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Pretreatment in Vortex Layer Apparatus Boosts Dark Fermentative Hydrogen Production from Cheese Whey

Federal State Autonomous Educational Institution of Higher Education, Lobachevsky State University of Nizhny Novgorod, 603950 Nizhny Novgorod, Russia
Federal State Budgetary Educational Institution of Higher Education “Nizhny Novgorod State University of Architecture and Civil Engineering”, St. Ilyinskaya 65, 603950 Nizhny Novgorod, Russia
Federal State Budgetary Scientific Institution “Federal Scientific Agroengineering Center VIM”, 1st Institutskiy Proezd 5, 109428 Moscow, Russia
“Fundamentals of Biotechnology” Federal Research Center, Russian Academy of Sciences, 119071 Moscow, Russia
Moscow State Academy of Veterinary Medicine and Biotechnology, 109472 Moscow, Russia
Authors to whom correspondence should be addressed.
Fermentation 2022, 8(12), 674;
Submission received: 7 November 2022 / Revised: 20 November 2022 / Accepted: 24 November 2022 / Published: 25 November 2022
(This article belongs to the Special Issue Anaerobic Fermentation and High-Value Bioproducts)


Dark fermentation (DF) is a promising process for mitigating environmental pollution and producing “green” H2. However, wider implementation and scaling of this technology is hampered by insufficient process efficiency. In this work, for the first time, the effect of innovative pretreatment of cheese whey (CW) in a vortex layer apparatus (VLA) on characteristics and DF of CW was studied. Pretreatment in VLA resulted in a heating of the CW, slight increase in pH, volatile fatty acids, iron, and reduction in fat, sugar, and chemical oxygen demand (COD). The biochemical hydrogen potential test and analysis of H2 production kinetics confirmed the significant potential of using VLA in enhancement of dark fermentative H2 production. The maximum potential H2 yield (202.4 mL H2/g COD or 3.4 mol H2/mol hexose) was obtained after pretreatment in VLA for 45 s and was 45.8% higher than the control. The maximum H2 production rate after 5 and 45 s of pretreatment was 256.5 and 237.2 mL H2/g COD/d, respectively, which is 8.06 and 7.46 times higher than in the control. The lag phase was more than halved as a function of the pretreatment time. The pretreatment time positively correlated with the total final concentration of Fe2+ and Fe3+ and negatively with the lag phase, indicating a positive effect of pretreatment in VLA on the start of H2 production.

1. Introduction

Anaerobic digestion (AD) is one of the most promising methods for processing organic waste and wastewater treatment, since this process produces biogas containing a valuable energy carrier in the form of methane. Another shortened anaerobic bioprocess is called dark fermentation (DF). DF comprises of the first two stages of AD–hydrolysis and acidogenesis–and can be used to produce high-added-value soluble metabolite products such as ethanol, lactate, short-chain fatty acids, and hydrogen-rich biogas [1]. Methanogenic archaea and some other microbial groups such as homoacetogens are hydrogen scavengers, so it is crucial to suppress their activity to ensure high H2 yields. Several strategies are known for this purpose, such as pre-treatment of the inoculum [2], maintaining a low hydraulic retention time (HRT) [3,4], operating at a pH below 7, etc. [5,6,7]. In the AD process, the HRT is typically higher, and the pH is higher than 7. DF is typically followed by AD to achieve maximum energy production and organics removal [8].
One of the limiting stages of AD and DF is hydrolysis, which can be accelerated by the pretreatment of feedstock. To enhance hydrolysis, physical, chemical, biological, and combined pretreatment methods are used [9]. Physical methods are the most studied and include thermal, ultrasonic, microwave, ultraviolet pretreatment methods [10] and high-pressure homogenization [11]. However, various studies show that combined pretreatment methods such as thermal alkaline treatment, thermal treatment and ozonation, thermal ultrasonic treatment, thermal acid treatment, alkaline treatment and ionizing radiation, and thermal ozone ultrasonic treatment are more effective than individual methods [12,13]. Thermal and thermochemical pretreatment can lead to the formation of undesirable by-products, such as furfural and 5-hydroxymethylfurfural, which inhibit the activity of microorganisms–in particular hydrogen-producing bacteria [14]. Prabakar et al. [15] reported that the choice of the appropriate pretreatment is largely determined by the nature and properties of the feedstock. The energy feasibility of a feedstock pretreatment method depends on the degree of feedstock disintegration and biofuel (hydrogen and/or methane) production and the energy and environmental benefits associated with anaerobic digestion technologies. To broadly analyze the upscale feasibility of the pretreatment techniques, it is also necessary to include an environmental impact assessment. Thus, efforts should be made in life cycle assessment (LCA) of feedstock pretreatment methods [16].
The VLA is a promising method for the pretreatment of feedstock prior to AD. The effectiveness of this type of pretreatment for increasing the yield of biogas has been shown in a number of our previous works [17,18,19,20]. Pretreatment of the organic fraction of municipal solid waste in VLA resulted in an increase in its solubility and bioavailability, which led to an increase in biogas yield [19]. The principle of VLA operation consists in a combined effect of electromagnetic, mechanical, electrolysis, and cavitation impacts on the feedstock during the rapid movement of ferromagnetic particles (steel needles or rods). In addition, due to the collision of the ferromagnetic particles, the pretreated feedstock is enriched with steel particles, the content of which may vary depending on the treatment duration and on the type of feedstock.
Metals in various forms (zerovalent, oxides, ions) are among the most common additives that are used to improve AD or DF. Iron is an integral component of ferredoxins and/or, together with nickel, is also a main component of the active site of hydrogenases. An increase in hydrogenase activity was observed with an increase in the FeSO4 concentration to 10.9 mg/L, providing a hydrogen yield of up to 41.6 L/d [21]. A FeSO4 concentration of 300 mg/L improved the hydrogen yield to 2.15 mol H2/mol glucose with the highest acetate content of 3.87 g/L in 24 h [22], while the addition of zero-valent iron during AD increased acetogenesis with propionate formation [23].
CW is the major by-product of the dairy industry [24]. It is considered as a potential feedstock for producing renewable biofuels in the process of AD or DF [25]. Lactose, the main component of CW, is a disaccharide consisting of one molecule of glucose and one molecule of galactose. The DF process is based on the activity of heterotrophic microorganisms capable of converting organic matter, primarily glucose, into hydrogen and soluble metabolite products (SMP), such as volatile fatty acids (VFA). Unless an electron transport chain is artificially introduced in the anaerobic environment, then the oxidation of glucose to VFA proceeds by dehydrogenation, which leads to the formation of a large amount of hydrogen [26,27]. In an anaerobic environment, lactose is rapidly hydrolyzed to form glucose and galactose (Equation (1)), the fermentation products of which are the same as those of glucose [28].
C12H22O11 (lactose) + H2O → C6H12O6 (glucose) + C6H12O6 (galactose)
However, to produce biohydrogen, galactose must first be converted to glucose-1-phosphate via the Leloir pathway (which requires energy input) and then metabolized to form various SMPs and hydrogen [29]. A previous study showed the biohydrogen production rate and yield by Clostridium beijerinckii from glucose were 321% and 47% higher than those from galactose [30]. This suggests galactose is less favored for biohydrogen production than glucose.
Proteins are not beneficial for the direct production of biohydrogen, but they can provide the necessary nitrogen source to improve the growth and the vital activity of acidogenic bacteria [31]. Protein monomers (amino acids) are unlikely to produce hydrogen under normal fermentation conditions [32]. At the same time, in their recent study, Xiao et al. [33] achieved improved biohydrogen production from protein wastewater by altering protein and amino acid structure through pretreatment at elevated pH 12 and DF at pH 10. Triacylglycerol, which is the main component of lipids, is composed of 10% glycerol and 90% long chain fatty acids (LCFAs) [34]. Glycerol is a readily available substrate for the production of biohydrogen (theoretical yield of biohydrogen is 3 mol H2/mol glycerol) [35], while LCFAs are poorly degraded during DF. Fermentation of LCFAs to shorter chain molecules is thermodynamically unfavorable and non-spontaneous (ΔG is positive) unless syntrophically associated with methanogenesis [36].
Ozone treatment, membrane filtration, acidification with lactic acid, neutralization with lime, and both microwave and ultrasonic radiation [37,38] have been used to improve the efficiency of AD of CW [39,40,41]. However, the main problem in DF of CW is still the rapid oxidation of sugars with the formation of VFAs, as a result of which the process of hydrogen formation proceeds less efficiently [42,43]. Co-fermentation with cow dung or alkali addition [44,45] is most commonly used to increase the buffer capacity. In connection with the combined impact on the feedstock described above, in this work, it was decided for the first time to test the pretreatment method in VLA to increase the stability and intensify the DF process due to the homogenization/destruction of the polymeric substances of CW, and the concomitant introduction of steel particles.
The aim of this study was to evaluate (1) changes in the physical and chemical properties of CW after pretreatment in VLA; (2) the iron content in the pretreated CW; (3) the biochemical hydrogen potential of the pretreated CW; (4) a possible relationship between the dissolved forms of iron and the kinetics of H2 production.

2. Materials and Methods

2.1. Feedstock and Inoculum

CW in the form of a solution was obtained by diluting 100 g of dry CW in 1 L of distilled water by stirring until complete dissolution of CW. The resulting CW solution (hereinafter referred to as the CW) had the characteristics presented in Table 1.
A pure culture of thermophilic anaerobic bacterium Thermoanaerobacterium thermosacharoliticum SP-H2 was used as an inoculum [1]. The culture was grown using lactose as a carbon source for adaptation to CW. As a rule, the rate of biochemical reactions is higher in the thermophilic mode. Secondly, this bacterium was found to be very tolerant of low pH values, for example, it dominated the microbial community (and was isolated from this microbial community) in the thermophilic DF reactor, which produced 2 mol H2/mol hexose at pH 4 [46]. Since pH in this work was not controlled (which will inevitably lead to acidification of the medium), T. thermosacharoliticum SP-H2 was preferred, as at low pH values, most hydrogen-producing bacteria yield less hydrogen because they change their metabolism to the solventogenesis pathway [47].

2.2. Pretreatment of CW in VLA

CW was pretreated in a VLA at a magnetic field frequency of 50 Hz and a power of 4 kW, as described previously [17]. Briefly, CW was loaded into a 500 mL non-magnetic stainless-steel flask, and then ferromagnetic particles (steel rods 20 × 1.5 mm, total weight 200 g) were loaded into the flask. The flask was mounted on the VLA, and CW was pretreated for 5, 20, 45, and 90 s.

2.3. Biochemical Hydrogen Potential Test

The mineral medium (24.7 mL) [1], used to prevent nutritional deficiency, 2.33 mL of the pretreated CW, and 3 mL of inoculum in the exponential growth phase (OD600 = 0.9) were added to 120 mL glass vials. CW was added to the control vials without pretreatment. To reduce lag phase [48], the initial pH was adjusted to 7.0 with 3 M HCl or 1 M NaOH. The flasks were thoroughly purged with argon, closed with rubber stoppers and aluminum caps, and incubated in the dark at 55 °C without mixing. After 8 days, when the cumulative production of H2 reached a plateau, the liquid phase was analyzed for the content of dissolved iron. All tests were carried out in duplicate.

2.4. Analytical Methods and Data Analysis

The amount and composition of VFAs in CW before and after treatment in VLA was determined on a GCMSQP2010 Ultra chromato-mass spectrometer (Shimadzu, MD, USA), as described previously [19]. The total solids (TS) content was determined after drying the samples to a constant weight at 105 °C. The non-volatile solids (NVS) content was determined by burning a dry sample in a muffle furnace at 600 °C. The content of volatile solids (VS) was calculated as the difference between TS and NVS. Chemical oxygen demand (COD) was determined by the bichromate method. The pH was measured using a WTW pH 3110 SET portable pH meter (WTW, Weilheim, Germany). The sugar content was determined by HPLC (LC-20AD Prominence (Shimadzu, Kyoto, Japan)). The fat content was determined by extraction with petroleum ether in a Soxhlet apparatus. To measure the concentrations of soluble forms of iron, spectrophotometric methods, Method 8008 (sum of Fe2+ and Fe3+) and Method 8146 (Fe2+) were used (Hach Lange, Düsseldorf, Germany). Measurements on a spectrophotometer DR 5000 (Hach Lange) were carried out in the liquid phase at the end of incubation after preliminary centrifugation at 10,000 rpm for 3 min. The amount and composition of biogas, pH, the concentrations of soluble metabolite products, and the kinetic parameters of biohydrogen production in the biochemical hydrogen potential test were determined according to [1].

2.5. Statistical Analysis

Statistical analysis was carried out using standard mathematical methods (Student’s t-test and calculation of the standard deviation) using the Microsoft Excel program. The data group was considered homogeneous if the mean square deviation σ did not exceed 10 per cent. The differences between the data groups were considered valid under the probability criterion p < 0.05. Spearman’s rank coefficients were calculated using IBM SPSS Statistics software version 26.0 and the heat map was built in Python version 3.8.3.

3. Results and Discussion

3.1. Changes in Physical and Chemical Properties of the Pretreated CW

The analysis of changes in the concentration of organic compounds is useful to evaluate the effect of pretreatment of the feedstock before AD [49]. The purpose of pretreatment is damage to compact organic structures, their disintegration, and solubilization, which increases the availability of the substrate for hydrolytic and acidogenic microorganisms [50]. Efficient conversion of organic compounds into VFA directly affects the rate and efficiency of AD [51]. The most commonly monitored pretreatment performance indicators include COD, TOC (total organic carbon), content of glucose and other sugars, etc. [52].
In the present study, changes in temperature, COD, pH, VFA, ethanol, lactose, glucose, TS, and VS of CW after pretreatment in VLA for 5, 20, 45, and 90 s were analyzed. An increase in the time of pretreatment led to heating of the CW, and with an increase in the treatment time of more than 20 s, the temperature of CW increased sharply (Figure 1). At the initial temperature of 30 °C, the temperature was 40 and 48 °C after 45 s and 90 s pretreatment. This feature of VLA may be beneficial to reduce the temperature shock for the anaerobic microbial community when the treated feedstock is fed to an anaerobic reactor and to increase the stability of the DF, which is especially important in the thermophilic mode of operation.
The greatest decrease in COD (by 11.7%) was observed after 45 s of treatment in VLA (Figure 1). However, taking into account the error in COD measurement (about 12%), changes in COD can be considered insignificant, within the measurement error. Mainardis et al. [37] found no correlation between UD input energy and soluble COD (CODsol) in CW. CODsol increased in fat-free CW by 14.5%, while UD pretreatment at 80 W resulted in a 2% increase in COD in fat-containing CW [37]. The greatest difference was observed between the control and 300 s UD pretreatment, after which CODsol increased by 11,700 ± 530 mg/L. Leano and Babel [53] showed a 40% reduction in COD after sonication of cassava wastewater. The hydrogen yield from cassava wastewater, pretreated with ultrasound for 45 min, was 0.913 mol H2/g COD [53].
Fats were detected only in the CW without pretreatment (20 mg/L) and after 5 s pretreatment (13.9 mg/L). With longer pretreatment times, no fats were detected. A possible reason for the absence of fats may be their decomposition or deposition on the surface of the VLA flask.
VS removal is a commonly used parameter to evaluate the waste reduction efficiency in the DF process [41,54]. The greatest decrease in TS and, accordingly, a proportional increase in NVS and a decrease in VS were observed after 45 s of pretreatment in VLA (Figure 2). Pretreatment in VLA for 5, 20, 45, and 90 s reduced the VS/TS ratio by 0.39%, 0.11%, 0.43%, and 1.16%, respectively. The decrease in COD after pretreatment in VLA was 5.88% (5 s), 3.43% (20 s), 11.76% (45 s), 7.84% (90 s).
Pretreatment in VLA contributed to a sharp increase in the ethanol content (Figure 3). The ethanol content compared to untreated CW after 45 and 90 s of pretreatment increased by 38.46 and 75.77%. The concentration of acetate after 5 and 20 s of pretreatment decreased, while after 45 and 90 s of pretreatment increased by 12.77% and 43.62%. The content of butyrate decreased by half after 5 s of pretreatment and did not change with increasing pretreatment time. The content of propionate increased after 5 s, 45 s and 90 s by 25%, 25% and 50%, while after 20 s it decreased by 8.33%. It should be noted that the 20 s pretreatment was less pronounced: the content of ethanol and acetate decreased by 8.97% and 24.47%, respectively, which correlates with the smallest decrease in the ratio of TS/VS by 0.11% and COD by 3.43%. Interestingly, the pH of CW after pretreatment in VLA increased, despite the increase in the content of VFAs.
The sugar content showed a trend towards a decrease in the content of lactose in CW by 17.27, 11.59, 22.53, and 26.29% and glucose by 23.33, 15.86, 25.83, and 31.67% after 5, 20, 45, and 90 s pretreatment (Table 2). The largest decrease in the concentration of glucose and lactose was observed after 90 s of pretreatment, which correlates with the largest increase in the concentration of ethanol, propionate, and acetate. Perhaps this is due to the processes of electrooxidation, which can occur during the movement and collision of ferromagnetic particles in a rotating magnetic field [55].

3.2. Dynamics of Biohydrogen Production

Results of the biochemical hydrogen potential test confirmed the positive effect of pretreatment in VLA on the dark fermentative hydrogen production from the CW. The hydrogen productivity increased with an increase in the pretreatment time in VLA to 45 s, at which the maximum cumulative yields of biohydrogen were achieved (Figure 4). However, for CW pretreated in VLA for 90 s, a significant decrease in biohydrogen productivity was observed, which can be explained by the possible accumulation of inhibitory compounds and/or radicals.
The concentration of biohydrogen in the composition of biogas was the highest in the control sample and reached 36% on the eigth day. However, the content of biohydrogen in biogas was the highest in the first days of incubation in vials containing pretreated CW. Thus, in vials with 45 s pretreated CW, the hydrogen content in biogas after the first day was 10 times higher (30.1 ± 1.5)% than in the control and remained stable for eight days of DF.

3.3. Dynamics of Soluble Metabolite Products

It is known that the biochemical/metabolic processes that occur during DF are largely dependent on pH and alkalinity. In the untreated CW, an increase in the content of VFAs was observed on the third day of incubation, and on the next day, the concentration of SMPs sharply increased, which led to a decrease in pH. The main products were formate, acetate, and butyrate (Figure 5). Pretreatment in VLA resulted in reducing the lag phase, which is already confirmed by the active accumulation of VFAs after the first day of incubation. At the same time, the VFA concentration remained relatively stable for eight days of incubation, except for CW after 90 s of pretreatment. Thus, the pH was maintained at the level of 4.7–6.1 for vials with the addition of the CW after 5, 20, and 45 s of pretreatment. However, the SMP composition in these treatments was different. Pretreatment for 20 and especially 45 s contributed to the relative stability of pH and SMP concentration. N-butyrate and acetate were the dominant SMPs. It is known that anaerobic degradation of lactose results in a high production of acids, converting it into butyrate [56]. The predominant production of butyrate, rather than acetate, is considered by many researchers to be the most advantageous pathway for the highest hydrogen yield [56]. Higher butyrate content at 20 and 45 s pretreatments correlated with the higher production of biohydrogen and its content in biogas (Figure 4). A small production of ethanol (up to 5.85 mM), which reduces H2 yield, was also observed in all treatments (Figure 5). The formation of propionic acid during DF was not observed. Similarly, Yang et al. [54] observed a decrease in the proportion of propionate when Fe2+ was added at a concentration of 100, 200, and 300 mg/L, and with a further increase in Fe2+ to 400 and 600 mg/L, propionate was not detected in the DF of grass. This may be because the addition of Fe2+ can reduce the redox potential in the fermentation system, which leads to a change in metabolism from propionate to acetate or butyrate production [51]. Lee et al. [57] also found that the addition of Fe2+ (1.3–1763.8 mg/L) can increase the efficiency of sucrose degradation in DF. However, Wang and Wan [58] found that the addition of Fe2+ (50–1500 mg/L) leads to a decrease in the efficiency of glucose degradation during DF. It is also interesting to note that the 90 s pretreatment resulted in a reduction in hydrogen production. This could be explained by a significant difference in the SMP composition; in particular, the accumulation of large amounts of lactic acid (up to 17 mM) was observed after the third day of the incubation. It is noteworthy that at the optimal pretreatment time (45 s), lactic acid formation was not observed. The formation of lactic acid is often accompanied by a significant decrease in the production of hydrogen [59]. At the same time, some studies show the possibility of obtaining hydrogen from lactic acid by the appropriate microbial community under optimal DF conditions [60].

3.4. Soluble Forms of Iron

Fe is the most commonly added trace element in DF due to its crucial role in the metabolism of hydrogen-producing bacteria and enzyme activity [15,61,62,63,64]. Fe is an essential element for the synthesis of hydrogenases (Fe, Fe-Fe, and Ni-Fe hydrogenases) and ferredoxin [61]. The addition of iron to an iron-deficient DF system can increase the soluble iron content, which can increase the growth and metabolism of microorganisms. However, most of the iron (zero valent) introduced into the anaerobic system is difficult to access for microorganisms [65].
The lower VFA concentration in the iron-supplemented AD reactor indicates that zerovalent iron (ZVI) promotes VFA conversion by improving the metabolic activity of anaerobic bacteria [66]. ZVI, a strong reducing material, is one of the most well-known additives for improving the AD of sewage and waste activated sludge due to its non-toxicity, abundance, low cost, and ease of production [67].
In an anaerobic system, ZVI reacts with water to form hydrogen (Equation (2)). As this reaction proceeds, the precipitation of Fe(OH)2 becomes favorable, and then Fe(OH)2 can turn into magnetite according to Equation (3) [68]:
Fe + 2H2O → Fe(OH)2 + H2
3Fe(OH)2 → Fe3O4 + H2 + 2H2O
Lee et al. [21] concluded that Fe2+ is an important component of biohydrogen production pathways because it increases the activity of hydrogenase, i.e., enzymes involved in molecular hydrogen synthesis. The sucrose degradation efficiencies were close to 1.0 when iron concentrations of the experimental mixtures were between 200 and 800 mg FeCl2/L [21]. In addition, some authors reported that increasing the concentration of Fe2+ promotes acetate-butyrate-type fermentation with the formation of biohydrogen [57,69]. The improvement in biohydrogen production due to iron supplementation (up to two times after ZVI addition [70]) can be explained by creating an optimal environment for acidogenic microorganisms [57,71]. In turn, since acidogenesis precedes methanogenesis, the qualitative and quantitative composition of SMPs after DF regulate the subsequent production of CH4.
As shown in Figure 6, the total iron content increased already after 5 s of pretreatment, and the final concentration of Fe2+ increased to the maximum (by a factor of 12.83) after 45 s pretreatment. Notably, the observed increase in the pH of CW pretreated in VLA is possibly associated with an increase in the iron content.

3.5. Kinetics of H2 Production and Correlation Analysis

Table 3 shows the calculated kinetic parameters of hydrogen production using the modified Gompertz equation [1]. The potential yield of hydrogen γ increased upon treatment in VLA. γ was the highest after treatment in VLA for 45 s and amounted to 202.4 mL H2/g COD, which was 45.8% more than without treatment. At the same time, with an increase in the treatment time to 90 s, a strong inhibition of the process occurred; γ practically did not differ from the control. The maximum rate of hydrogen production K also increased after pretreatment of CW in VLA, but the dependence was not as smooth as in the case of γ. The highest K values were observed after pretreatment in VLA for 5 and 45 s, 256.5 and 237.2 mL H2/g COD/d, respectively, which was 8.06 and 7.46 times higher than in the control without pretreatment in VLA. The lag phase of the process 𝜆 also decreased by more than two times, depending on the pretreatment time.
Figure 7 shows the heatmap of Spearman’s rank correlation coefficients [1] between the pretreatment time (VLA time), final (at the end of incubation) concentrations of dissolved forms of iron (Figure 6), and the kinetic parameters of H2 production (Table 3), calculated using the IBM SPSS Statistics. It can be noted that the sum of Fe2+ and Fe3+ had a statistically significant positive correlation with the pretreatment (VLA time). In addition, the lag phase had a statistically significant negative correlation with VLA time, indicating the positive effect of pretreatment in VLA on the onset of active hydrogen production. Of the two soluble forms of iron, Fe3+ correlated positively with K, and negatively with 𝜆, while Fe2+ had no relation to H2 production kinetics.

3.6. Recent Progress in Various Pretreatment Methods: Comparison with VLA and Limitations

The most recent research on various types of pretreatment methods to enhance dark fermentative H2 production is summarized in Table 4. It can be concluded that thermal and chemical methods for the feedstock pretreatment were quite effective. Thus, a 20% higher H2 yield and twofold improvement in bioenergy recovery were achieved by thermal pretreatment (121 °C, 15 psi for 15 min) of starchy wastewater supplemented with groundnut de-oiled cake [72]. An increase in the potential yield of H2 and the maximum rate of H2 production by 21 and 52%, respectively, was achieved after pretreatment of microalgae with free ammonia (240–530 mg NH3-N/L for 1 day) [73]. A more noticeable increase in H2 yield was observed after oxidative pretreatment of food waste with potassium ferrate (2.2 times increase) [74] and waste-activated sludge with calcium peroxide (13.7 times higher) [75]. Enhancement of H2 production with combined chemical and thermal methods has been also remarkable. For example, a 10 times higher maximum H2 yield was achieved after acid and heat pretreatment of deoiled microalgae biomass [76], while freezing with nitrite pretreatment of waste activated sludge resulted in a 5.5–13.4 times increase in H2 yield [77]. From Table 4, it can be concluded that VLA pretreatment shows very competitive results in improving the dark fermentative H2 production. It could make a significant contribution to the wider adoption and scaling of DF technology, which is currently hampered by the inefficiency of the process.
A limitation of this study is that H2 production improvements were achieved only in batch mode, and the obtained findings can be very different in a continuous operation. The second limitation is that the continuous operation of VLA can also result in different CW characteristics, for example total iron content, etc., which can lead to unexpected results. In addition, considerations of environmental impact and global sustainability, energy demands of VLA, and its influence on the process of scaling were not taken into account. Therefore, future studies are needed to assess the feasibility of using VLA in continuous DF process, as well as LCA with the use of sensitivity and uncertainty analysis to evaluate the energy conversion performance and environmental impact of industrial production of biohydrogen [78].

4. Conclusions and Prospects

The pretreatment of CW in VLA resulted in (1) an increase in pH and volatile fatty acids and iron content and (2) a decrease in COD, and fat and sugar content. The pretreatment in VLA led to the heating of CW (up to 48 °C after 90 s pretreatment), which can be beneficial in reducing the temperature shock in the anaerobic microbial community under thermophilic conditions of AD.
The content of total dissolved iron had a statistically positive correlation with the pretreatment time. The positive effect of increasing the treatment time in VLA on the onset of active hydrogen production is confirmed by the statistically negative correlation of the lag phase with the pretreatment time. Of the two soluble forms of iron, Fe3+ correlated positively with K, and negatively with 𝜆, while Fe2+ had no relation to H2 kinetics.
According to the biochemical hydrogen potential test and analysis of H2 production kinetics, the optimal pretreatment time was 45 s. This was confirmed by significant improvement in potential H2 yield and maximum H2 production rate and decrease in lag phase. However, 90 s pretreatment worsened H2 production, possibly due to the accumulation of inhibitory compounds and/or radicals. The main soluble metabolite products were butyrate and acetate. The predominant formation of butyrate correlated with a higher H2 production and its concentration in biogas, while an increased production of other products, such as lactic and formic acids, on the contrary, was observed at a lower DF efficiency.
Overall, this study showed the significant potential of using VLA in the enhancement of the dark fermentative H2 production and sustainable biorefineries. Compared to recent research in chemical, thermal, and combined substrate pretreatment, VLA is showing very competitive results. Future research will focus on continuous DF and AD of VLA-pretreated CW followed by LCA with the use of sensitivity and uncertainty analysis to evaluate the energy conversion performance and environmental impact of industrial production of biohydrogen and biohythane (a mixture of biohydrogen and biomethane) through the two-stage AD of VLA-pretreated CW. In the interpretation step of LCA, studies conducted on bioenergy and bioproduct systems seek to justify the production and utilization of these products, which might lead to incorrect recommendations and strategies. Future studies should focus on these limitations to help achieve more tangible sustainability in producing bioenergy and bioproducts.

Author Contributions

Conceptualization, E.R.M. and Y.V.L.; methodology, Y.V.L. and I.V.K.; software, A.A.K.; validation, Y.V.L. and I.V.K.; formal analysis, P.D.B., E.R.M. and E.A.Z.; investigation, I.V.K., P.D.B., E.A.Z. and A.V.V.; resources, E.R.M.; writing—original draft preparation, E.R.M.; writing—review and editing, E.R.M. and Y.V.L.; visualization, A.V.V.; project administration, E.R.M.; funding acquisition, E.R.M. All authors have read and agreed to the published version of the manuscript.


The study was supported by the grant of the Russian Science Foundation No. 21-79-10153, (accessed on 1 November 2022). P.D.B., E.A.Z., A.V.V. and Y.V.L. were supported by the Ministry of Science and Higher Education of the Russian Federation.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Not applicable.

Conflicts of Interest

The authors declare no conflict of interest.


  1. Litti, Y.V.; Potekhina, M.A.; Zhuravleva, E.A.; Vishnyakova, A.V.; Gruzdev, D.S.; Kovalev, A.A.; Kovalev, D.A.; Katraeva, I.V.; Parshina, S.N. Dark fermentative hydrogen production from simple sugars and various wastewaters by a newly isolated thermoanaerobacterium thermosaccharolyticum SP-H2. Int. J. Hydrogen Energy 2022, 47, 24310–24327. [Google Scholar] [CrossRef]
  2. Mikheeva, E.R.; Katraeva, I.V.; Kovalev, A.A.; Kovalev, D.A.; Nozhevnikova, A.N.; Panchenko, V.; Fiore, U.; Litti, Y.V. The start-up of continuous biohydrogen production from cheese whey: Comparison of inoculum pretreatment methods and reactors with moving and fixed polyurethane carriers. Appl. Sci. 2021, 11, 510. [Google Scholar] [CrossRef]
  3. Mikheeva, E.R.; Katraeva, I.V.; Vorozhtsov, D.L.; Kovalev, D.A.; Kovalev, A.A.; Grigoriev, V.S.; Litti, Y.V. Dark fermentative biohydrogen production from confectionery wastewater in continuous-flow reactors. Int. J. Hydrogen Energy 2022, 47, 22348–22358. [Google Scholar] [CrossRef]
  4. Kovalev, A.A.; Kovalev, D.A.; Nozhevnikova, A.N.; Zhuravleva, E.A.; Katraeva, I.V.; Grigoriev, V.S.; Litti, Y.V. Effect of low digestate recirculation ratio on biofuel and bioenergy recovery in a two-stage anaerobic digestion process. Int. J. Hydrogen Energy 2021, 46, 39688–39699. [Google Scholar] [CrossRef]
  5. Florio, C.; Pirozzi, D.; Ausiello, A.; Micoli, L.; Pasquale, V.; Toscano, G.; Turco, M.; Dumontet, S. Effect of inoculum/substrate ratio on dark fermentation for biohydrogen production from organic fraction of municipal solid waste. Chem. Eng. Trans. 2017, 57, 175–180. [Google Scholar] [CrossRef]
  6. Ausiello, A.; Micoli, L.; Turco, M.; Toscano, G.; Florio, C.; Pirozzi, D. Biohydrogen production by dark fermentation of Arundo donax using a new methodology for selection of H2-producing bacteria. Int. J. Hydrogen Energy 2017, 42, 30599–30612. [Google Scholar] [CrossRef]
  7. Castelló, E.; Ferraz-Junior, A.D.N.; Andreani, C.; Anzola-Rojas, M.D.P.; Borzacconi, L.; Buitrón, G.; Carrillo-Reyes, J.; Gomes, S.D.; Maintinguer, S.I.; Moreno-Andrade, I.; et al. Stability problems in the hydrogen production by dark fermentation: Possible causes and solutions. Renew. Sustain. Energy Rev. 2019, 119, 109602. [Google Scholar] [CrossRef]
  8. Kovalev, A.; Mikheeva, E.; Katraeva, I.; Kozlov, A.; Yu, V.L. Bioenergy recovery from two-stage mesophilic-thermophilic anaerobic digestion of cheese whey. Int. J. Hydrogen Energy 2022. [Google Scholar] [CrossRef]
  9. Banu, J.R.; Merrylin, J.; Usman, T.M.; Kannah, R.Y.; Gunasekaran, M.; Kim, S.-H.; Kumar, G. Impact of pretreatment on food waste for biohydrogen production: A review. Int. J. Hydrogen Energy 2019, 45, 18211–18225. [Google Scholar] [CrossRef]
  10. Moodley, P.; Trois, C. Lignocellulosic Biorefineries: The Path Forward; Academic Press: Cambridge, MA, USA, 2021; pp. 21–42. [Google Scholar] [CrossRef]
  11. Aslanzadeh, S.; Ishola, M.M.; Richards, T.; Taherzadeh, M.J. An overview of existing individual unit operations. In Biorefineries: Integrated Biochemical Processes for Liquid Biofuels; Elsevier: Amsterdam, The Netherlands, 2014; pp. 3–36. [Google Scholar] [CrossRef]
  12. Wang, J.; Yin, Y. Sewage sludge for hydrogen production. In Green Energy and Technology; Springer: Berlin/Heidelberg, Germany, 2017; pp. 339–433. [Google Scholar] [CrossRef]
  13. Baek, G.; Kim, J.; Kim, J.; Lee, C. Role and potential of direct interspecies electron transfer in anaerobic digestion. Energies 2018, 11, 107. [Google Scholar] [CrossRef] [Green Version]
  14. Sambusiti, C.; Bellucci, M.; Zabaniotou, A.; Beneduce, L.; Monlau, F. Algae as promising feedstocks for fermentative biohydrogen production according to a biorefinery approach: A comprehensive review. Renew. Sustain. Energy Rev. 2015, 44, 20–36. [Google Scholar] [CrossRef]
  15. Prabakar, D.; Manimudi, V.T.; Suvetha, S.S.; Sampath, S.; Mahapatra, D.M.; Rajendran, K.; Pugazhendhi, A. Advanced biohydrogen production using pretreated industrial waste: Outlook and prospects. Renew. Sustain. Energy Rev. 2018, 96, 306–324. [Google Scholar] [CrossRef]
  16. Balasundaram, G.; Vidyarthi, P.K.; Gahlot, P.; Arora, P.; Kumar, V.; Kumar, M.; Kazmi, A.; Tyagi, V.K. Energy feasibility and life cycle assessment of sludge pretreatment methods for advanced anaerobic digestion. Bioresour. Technol. 2022, 357, 127345. [Google Scholar] [CrossRef]
  17. Mikheeva, E.; Katraeva, I.; Kovalev, A.; Litti, Y.V. Effects of pretreatment in a vortex layer apparatus on the properties of confectionery wastewater and its dark fermentation. Int. J. Hydrogen Energy 2022, 47, 23165–23174. [Google Scholar] [CrossRef]
  18. Kovalev, A.A.; Kovalev, D.A.; Litti, Y.V.; Katraeva, I.V. The synergistic effect of the thickened digestate treatment in the vortex layer apparatus prior to its recirculation into the reactor on the characteristics of anaerobic bioconversion of organic waste. J. Phys.: Conf. Ser. 2020, 1652, 012014. [Google Scholar] [CrossRef]
  19. Mikheeva, E.R.; Katraeva, I.V.; Vorozhtsov, D.L.; Litti, Y.V.; Nozhevnikova, A.N. Efficiency of two-phase anaerobic fermentation and the physicochemical properties of the organic fraction of municipal solid waste processed in a vortex-layer apparatus. Appl. Biochem. Microbiol. 2020, 56, 736–742. [Google Scholar] [CrossRef]
  20. Kovalev, A.A.; Kovalev, D.A.; Grigoriev, V.S.; Litti, Y.V. The vortex layer apparatus as a source of low-grade heat in the process of pretreatment of the substrate before anaerobic digestion. IOP Conf. Ser. Earth Environ. Sci. 2021, 938, 012004. [Google Scholar] [CrossRef]
  21. Lee, D.-Y.; Li, Y.-Y.; Oh, Y.-K.; Kim, M.-S.; Noike, T. Effect of iron concentration on continuous H2 production using membrane bioreactor. Int. J. Hydrogen Energy 2009, 34, 1244–1252. [Google Scholar] [CrossRef]
  22. Jiang, D.; Fang, Z.; Chin, S.-X.; Tian, X.; Su, T.-C. Biohydrogen production from hydrolysates of selected tropical biomass wastes with clostridium butyricum. Sci. Rep. 2016, 6, 27205. [Google Scholar] [CrossRef] [Green Version]
  23. Salama, E.-S.; Saha, S.; Kurade, M.B.; Dev, S.; Chang, S.W.; Jeon, B.-H. Recent trends in anaerobic co-digestion: Fat, oil, and grease (FOG) for enhanced biomethanation. Prog. Energy Combust. Sci. 2018, 70, 22–42. [Google Scholar] [CrossRef]
  24. Thakur, H.; Dhar, A.; Powar, S. Biogas production from anaerobic co-digestion of sewage sludge and food waste in continuously stirred tank reactor. Results Eng. 2022, 16, 100617. [Google Scholar] [CrossRef]
  25. Ghimire, A.; Frunzo, L.; Pirozzi, F.; Trably, E.; Escudie, R.; Lens, P.N.; Esposito, G. A review on dark fermentative biohydrogen production from organic biomass: Process parameters and use of by-products. Appl. Energy 2015, 144, 73–95. [Google Scholar] [CrossRef]
  26. Peterson, W.H.; Fred, E.B.; Anderson, J.A. The fermentation of glucose, galactose, and mannose by lactobacillus pentoaceticus, n. sp. J. Biol. Chem. 1920, 42, 273–287. [Google Scholar] [CrossRef]
  27. Sun, Y.; He, J.; Yang, G.; Sun, G.; Sage, V. A review of the enhancement of bio-hydrogen generation by chemicals addition. Catalysts 2019, 9, 353. [Google Scholar] [CrossRef] [Green Version]
  28. Montecchio, D.; Yuan, Y.; Malpei, F. Hydrogen production dynamic during cheese whey dark fermentation: New insights from modelization. Int. J. Hydrogen Energy 2018, 43, 17588–17601. [Google Scholar] [CrossRef]
  29. Park, J.-H.; Lee, S.-H.; Yoon, J.-J.; Kim, S.-H.; Park, H.-D. Predominance of cluster I clostridium in hydrogen fermentation of galactose seeded with various heat-treated anaerobic sludges. Bioresour. Technol. 2014, 157, 98–106. [Google Scholar] [CrossRef]
  30. Pan, C.-M.; Fan, Y.-T.; Zhao, P.; Hou, H.-W. Fermentative hydrogen production by the newly isolated Clostridium beijerinckii Fanp3. Int. J. Hydrogen Energy 2008, 33, 5383–5391. [Google Scholar] [CrossRef]
  31. Xia, A.; Cheng, J.; Ding, L.; Lin, R.; Song, W.; Zhou, J.; Cen, K. Enhancement of energy production efficiency from mixed biomass of Chlorella pyrenoidosa and cassava starch through combined hydrogen fermentation and methanogenesis. Appl. Energy 2014, 120, 23–30. [Google Scholar] [CrossRef]
  32. Xia, A.; Cheng, J.; Ding, L.; Lin, R.; Song, W.; Su, H.; Zhou, J.; Cen, K. Substrate consumption and hydrogen production via co-fermentation of monomers derived from carbohydrates and proteins in biomass wastes. Appl. Energy 2015, 139, 9–16. [Google Scholar] [CrossRef]
  33. Xiao, N.; Chen, Y.; Chen, A.; Feng, L. Enhanced Bio-hydrogen Production from Protein Wastewater by Altering Protein Structure and Amino Acids Acidification Type. Sci. Rep. 2014, 4, 3992. [Google Scholar] [CrossRef] [Green Version]
  34. Xia, A.; Jacob, A.; Herrmann, C.; Murphy, J.D. Fermentative bio-hydrogen production from galactose. Energy 2016, 96, 346–354. [Google Scholar] [CrossRef]
  35. Maru, B.T.; Bielen, A.A.M.; Constantí, M.; Medina, F.; Kengen, S.W.M. Glycerol fermentation to hydrogen by thermotoga maritima: Proposed pathway and bioenergetic considerations. Int. J. Hydrogen Energy 2013, 38, 5563–5572. [Google Scholar] [CrossRef]
  36. Oh, S.T.; Martin, A.D. Long chain fatty acids degradation in anaerobic digester: Thermodynamic equilibrium consideration. Process. Biochem. 2010, 45, 335–345. [Google Scholar] [CrossRef]
  37. Mainardis, M.; Flaibani, S.; Trigatti, M.; Goi, D. Techno-economic feasibility of anaerobic digestion of cheese whey in small Italian dairies and effect of ultrasound pre-treatment on methane yield. J. Environ. Manag. 2019, 246, 557–563. [Google Scholar] [CrossRef]
  38. Kazimierowicz, J.; Zieliński, M.; Bartkowska, I.; Dębowski, M. Effect of acid whey pretreatment using ultrasonic disintegration on the removal of organic compounds and anaerobic digestion efficiency. Int. J. Environ. Res. Public Health 2022, 19, 11362. [Google Scholar] [CrossRef]
  39. Gannoun, H.; Khelifi, E.; Bouallagui, H.; Touhami, Y.; Hamdi, M. Ecological clarification of cheese whey prior to anaerobic digestion in upflow anaerobic filter. Bioresour. Technol. 2008, 99, 6105–6111. [Google Scholar] [CrossRef]
  40. Zieliński, M.; Dębowski, M.; Kazimierowicz, J. Microwave radiation influence on dairy waste anaerobic digestion in a multi-section hybrid anaerobic reactor (M-SHAR). Processes 2021, 9, 1772. [Google Scholar] [CrossRef]
  41. Skripsts, E.; Dubrovskis, V.; Jasko, J.; Zabarovskis, E.; Kotelenecs, V. Investigation of biogas production of cheese whey in processing with ozone before anaerobic digestion. Eng. Rural. Dev. 2011, 10, 377–381. [Google Scholar]
  42. Escalante, H.; Castro, L.; Amaya, M.P.; Jaimes, L.; Jaimes-Estévez, J. Anaerobic digestion of cheese whey: Energetic and nutritional potential for the dairy sector in developing countries. Waste Manag. 2018, 71, 711–718. [Google Scholar] [CrossRef]
  43. da Silva, A.N.; Macêdo, W.V.; Sakamoto, I.K.; Pereyra, D.D.L.A.D.; Mendes, C.O.; Maintinguer, S.I.; Filho, R.A.C.; Damianovic, M.; Varesche, M.B.A.; de Amorim, E.L.C. Biohydrogen production from dairy industry wastewater in an anaerobic fluidized-bed reactor. Biomass Bioenergy 2018, 120, 257–264. [Google Scholar] [CrossRef]
  44. Prapinagsorn, W.; Sittijunda, S.; Reungsang, A. Co-digestion of napier grass and its silage with cow dung for bio-hydrogen and methane production by two-stage anaerobic digestion process. Energies 2017, 11, 47. [Google Scholar] [CrossRef] [Green Version]
  45. Ghimire, A.; Luongo, V.; Frunzo, L.; Pirozzi, F.; Lens, P.N.; Esposito, G. Continuous biohydrogen production by thermophilic dark fermentation of cheese whey: Use of buffalo manure as buffering agent. Int. J. Hydrogen Energy 2017, 42, 4861–4869. [Google Scholar] [CrossRef]
  46. Kovalev, A.A.; Kovalev, D.A.; Zhuravleva, E.A.; Katraeva, I.V.; Panchenko, V.; Fiore, U.; Litti, Y.V. Two-stage anaerobic digestion with direct electric stimulation of methanogenesis: The effect of a physical barrier to retain biomass on the surface of a carbon cloth-based biocathode. Renew. Energy 2021, 181, 966–977. [Google Scholar] [CrossRef]
  47. Bundhoo, M.Z.; Mohee, R. Inhibition of dark fermentative bio-hydrogen production: A review. Int. J. Hydrogen Energy 2016, 41, 6713–6733. [Google Scholar] [CrossRef]
  48. Ferchichi, M.; Crabbe, E.; Gil, G.-H.; Hintz, W.; Almadidy, A. Influence of initial pH on hydrogen production from cheese whey. J. Biotechnol. 2005, 120, 402–409. [Google Scholar] [CrossRef] [PubMed]
  49. Arenas, C.B.; González, R.; González, J.; Cara, J.; Papaharalabos, G.; Gómez, X.; Martínez, E.J. Assessment of electrooxidation as pre- and post-treatments for improving anaerobic digestion and stabilisation of waste activated sludge. J. Environ. Manag. 2021, 288, 112365. [Google Scholar] [CrossRef] [PubMed]
  50. Bhat, A.P.; Gogate, P.R. Cavitation-based pre-treatment of wastewater and waste sludge for improvement in the performance of biological processes: A review. J. Environ. Chem. Eng. 2020, 9, 104743. [Google Scholar] [CrossRef]
  51. Chen, Z.; Rao, Y.; Usman, M.; Chen, H.; Białowiec, A.; Zhang, S.; Luo, G. Anaerobic fermentation of hydrothermal liquefaction wastewater of dewatered sewage sludge for volatile fatty acids production with focuses on the degradation of organic components and microbial community compositions. Sci. Total Environ. 2021, 777, 146077. [Google Scholar] [CrossRef]
  52. Hitit, Z.Y.; Hallenbeck, P.C. Analytical procedures, data reporting and selected reference values for biological hydrogen production. Biomass Bioenergy 2021, 147, 106014. [Google Scholar] [CrossRef]
  53. Leaño, E.; Babel, S. Effects of pretreatment methods on cassava wastewater for biohydrogen production optimization. Renew. Energy 2012, 39, 339–346. [Google Scholar] [CrossRef]
  54. Yang, G.; Wang, J. Enhancement of biohydrogen production from grass by ferrous ion and variation of microbial community. Fuel 2018, 233, 404–411. [Google Scholar] [CrossRef]
  55. Chatterjee, C.; Pong, F.; Sen, A. Chemical conversion pathways for carbohydrates. Green Chem. 2014, 17, 40–71. [Google Scholar] [CrossRef]
  56. Dereli, R.K.; Loverdou, L.; van der Zee, F.P.; van Lier, J.B. A systematic study on the effect of substrate acidification degree and acidogenic biomass on sludge filterability. Water Res. 2015, 82, 94–103. [Google Scholar] [CrossRef]
  57. Lee, Y.J.; Miyahara, T.; Noike, T. Effect of iron concentration on hydrogen fermentation. Bioresour. Technol. 2001, 80, 227–231. [Google Scholar] [CrossRef]
  58. Wang, J.; Wan, W. Effect of Fe2+ concentration on fermentative hydrogen production by mixed cultures. Int. J. Hydrogen Energy 2008, 33, 1215–1220. [Google Scholar] [CrossRef]
  59. Park, J.-H.; Lee, S.-H.; Ju, H.-J.; Kim, S.-H.; Yoon, J.-J.; Park, H.-D. Failure of biohydrogen production by low levels of substrate and lactic acid accumulation. Renew. Energy 2016, 86, 889–894. [Google Scholar] [CrossRef]
  60. García-Depraect, O.; Castro-Muñoz, R.; Muñoz, R.; Rene, E.R.; León-Becerril, E.; Valdez-Vazquez, I.; Kumar, G.; Reyes-Alvarado, L.C.; Martínez-Mendoza, L.J.; Carrillo-Reyes, J.; et al. A review on the factors influencing biohydrogen production from lactate: The key to unlocking enhanced dark fermentative processes. Bioresour. Technol. 2020, 324, 124595. [Google Scholar] [CrossRef]
  61. Yin, Q.; Miao, J.; Li, B.; Wu, G. Enhancing electron transfer by ferroferric oxide during the anaerobic treatment of synthetic wastewater with mixed organic carbon. Int. Biodeterior. Biodegrad. 2017, 119, 104–110. [Google Scholar] [CrossRef]
  62. Zhuravleva, E.A.; Shekhurdina, S.V.; Kotova, I.B.; Loiko, N.G.; Popova, N.M.; Kryukov, E.; Kovalev, A.A.; Kovalev, D.A.; Litti, Y.V. Effects of various materials used to promote the direct interspecies electron transfer on anaerobic digestion of low-concentration swine manure. Sci. Total Environ. 2022, 839, 156073. [Google Scholar] [CrossRef]
  63. Gadhe, A.; Sonawane, S.S.; Varma, M.N. Influence of nickel and hematite nanoparticle powder on the production of biohydrogen from complex distillery wastewater in batch fermentation. Int. J. Hydrogen Energy 2015, 40, 10734–10743. [Google Scholar] [CrossRef]
  64. Zhao, Z.; Zhang, Y.; Li, Y.; Quan, X.; Zhao, Z. Comparing the mechanisms of ZVI and Fe3O4 for promoting waste-activated sludge digestion. Water Res. 2018, 144, 126–133. [Google Scholar] [CrossRef] [PubMed]
  65. Cai, Y.; Zhao, X.; Zhao, Y.; Wang, H.; Yuan, X.; Zhu, W.; Cui, Z.; Wang, X. Optimization of Fe2+ supplement in anaerobic digestion accounting for the Fe-bioavailability. Bioresour. Technol. 2018, 250, 163–170. [Google Scholar] [CrossRef] [PubMed]
  66. Yuan, T.; Ko, J.H.; Zhou, L.; Gao, X.; Liu, Y.; Shi, X.; Xu, Q. Iron oxide alleviates acids stress by facilitating syntrophic metabolism between syntrophomonas and methanogens. Chemosphere 2020, 247, 125866. [Google Scholar] [CrossRef] [PubMed]
  67. Hwang, Y.; Sivagurunathan, P.; Lee, M.-K.; Yun, Y.-M.; Song, Y.-C.; Kim, D.-H. Enhanced hydrogen fermentation by zero valent iron addition. Int. J. Hydrogen Energy 2019, 44, 3387–3394. [Google Scholar] [CrossRef]
  68. Wei, J.; Hao, X.; van Loosdrecht, M.C.; Li, J. Feasibility analysis of anaerobic digestion of excess sludge enhanced by iron: A review. Renew. Sustain. Energy Rev. 2018, 89, 16–26. [Google Scholar] [CrossRef]
  69. Durán-Padilla, V.R.; Davila-Vazquez, G.; Chavez-Vela, N.A.; Tinoco-Valencia, J.R.; Jáuregui-Rincón, J. Iron effect on the fermentative metabolism of Clostridium acetobutylicum ATCC 824 using cheese whey as substrate. Biofuel Res. J. 2014, 1, 129–133. [Google Scholar] [CrossRef]
  70. Zhu, H.; Seto, P.; Parker, W.J. Enhanced dark fermentative hydrogen production under the effect of zero-valent iron shavings. Int. J. Hydrogen Energy 2014, 39, 19331–19336. [Google Scholar] [CrossRef]
  71. Taherdanak, M.; Zilouei, H.; Karimi, K. Investigating the effects of iron and nickel nanoparticles on dark hydrogen fermentation from starch using central composite design. Int. J. Hydrogen Energy 2015, 40, 12956–12963. [Google Scholar] [CrossRef]
  72. Mahata, C.; Dhar, S.; Ray, S.; Das, D. Effect of thermal pretreated organic wastes on the dark fermentative hydrogen production using mixed microbial consortia. Fuel 2020, 284, 119062. [Google Scholar] [CrossRef]
  73. Wang, Q.; Gong, Y.; Liu, S.; Wang, D.; Liu, R.; Zhou, X.; Nghiem, L.D.; Zhao, Y. Free ammonia pretreatment to improve bio-hydrogen production from anaerobic dark fermentation of microalgae. ACS Sustain. Chem. Eng. 2019, 7, 1642–1647. [Google Scholar] [CrossRef]
  74. Kuang, Y.; Zhao, J.; Gao, Y.; Lu, C.; Luo, S.; Sun, Y.; Zhang, D. Enhanced hydrogen production from food waste dark fermentation by potassium ferrate pretreatment. Environ. Sci. Pollut. Res. 2020, 27, 18145–18156. [Google Scholar] [CrossRef] [PubMed]
  75. Wang, D.; Zhang, D.; Xu, Q.; Liu, Y.; Wang, Q.; Ni, B.-J.; Yang, Q.; Li, X.; Yang, F. Calcium peroxide promotes hydrogen production from dark fermentation of waste activated sludge. Chem. Eng. J. 2019, 355, 22–32. [Google Scholar] [CrossRef]
  76. Singh, H.; Rout, S.; Das, D. Dark fermentative biohydrogen production using pretreated Scenedesmus obliquus biomass under an integrated paradigm of biorefinery. Int. J. Hydrogen Energy 2022, 47, 102–116. [Google Scholar] [CrossRef]
  77. Liu, X.; He, D.; Wu, Y.; Xu, Q.; Wang, D.; Yang, Q.; Liu, Y.; Ni, B.-J.; Wang, Q.; Li, X. Freezing in the presence of nitrite pretreatment enhances hydrogen production from dark fermentation of waste activated sludge. J. Clean. Prod. 2020, 248, 119305. [Google Scholar] [CrossRef]
  78. Aghbashlo, M.; Hosseinzadeh-Bandbafha, H.; Shahbeik, H.; Tabatabaei, M. The role of sustainability assessment tools in realizing bioenergy and bioproduct systems. Biofuel Res. J. 2022, 9, 1697–1706. [Google Scholar] [CrossRef]
  79. Song, W.; Ding, L.; Liu, M.; Cheng, J.; Zhou, J.; Li, Y.-Y. Improving biohydrogen production through dark fermentation of steam-heated acid pretreated alternanthera philoxeroides by mutant enterobacter aerogenes ZJU1. Sci. Total Environ. 2020, 716, 134695. [Google Scholar] [CrossRef]
Figure 1. Change in COD and temperature of CW after pretreatment in VLA.
Figure 1. Change in COD and temperature of CW after pretreatment in VLA.
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Figure 2. Change in the content of total and non-volatile solids depending on the pretreatment time.
Figure 2. Change in the content of total and non-volatile solids depending on the pretreatment time.
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Figure 3. The content of VFA and ethanol in the CW depending on the pretreatment time.
Figure 3. The content of VFA and ethanol in the CW depending on the pretreatment time.
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Figure 4. Dynamics of the cumulative hydrogen production per g COD (A) and mol of hexose (B), and hydrogen concentration in biogas (C).
Figure 4. Dynamics of the cumulative hydrogen production per g COD (A) and mol of hexose (B), and hydrogen concentration in biogas (C).
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Figure 5. Dynamics of soluble metabolite products (SMP) and pH during DF of CW, pretreated in VLA: (A)—0 s, (B)—5 s, (C)—20 s, (D)—45 s, (E)—90 s.
Figure 5. Dynamics of soluble metabolite products (SMP) and pH during DF of CW, pretreated in VLA: (A)—0 s, (B)—5 s, (C)—20 s, (D)—45 s, (E)—90 s.
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Figure 6. Concentration of total Fe, Fe2+, and Fe3+ after pretreatment in VLA.
Figure 6. Concentration of total Fe, Fe2+, and Fe3+ after pretreatment in VLA.
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Figure 7. A heatmap of Spearman’s correlation coefficients between VLA time, dissolved forms of iron and kinetic parameters of H2 production. *–Statistically significant values at p < 0.05.
Figure 7. A heatmap of Spearman’s correlation coefficients between VLA time, dissolved forms of iron and kinetic parameters of H2 production. *–Statistically significant values at p < 0.05.
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Table 1. Characteristics of CW.
Table 1. Characteristics of CW.
COD, g/L93.8 ± 11.1
pH6.3 ± 0.2
Fat, mg/L20.7 ± 2.3
Lactose, g/L46.6 ± 10.2
Glucose, g/L6.4 ± 1.2
TS, %9.9 ± 0.8
NVS, %TS3.9 ± 0.8
VS, % TS96.1 ± 0.8
Fetotal, g/L0.22 ± 0.01
Acetate, mg/L470 ± 25
Propionate, mg/L60 ± 3
Butyrate, mg/L20 ± 1
Ethanol, mg/L390 ± 20
Table 2. The content of sugars in CW after pretreatment in VLA.
Table 2. The content of sugars in CW after pretreatment in VLA.
Concentration, mg/LPretreatment Time, s
Lactose46.6 ± 10.238.5 ± 1.541.2 ± 0.236.2 ± 1.834.3 ± 3.6
Glucose6.4 ± 1.24.6 ± 0.15.1 ± 1.54.5 ± 1.14.1 ± 0.7
Table 3. Kinetic parameters of biohydrogen production.
Table 3. Kinetic parameters of biohydrogen production.
Pretreatment TimePotential Hydrogen Yield γ, mL H2/g CODMaximum Hydrogen Production Rate K, mL H2/g COD/dLag Phase 𝜆, dR2
0 (Control)138.831.80.820.99
Table 4. Enhancement of H2 production through various types of feedstock pretreatment.
Table 4. Enhancement of H2 production through various types of feedstock pretreatment.
TypePretreatment MethodFeedstockDF Temperature, °CMicroorganismH2 Production ImprovementsReference
ThermalHeatStarchy wastewater supplemented with groundnut de-oiled cake37.5Anaerobically digested sludge, dominated by Clostridium sp. and Eubacterium sp.20% higher H2 yield, twofold improvement in bioenergy recovery[72]
ChemicalFree ammoniaMicroalgae37Anaerobically digested sludgeUp to 21% increase in the potential H2 yield, up to 52% increase in the maximum H2 production rate[73]
Potassium ferrateFood waste35Sewage sludge2.2-fold increase in H2 yield, lag-phase shortened from 120 to 96 h[74]
Calcium peroxideWaste activated sludge35Mixed consortia13.7 times higher maximum H2 yield[75]
Combined thermal and chemicalAcid and heatDeoiled Scenedesmus obliquus biomass37Mixed consortia10 times higher maximum H2 yield[76]
Steam and acidAlternanthera philoxeroides37Enterobacter aerogenes ZJU1 mutagenized by 60Co-γ irradiation59.9% increase in H2 yield[79]
Freezing with nitriteWaste activated sludge35Waste activated sludge5.5–13.4 times increase in H2 yield[77]
PhysicalVLACW55T. thermosacharoliticum SP-H2Up to 45.8% increase in the potential H2 yield, up to 8.06 times the maximum H2 production rate, more than a twofold reduction in lag-phaseThis study
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Mikheeva, E.R.; Katraeva, I.V.; Kovalev, A.A.; Biryuchkova, P.D.; Zhuravleva, E.A.; Vishnyakova, A.V.; Litti, Y.V. Pretreatment in Vortex Layer Apparatus Boosts Dark Fermentative Hydrogen Production from Cheese Whey. Fermentation 2022, 8, 674.

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Mikheeva ER, Katraeva IV, Kovalev AA, Biryuchkova PD, Zhuravleva EA, Vishnyakova AV, Litti YV. Pretreatment in Vortex Layer Apparatus Boosts Dark Fermentative Hydrogen Production from Cheese Whey. Fermentation. 2022; 8(12):674.

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Mikheeva, Elza R., Inna V. Katraeva, Andrey A. Kovalev, Polina D. Biryuchkova, Elena A. Zhuravleva, Anastasia V. Vishnyakova, and Yuriy V. Litti. 2022. "Pretreatment in Vortex Layer Apparatus Boosts Dark Fermentative Hydrogen Production from Cheese Whey" Fermentation 8, no. 12: 674.

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