Biological production of hydrogen from crude glycerol under thermophilic conditions

ABSTRACT

Glycerol is the main residue in biodiesel production. A viable alternative is its application in anaerobic digestion for hydrogen production. Thermophilic conditions can favor biogas generation from glycerol, by anaerobic digestion, with advantages in microorganism selectivity and low biogas solubility in liquid phase. Therefore, this study evaluated the bioconversion potential of crude glycerol into H₂, using a thermophilic granular sludge, in anaerobic batch reactors operated at 55°C, at initial pH 5.5 or 6.0 in 5 experiments containing (g L^-1) glycerin (10.0) or crude glycerol (5.8) in different culture media with additions of peptone; meat extract and yeast extract. High glycerin removal (92.4–97.5%) was observed in all experiments, resulting in hydrogen production of (mmol L⁻¹) 47.81, 24.19, 51.48, 43.17 and 15.43; yields of (mol H₂ mol glycerin consumed^-1) 1.36; 0.75; 1.48; 1.30 and 0.48 in the experiments 1, 2, 3, 4, and 5, respectively. Metagenomic analysis revealed that thermophilic bacteria from the genera Coprothermobacter and Thermoanaerobacterium were favored by the imposed operating conditions, acting synergistically in the thermophilic fermentative metabolic pathways of H₂ generation from crude glycerol. Thermophilic anaerobic digestion of crude glycerol is an alternative for its treatment, resulting in the generation of biofuels such as H₂.

Keywords:
Bioenergy; Biohydrogen; Anaerobic digestion; Fermentation; Thermophilic bacteria

1. INTRODUCTION

The search for renewable fuels has boosted biodiesel production. Brazilian Government incentives have driven its production. Since 2008, the addition of the biodiesel into diesel became mandatory at 2% (B2) to the Brazilian energy matrix. Additions occurred over the years, reaching 3% (B3) in 2009, 4% (B4) from July 2009, 5% (B5) in 2011 and 2012, and 6% (B6) in 2014 [¹]. These additions have occurred gradually over the years, and since April 2025, 15% biodiesel has been added to national diesel [²].

Transesterification is the main method for obtaining biodiesel from vegetable oils, animal fats, and recyclable household oils. However, for every 100 kg of biodiesel produced, 10 kg of crude glycerol is generated, which contains contaminants such as methanol, soaps, and salts [³, ⁴]. A sustainable alternative for its reuse in biodiesel production plants would be its application in anaerobic digestion for generating biofuels such as hydrogen. Furthermore, crude glycerol is beneficial for anaerobic digestion because it significantly increases biogas production (methane and hydrogen) [⁵].

Temperature is considered one of the main influencing factors in hydrogen production via fermentation. Such reactions can be conducted in mesophilic (25–40 ºC), thermophilic (40–65 ºC), extreme thermophilic (65–80 ºC), and hyperthermophilic (> 80 ºC) temperature ranges [⁶]. The thermophilic condition has received more attention due to the high yield of hydrogen produced from the fermentation of wastewater containing whey [⁷, ⁸], sugarcane vinasse [⁹], among others.

Thermophilic fermentation has some advantages regarding H₂ production when compared to mesophilic condition. Metabolism under thermophilic conditions is favored by the partial pressure of hydrogen and some thermophilic H₂-producing microorganisms are tolerant of this condition. Another advantage is the greater thermodynamic favorability in chemical and biological reactions, with a predominance of the acetate metabolic pathway as opposed to the ethanol or lactate [¹⁰]. Furthermore, thermophilic conditions exhibit low H₂ solubility in the aqueous phase and better liquid-gas transfer conditions to the gas phase [¹¹]. In this sense, bioenergy production at elevated temperatures could increase H₂ production, selecting microorganisms with higher efficiencies than mesophilic ones [¹²].

However, the extra energy costs to maintain temperatures under thermophilic conditions are approximately 1–2% higher than under mesophilic conditions [¹⁰]. This margin could be easily compensated by feeding the reactors with industrial waste generated at high temperatures, such as in transesterification plants from oils and fats to biodiesel production, promoting the hydrolysis of organic compounds and facilitating their degradation by the microbial community in bioenergy generation [¹³]. Thermophilic temperatures are also capable of removing pathogenic microorganisms and preventing H₂-consuming bacteria (homoacetogenic and sulfate-reducing) [¹⁰].

Furthermore, studies on the applications of crude glycerol in hydrogen generation under thermophilic conditions are scarce. Its application is important since the biodiesel production process can occur at high temperatures. SITTIJUNDA and REUNGSANG [¹⁴] analyzed how the composition of the culture media affected the thermophilic production of bio-hydrogen from crude glycerol. Different concentrations of crude glycerol, urea, and disodium hydrogen phosphate (Na₂HPO₄) were tested. The optimal culture medium composition was of (g L^-1) residual glycerol (20.33), urea (0.16) and Na₂HPO₄ (3.97) with the maximum yield of 1470.19 mL H₂ L^-1. The authors also investigated the production of hydrogen from pure glycerol and crude glycerol under thermophilic conditions (55 ± 4°C) in a UASB reactor, observing the effect of the organic loading rate (OLR) (25, 37.5, 50, 62.5, and 75 g L⁻¹ d⁻¹). Maximum hydrogen yields of 2.90 and 2.05 mol H₂ mol⁻¹ glycerol were achieved with an OLR of 62.5 g L^-1. d^-1 of pure and crude glycerol, respectively [¹⁵].

KANCHANASUTA and SILLAPARASSAMEE [¹⁶] investigated two-stage thermophilic hydrogen fermentation and the mesophilic methanogenic process, using effluent from the co-digestion of palm oil (PO) and crude glycerol (CG) decanter cake. For the first thermophilic stage, the hydrogen generating reactor with 2% w/v total solids (TS) of PO and crude glycerol was operated for 4 days of hydraulic retention time (HRT), with a variable load of 0.75–1.5% CG. The maximum production rate and hydrogen yield in the first thermophilic stage obtained from the co-digestion condition with 1.5% GB were 461 mL of H₂ L^-1 d and 23 L H₂ kg⁻¹ of added ST, respectively.

ROMERO-GÜIZA et al. [¹⁷] investigated the temperature-phased (hyper-thermophilic/mesophilic) anaerobic co-digestion of crude glycerol (CG) and sewage sludge (SS) in semi-continuous reactors generating methane. The authors verified biogas production had better performance in methane yield (increased to 15%), VS removal (increased to 5.6%) and final sludge dewatering (qualitatively) in comparison with conventional mesophilic anaerobic co-digestion. Moreover, energy balance showed that the proposed system reaches up to 15% extra power and 68% extra heat production in comparison to the conventional mesophilic anaerobic system.

ALMEIDA et al. [¹⁸] studied the effect of the organic loading rate (OLR) 60, 90, and 120 g Chemical Oxygen Demand (COD. L^-1. d^-1) in a thermophilic fluidized bed reactor, from cheese whey and glycerol fermentation as cosubstrates (50% cheese whey and 50% glycerol on a COD basis) operated at 55°C. The authors verified hydrogen production of 3.9 L H₂. L^-1. d^-1 and hydrogen yield of 1.7 mmol H₂. gCOD^-1 in OLR of 90 g COD. L^-1. d^-1. The highest hydrogen production was observed with the genera of Thermoanaerobacterium (34.9%), Pseudomonas (14.5%), and Clostridium (4.7%) during the operation of the reactor. Cofermentation favored hydrogen production at higher OLRs than cheese whey single fermentation. AUGUSTO et al. [¹⁹]. Evaluated hydrogen production at thermophilic condition (45–60°C) bioreactor (3L) filled with glycerol (0–4.7 g/L). The H₂ generation decreased with increasing glycerol concentrations (from 89.7 mmol-H₂ L^-1 d^-1 to 2.7 mmol-H₂ L^-1 d^-1 at 100% glycerol). The authors confirmed that the temperature influenced microbial diversity, with moderate thermophilic conditions (45°C) favoring a diverse consortium (including Thermoanaerobacterium, Clostridium sensu stricto and Oxobacter), while higher temperatures (60°C) led to the dominance of Thermoanaerobacterium.

Thermophilic processes exhibit low H₂ solubility in the aqueous phase and better liquid-gas transfer conditions for H₂ [¹¹]. In this sense, bioenergy production at elevated temperatures could increase the production of H₂, selecting producing microorganisms with higher efficiencies than mesophilic ones [¹²]. Furthermore, operation at elevated temperatures can improve fermentation performance, promote the hydrolysis of organic compounds and facilitate degradation by the microbial community in bioenergy generation [¹³].

In this sense, the objectives of this study were to compare the production of bioH₂ in anaerobic batch reactors filled with glycerin or crude glycerol under thermophilic conditions, in different experiments. The results obtained of them were adjusted in the mathematical model and the anaerobic consortia involved in the anaerobic digestion of hydrogen were determinate in metagenomic analysis.

2. MATERIALS AND METHODS

2.1. Inoculum source

Inoculum 1: Thermophilic granular sludge from a UASB (Upflow Anaerobic Sludge Blanket) reactor treating vinasse (São Martinho Plant – Pradópolis-SP-Brazil), pre-treated (pH 3.0 for 24 h) with the aim of inhibiting H₂-producing microorganisms [²⁰].

Inoculum 2: It was obtained by enrichment of inoculum 1, with the addition of 1 mL of inoculum 1, in anaerobic batch reactors (100 mL), in culture medium (50 mL): glycerin (10 g L^-1), yeast extract (5 g L^-1), meat extract (5 g L^-1) and peptone (5 g L^-1), headspace (50 mL) filled with N₂ (99.99%) under constant flow for 15 minutes, initial pH 5.5, at 55°C for 72 hours. After that, the biomass was submitted to serial dilutions with the same culture medium and initial pH 5.5, to obtain selection of the hydrogen producing microorganisms.

2.2. Substrates

2.2.1. Glycerin

Commercially obtained, with 99% purity (Synth).

2.2.2. Crude glycerol

Originating from biodiesel production by transesterification process from soybean oil (BioBrotas, Brotas-SP). This substrate had 1130 g COD L^-1, 80.63% free glycerol, soap content of 8.01% and 2.8% non-glycerol organic matter, pH 5.3 and 11.4% moisture.

2.3. Operation of anaerobic batch reactors

2.3.1. Experimental setup

The anaerobic batch reactors (500 mL) were operated in triplicate, with working volume of 300 mL, containing 210 mL of culture medium, the headspace (200 mL) was flushed with N₂ (99.99%) under constant flow for 15 minutes, closed with butyl cap and plastic screw cap, maintained in static mode, at 55°C. Five distinct tests were performed in triplicate on anaerobic batch reactors, as described below. A schematic representation of the experimental design is shown in Figure 1.

Figure 1
Schematic representation of the experimental design for hydrogen production from glycerin and crude glycerol under thermophilic conditions, including reactor setup, operational conditions, and the five experimental configurations evaluated.

1. Experiment 1: Inoculum 1 [30% (v/v)] + culture medium (g L^-1): glycerin (10.0), yeast extract (5.0), meat extract (5.0) and peptone (5.0), at initial pH 5.5, for 57 hours.

2. Experiment 2: Inoculum 2 [30% (v/v)] + culture medium consisting of (g L^-1): glycerin (10.0), peptone (10.0), meat extract (10.0), sodium chloride (5.0), sodium acetate (3.0), yeast extract (3.0) and L-cysteine hydrochloride (0.5), at initial pH 6.0, for 96 hours.

3. Experiment 3: Inoculum 1 [30% (v/v)] + culture medium (g L^-1): crude glycerol (5.8), peptone (10.0), meat extract (10.0), sodium chloride (5.0), sodium acetate (3.0), yeast extract (3.0) and L-cysteine hydrochloride (0.5), at initial pH 5.5, for 86 hours.

4. Experiment 4: Inoculum 2 [30% (v/v)] + culture medium (g L^-1): crude glycerol (5.8), peptone (10.0), meat extract (10.0), sodium chloride (5.0), sodium acetate (3.0), yeast extract (3.0) and L-cysteine hydrochloride (0.5), at initial pH 6.0, for 96 hours.

5. Experiment 5: Inoculum 1 [30% (v/v)] + culture medium (g L^-1): crude glycerol (5.8), yeast extract (5.0), meat extract (5.0) and peptone (5.0), at initial pH 5.5, for 96 hours.

In all tests (Experiments 1 to 5), H₂ production, pH, glycerol removal and COD removal were analyzed.

2.4. Physicochemical and chromatographic analyses

2.4.1. pH

The initial pH was adjusted during the assembly of the reactors, with additions of HCl (1M) or NaOH (1M) [²¹]. The analyses were made at initial and at final operation of the tests (experiments 1 to 5).

2.4.2. COD (chemical oxygen demand)

The samples (1.25 mL) were added with 0.75 mL of the digestion solution (potassium dichromate and mercury sulfate) and 1.75 mL of the sulfuric acid and silver sulfate solution. The tube was closed with a plastic screw cap. The samples were then placed in a COD digester (Nanocolor® VARIO C2) for 2 hours at 150°C. Subsequently, a spectrophotometric reading was performed at 620 nm [²¹].

2.4.3. Free glycerol (glycerin)

The removal of free glycerol from crude glycerol was quantified according to BONDIOLI and Della Bella [²²]. The analysis consisted of oxidizing the free glycerin present in the sample, in the presence of sodium metaperiodate, resulting in the formaldehyde, which, in turn, reacts with acetyl ketone in the presence of ammonium acetate, leading to the formation of 3,5-diacetyl-1,4-dihydrolutidine (Hantzsch reaction), quantified by spectrophotometry at 410 nm [²²].

2.4.4. Soap content

The soap content was determined by titration and expressed as sodium oleate. A mixture of 60 mL of acetone and 0.15 mL of 0.5% (w/v) bromophenol blue (prepared in 95% ethanol) was prepared and neutralized with 0.01 M NaOH. The solution was then mixed with 10 g of crude glycerol (CG) sample and heated in a water bath at 70 ± 1 °C for 1 min. Subsequently, the mixture was titrated with 0.1 M hydrochloric acid solution.

2.4.5. Non-glycerol organic matter

Non-glycerol organic matter was determined by acidifying the crude glycerol sample to pH 2.0 to promote phase separation of non-glycerol organic compounds into a distinct phase, followed by centrifugation at 9000 rpm for 10 min.

2.4.6. Moisture

The water content was determined by volumetric Karl Fischer titration according to the method described [²³].

2.4.7. Hydrogen production

Hydrogen production was measured by a displacement system adapted [²⁴] in all tests performed. The reactors were coupled to a gas scrubbing system containing a flask with a 15% sodium hydroxide (NaOH) solution to trap CO₂ that could be generated, and a flask containing distilled water. The reactors were connected by means of a “needle-hose” to the NaOH solution flask, with automatic depressurization of the incubation flask occurring due to the displacement of biogas generated in the headspace of the reactors, causing the displacement of water from the subsequent flask.

Sporadic analyses of biogas composition were performed using a Shimadzu GC 2014 and Shimadzu GC-2010 gas chromatograph equipped with a thermal conductivity detector (TCD) and a Carboxen 1010 Plot column (30 m × 0.53 mm), using argon as the carrier gas. The chromatographic conditions were: Injector temperature: 220°C; Column temperature: temperature ramp, starting at 120°C and ending at 230°C, at intervals of 40°C and 50°C; Detector temperature: 230°C; Carrier gas flow rate: 1.9 mL min⁻¹ [²⁵].

2.4.8. Adjustment of experimental data

The maximum cumulative hydrogen production rate from anaerobic batch reactors was obtained by non-linear sigmoidal adjustment of the modified Gompertz function described as research [²⁶], to evaluate hydrogen generation kinetics according to Equation (1):

(1) $$\mathit{P}(\mathit{t})=\mathit{P}\times \mathit{exp}\{-\mathit{exp}[\frac{\mathit{R}\mathit{m}\times \mathit{e}}{\mathit{p}}(\mathit{\lambda }-\mathit{t})+1]\}$$

where P is the hydrogen production potential (amplitude of the sigmoidal curve) in mmol L^-1 culture, Rm is the maximum hydrogen production rate (mmol L^-1 culture. h), λ is the initial time (h) of H₂ generation, and e is the Euler constant (2.718281828). The experimental setup was performed in the five experiments.

2.4.9. Metagenomic analysis

The DNA extraction process was performed on samples from inoculum 1 and 2 as described by [²⁷], with modifications, and stored at -20ºC until shipment to GENONE® (Rio de Janeiro-RJ, Brazil) for identification using the Illumina MiSeq NGS platform [²⁷] Subsequently, PCR amplification of the submitted DNA samples was performed using primers 515F (5ʹ-barcode-GTGCCAGCMGCCGCGG-3ʹ) and 806R (5ʹ-GGACTA CHVGGGTWTCTAAT-3ʹ) to amplify the V3-V4 regions of the 16S ribosomal RNA gene using the GeneAmp PCR System (ABI company, USA).

3. RESULTS AND DISCUSSION

It was verified, under all imposed conditions, high glycerin consumption occurred with consequent hydrogen generation. Experiment 1 produced 47.81 mmol H2 L-1, with a maximum production rate of 1.22 mmol H2 L-1 h-1. In test 2, generations of 24.19 mmol H2 L-1 and 1.22 mmol H2 L-1 h-1 were observed. The modified Gompertz model showed an excellent fit to the experimental data, with coefficients of determination (R²) equal to 0.99 for all experiments, demonstrating the suitability of the model to describe hydrogen production kinetics under the evaluated conditions (Table 1, Figure 2).

Table 1
Experiments results and values presented as mean ± standard deviation (n = 3), when available.

EXPERIMENTS	1	2	3	4	5
P (mmol H2 L-1)	47.81 ± 0.95	24.19 ± 2.22	51.48 ± 1.04	43.17 ± 1.18	15.43
Rm (mmol H2 L-1 h-1)	1.22	1.22	3.84	3.22	1.15
λ (h)	0.00	8.00	0.00	8.00	0.00
Glycerin removal (%)	97.5 ± 0.17	93.7 ± 0.05	94.4	92.4	93.0
Yield (mol H2 mol/glycerin consumed-1)	1.36	0.75	1.48	1.30	0.48
Yield (mmol H2 g-1/CODadd)	1.7	0.8	1.3	1.2	0.5
COD consumption (%)	44.9	19.1	10.3	6.6	59.9
Final pH	6.1	5.2	5.7	5.9	5.4

Figure 2
Cumulative hydrogen production fitted by the modified Gompertz model for all experimental conditions. Differences in lag phase (λ), maximum production rate (Rm), and maximum hydrogen production (P) are observed among the experiments.

Furthermore, H2 production in Experiment 1 was immediate. However, for Experiment 2, initial H2 generation was observed after 8 hours of operation. This fact may be related to the higher glycerin concentrations imposed (Table 1, Figure 3).

Figure 3
Temporal profile of hydrogen, glycerin, COD and pH of the anaerobic batch reactors in the tests: (a) Experiment 1, (b) Experiment 2, (c) Experiment 3, (d) Experiment 4 and (e) Experiment 5.

High rates in H₂ generation of 3.84 and 3.22 mmol H₂ L^-1 h^-1 in tests 3 and 4, operated with inoculum 1 and inoculum 2, respectively, proved that the addition of nutrients such as sodium acetate and L-cysteine hydrochloride were essential for the generation of hydrogen from crude glycerol [²⁸, ²⁹]. Controversially, in test 5 low hydrogen yields were observed of 0.48 mol H₂ mol glycerin consumed^-1. The presence of microorganism’s hydrogen consumers, such as methanogenic archaea, justified the high COD consumption of 59.9%, responsible for the traces of methane generation verified in headspace of these reactors [³⁰, ³¹].

The pH had low variation throughout the tests. The formulation of the culture media in all experiments with yeast extract could act as a natural buffer in microbiological media by resisting pH changes, primarily due to its high concentration of amino acids, peptides, and proteins. It provides, along with essential nutrients, protection against stress conditions caused by organic acid accumulation during yeast fermentation, thereby promoting consistent growth [³²]. For experiment 4 (reactors operated with inoculum 2), pH stability was observed up to 8 hours, probably due to the adaptation of the bacteria to the nutritional conditions, also resulting in reduced H₂ production. After 20 hours of operation, there was an increase in H₂ production and, consequently, a drop in pH.

In Experiments 3 and 5 (reactors operated with inoculum 1), the pH showed little variation. The pH increased in Experiment 1, varying from 5.50 ± 0.05 to 6.09 ± 0.3 at the end of the operation. Conversely, in Experiment 2, a decrease in pH from 6.0 ± 0.01 to 5.23 ± 0.02 was observed. Some nitrogen compounds that were added to compose the culture media, such as meat extract and meat peptone, could have caused the formation of ammonia, as a buffering agent, under anaerobic conditions [⁴]. ADAMES et al. [³³] observed a drop in pH in series horizontal anaerobic reactors with fixed bed (HARFB) filled with 1% of crude glycerol and domestic sewage; the substrate was supplemented with 1 g L⁻¹ NaHCO₃, and the pH remained for about 10 days. The authors concluded that the domestic sewage used with crude glycerol as a substrate cannot supply the alkalinity to keep the reactor’s pH stable/controlled (Table 1, Figure 3).

Glycerin consumption was high in all experiments, ranging from 92.4% to 97.5%, confirming that the imposed concentrations were not inhibitory. These results were similar to those reported with crude glycerol [³⁴], E. aerogenes in a complex culture medium (5 g L-1 of yeast extract and 5 g L-1 of tryptone) and pH 6.8. SARMA et al. [³⁵] tested crude glycerol (10 g L-1) from transesterification of meat processing waste and restaurant oils and they obtained glycerin consumption ranging from 60% to 93%, in anaerobic batch reactors operated at initial pH 6.0, with E. aerogenes under sterile conditions, in wastewater from breweries, starch industries, apple pomace, and slaughterhouses. (Table 1, Figure 3).

It was verified that in the headspace of the reactors operated with inoculum 1 (experiments 1 and 5), the biogas composition consisted of a mixture of H2 (47.0 mmol/L) and traces of CH4 (3.2 mmol/L)). The inoculum 1, originally methanogenic, came from a UASB reactor treating vinasse and generating methane, as previously described. This evidence justified the high COD consumption for these experiments, which was 44.9% and 59.9%, respectively. SOARES et al. [³⁶] verified the significant effect of the addition of yeast extract (1g/L) on trace of methane generation (7.3 mmolL) in batch reactors filled with sugarcane bagasse applied in hydrogen production operated at thermophilic conditions (50°C), such as the present study. Conversely, in the headspace of the reactors operated with inoculum 2 (Experiments 2 and 4), only H2 production occurred. It was verified that the pretreatment and enrichment by serial dilutions selected hydrogen-generating microorganisms.

The acid pretreatment applied in this study was not sufficient to inhibit methanogenic archaea, thus favoring them and interfering with the observed results. SÁ et al. [⁶] tested various pretreatments (acid, basic, and thermal) on methanogenic inocula aiming at hydrogen generation from glucose for 120 h and did not observe effectiveness with the acid pretreatment, as in the present study. The authors observed that the inoculum pretreated with heat provided the highest yield of H2 production over 95 h of fermentation, and the maximum yield (4.62 mol H2 mol-1 sucrose) was obtained in 72 h (Table 1; Figure 3).

Differences in H₂ generation were observed in experiments 3, 4, and 5 fed with crude glycerol, as described below. The experiments 3 and 4, fed with the same composition in the culture medium it was verified: maximum H₂ production (mmol H₂ L^-1) 51.48 and 43.51; maximum production rates (mmol H₂ L^-1 h^-1) 3.84 and 3.22; yields (mol H₂ mol glycerol consumed^-1) 1.48 and 1.30, respectively. Conversely, experiment 5, fed with a culture medium with fewer nutrients than experiments 3 and 4, it was observed: 15.43 mmol H₂ L^-1, 1.15 mmol H₂ L^-1 h^-1 and 0.48 mol H² mol glycerol consumed^-1. These differences confirmed that the composition of the culture medium altered the metabolic pathway, generating a mixture of hydrogen and methane.

Sittijunda and Reungsang[14] analyzed bio-H₂ production from 20.33 g L^-1 of crude glycerol, using thermophilic mixed culture in anaerobic batch reactors operated at 55°C in different culture media and they obtained lower yields (0.3 mol H₂ mol glycerol consumed^-1). Similarly, VIANA et al. [³⁷] investigated hydrogen and methane production from crude glycerol in two-stage UASB reactors and also observed lower hydrogen yields. Then, according to the present study, these findings confirm that both culture medium composition and operational conditions strongly influence biohydrogen production under thermophilic conditions (Table 1; Figure 3).

CHOOKAEW et al. [³⁸] and O-THONG et al. [³⁹] investigated hydrogen production from crude glycerol by the thermotolerant bacterium Klebsiella sp. TR17 in anaerobic reactors at 40°C, initial pH 8.0 with 11.14 g L⁻¹ of crude glycerol obtaining hydrogen yields (0.26 mol H₂ mol glycerol⁻¹) lower than those in the present study. Previous studies [⁴] noticed that H₂ production in anaerobic batch reactors containing 20 g COD L⁻¹ crude glycerol from used cooking oil, in a culture medium, at an initial pH of 5.5, at 37°C, obtained results lower than the present study, which were 22.38 mmol H₂ L^-1, glycerol consumption of 45.74% and yields of 1.75 mol H₂ mol glycerol consumed^-1.

ADAMES et al. [⁴⁰] verified high conversion of crude glycerol into H₂, in series horizontal anaerobic reactors with fixed bed (HARFB) operated at pH 5.5, in mesophilic conditions, with daily production of crude Glycerol codigested with sanitary sludge in reactors R1 and R2 filled with 1.5% CG and 2% CG, respectively. The highest hydrogen production was (L H₂/m³ day) 277.88 (R1) and 84.43 (R2).

Metagenomic analyses performed on inoculum 1 revealed relative abundances of Bacteria (94%) and Archaea (6%) domains. Even with the applied acid pretreatment, methanogenic archaea remained, which likely produced methane during the experiments, as previously verified and described. Relative abundances of the genera Coprothermobacter, Acetomicrobium, and Lactobacillus were 60%, 24%, and 2%, respectively. The genus Coprothermobacter is generally associated with the consumption of protein-rich substrates in biodigesters with hydrogen production, due to its proteolytic properties. Furthermore, hydrogen is likely one of the most important carriers in interspecies electron transfer between Coprothermobacter spp. and methanogenic archaea. Therefore, Coprothermobacter spp. it may be involved in biological processes of degradation of organic substrates such as crude glycerol into volatile fatty acids and alcohols, in addition to hydrogenotrophic methanogenesis, in anaerobic digesters [⁴¹], as verified in the present study (Figure 4).

Figure 4
Taxonomic classification of genera from the anaerobic consortium: comparison between the inocula 1 and 2.

Conversely, in inoculum 2, relative abundances of Bacteria Domain (100%) were observed, confirming the efficiency of enrichment by serial dilutions after pre-treatment. High relative abundances of Thermoanaerobacterium thermosaccharolyticum (91%) were observed. This species was verified in thermophilic reactors operating in the range of 45 to 60°C fed with sucrose, xylose, and starch at pH ranging from 4.5 to 8, with yields of 2.53 mol H₂ mol^-1 hexose and 12.12 mmol H₂ L^-1 h^-1 [⁸, ¹⁰] as observed in the present study. Bacteria belonging to the genus Thermoanaerobacterium have been observed in studies of thermophilic H₂ production from various substrates, such as food waste [⁴²], sugarcane vinasse [⁴³] and cheese whey [⁸]. However, in the present study, this genus was found in both samples, confirming that the imposed thermophilic condition favored the maintenance of this genus (Figure 4).

Relative abundances of 24% were observed for the genus Anaerobaculum, belonging to the phylum Synergistes, in inoculum 1. This genus includes thermophilic anaerobic bacteria, bacilli morphologies, Gram-negative, non-motile, non-spore-forming, with a high capacity to produce hydrogen from glucose, and they are also found in anaerobic digesters [⁴³]. MAUNE and Tanner [⁴⁴] isolated the genus Anaerobaculum from oils in waters in Alaska (USA). The authors observed that the isolate grew on different nutrients such as tryptone, glucose, maltose, malonate, among others. However, its growth was inhibited in the presence of glycerol. Probably, inoculum 1 from a thermophilic UASB reactor contained such anaerobic bacteria belonging to the genus Anaerobaculum and it was favored by the nutrients added to the culture media, mainly meat extracts, yeast, and peptone (Figure 3).

The genus Methanobacteirum was verified in relative abundances of 22% in inoculum 1 in this study. This genus includes hydrogenotrophic methanogenic archaea. YANG et al. [⁴⁵] obtained methane generation with thermophilic methanogenic inoculum in reactors operated at 55°C from synthetic waste containing glycerol. This genus was probably involved in the methane generation observed in experiments 1 and 5, as described previously (Figure 3).

The genera Lactobacillus and Nitratireductor were verified in low relatively abundances in both inocula (<2%). The genus Lactobacillus includes anaerobic bacteria that can perform homofermentative metabolism when lactate is the only coproduct generated from glycerol fermentation and heterofermentative metabolism when other coproducts include lactate, acetate, CO₂, and ethanol [⁴⁶]. They are commonly present in biodigesters fed different organic residues, including crude glycerol [⁴⁷]. Bacteria species from Nitratireductor genus are capable to metabolize crude glycerol as a carbon source, converting it into value-added products like lipids (triacylglycerol) for potential use in biofuel production [⁴⁸]. The synergy between these two groups suggested the metabolic pathways for H₂ in crude glycerol fermentation in the present study (Figure 4).

4. CONCLUSION

The thermal pretreatment associated with serial dilution in selective culture media enabled the selection of anaerobic consortia capable of producing hydrogen under thermophilic conditions and tolerating higher concentrations of crude glycerol and its associated inhibitors.

High glycerol removal efficiencies (92.4–97.5%) confirmed that thermophilic conditions favored substrate degradation and hydrogen production. Lower hydrogen yields observed in Experiment 5 were associated with the presence of hydrogen-consuming microorganisms, which also contributed to higher COD removal and methane traces. Higher hydrogen production rates observed in Experiments 3 and 4 demonstrated that nutrient supplementation (e.g., sodium acetate and L-cysteine hydrochloride) plays a key role in optimizing hydrogen production from crude glycerol.

Metagenomic analysis revealed the predominance of thermophilic bacteria such as Coprothermobacter and Thermoanaerobacterium, which likely acted synergistically in hydrogen-producing metabolic pathways. These findings demonstrate that crude glycerol can be effectively used for biohydrogen production under thermophilic conditions without the need for costly purification processes.

5. ACKNOWLEDGMENTS

This study was supported by financially supported by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP Processes 2017/16795-3; 2017/22401-8 and 2022/15706-5).

6. DATA AVAILABILITY

The data supporting the findings of this study are available from the corresponding author upon reasonable request.

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Publication Dates

• Publication in this collection
  21 Aug 2026
• Date of issue
  2026

History

• Received
  20 Dec 2025
• Accepted
  09 June 2026