diesel replacement and bioCH₄ production, and further injection into the gas grid).
• The energy potential of 1 ton of cleaned sugarcane is approximately 1718 MCal, which is characterized by the fractions of sugars (153 kg, 608 Mcal), bagasse with 50% moisture (216 kg, 598 Mcal), and trash with 15% moisture (165 kg, 512 Mcal) [282]. Thus, the maximum theoretical energy recoverable from sugarcane through 1G ethanol production could reach roughly 35%, when assuming the conversion of the total fraction of sugars and without taking into consideration energy losses due to parallel metabolic pathways and yeast growth. However, in practical aspects, the ethanol productivity (in which all “metabolic losses” are included) currently obtained in 1G sugarcane biorefineries (82 L TC⁻¹ [5]) leads to an effective energy recovery below 25% of the total energy in sugarcane (416 Mcal) when considering a LHV of 21.22 MJ L⁻¹ for ethanol [62]. Conversely, van Haandel and Catunda [46] and Wilkie [269] indicated that biodigestion may lead to a more efficient use of the sugars present in sugarcane. The direct conversion of the juice into biogas–CH₄ would recover approximately 50% of the total energy present in the sugarcane. Fuess et al. [281] associated an energy return on investment (EROI) ratio to the direct biogas production (and further conversion into electricity) from juice and juice/bagasse as four times higher than the one observed in ethanol production. In practical terms, the results indicate that producing energy from biogas is more efficient than using ethanol, which fully suits one of the UN Sustainable Development Goals (#7: Ensure access to affordable, reliable, sustainable, and modern energy for all [283]). The further conversion of bagasse into biogas in anaerobic systems, either via solid-state biodigestion [284,285] or via sugar-rich hydrolisates [10,11,12,286] (Figure 5), could enhance the energy recovery to 60–80% [46,269], while the integration of 1G and 2G sugarcane biorefineries may increase the energy recovery through ethanol production to only 30–34% (i.e., 518–589 Mcal, considering global ethanol productivities in the range of 102–116 L TC⁻¹ [5]). Some energy recovery improvements in 2G ethanol plants could be achieved through the biodigestion of 2G vinasse and pentose liquor (HSW streams depicted in Figure 1b), as proposed elsewhere [66].
• The better performance of biodigestion compared to yeast-driven fermentation in terms of energy extraction directly affects the energy potential of the produced biofuels, because the LHV of CH₄ (50.00 MJ kg⁻¹ [223]) is approximately double the value observed for ethanol (26.90 MJ kg⁻¹ [223]). Moreover, biogas applications (either purified or non-purified) are diverse (Figure 5), insofar as the layout of the biorefineries could be designed according to the most profitable option, i.e., the production of biochemicals (through acidogenesis), electricity generation from biogas–CH₄, and bioCH₄ production. In practical terms, biogas-based biorefineries are much more flexible than those producing ethanol as the main biofuel [270,281], which fully suits the biorefining concept presented elsewhere [41]: “the sustainable processing of biomass into a spectrum of marketable products, which means: energy, materials, chemicals, food and feed”. Naturally, the implementation of sugarcane biorefineries using biodigestion as the core process does not exclude industrial plants based on the traditional production of sugar and/or ethanol, in which the biodigestion of vinasse should necessarily be incorporated. In short, the aforementioned aspects could be carefully considered in future greenfield projects of the sugar and ethanol sector to consider biodigestion as an efficient step for exploiting sugarcane, either directly or from the residual fractions.

Finally, an equally huge amount of opportunities results from exploiting the co-digestion of sugarcane-derived residues, as substantially studied from the middle of the last decade. For instance, numerous experimental studies, usually dealing with the co-processing of vinasse and filter cake, are currently available [8,13,287,288]. There are also studies addressing techno-economic analysis of biogas use in such co-digestion-based alternatives [192,289]. Thus, a successful future is expected for biodigestion within the context of sugarcane processing plants, as also motivated by the creation of the Brazilian National Biofuel Policy (RenovaBio) and other correlated policy acts, in which biofuel production from biodigestion plants is one of the key aspects considered to diversify the Brazilian energy matrix. Additional references [290,291,292] are found in Table S2, while references [293,294,295] are cited in Tables S3 and S4, respectively.