Comparison and Evaluation of Hydrogen and Methane Productions from Hydrothermal Pretreated Sugarcane Bagasse by Two Microbial Consortium

Special Article - Environmental Microbiology

Austin Clin Microbiol. 2017; 2(1): 1009.

Comparison and Evaluation of Hydrogen and Methane Productions from Hydrothermal Pretreated Sugarcane Bagasse by Two Microbial Consortium

Rabelo CABS*, Soares LA and Varesche MBA

Department of Hydraulics and Sanitation, University of São Paulo, Brazil

*Corresponding author: Camila Rabelo ABS, Department of Hydraulics and Sanitation, University of São Paulo, School of Engineering of São Carlos, Av. João Dagnone, 1100, Jd. Santa Angelina, 13563-120 São Carlos, SP, Brazil

Received: May 08, 2017; Accepted: May 26, 2017; Published: June 02, 2017


The present study aimed to compare the hydrogen and methane productions by microbial consortium designated MC1 (Clostridium, Bacillus, Bacteroides and Paenibacillus genus) and MC2 (Clostridium, Raoultella, Klebsiella and Desulfovibrio genus). Both tests used hydrothermally pretreated Sugarcane Bagasse (SCB) as substrate in mesophilic conditions (37°C). The maximum hydrogen productions were 5.33mmol/L for MC1 and 2.45mmol/L for MC2. Methane was produced only by the MC2, reaching 53.65mmol/L. Thus, the culture of MC1 can be used as a source of fermentative hydrogen producer while the MC2 can be a promising source of methanogenic microorganisms which can improve the biogas production.

Keywords: Bioconversion; Hydrogen; Methane; Hydrothermal pretreatment; Cellulolytic substrate


The hyper-consumption of non-renewable resources, mainly fossil fuels, has resulted in unprecedented levels of greenhouse gas emissions which are related to carbon dioxide emissions, considered the major cause of current global warming and climate change [1]. Therefore, research and development on biological hydrogen production using microorganisms has advanced, which may relieve the pressure caused by carbon dioxide emissions and the depletion of fossil fuel resources [2].

Hydrogen must be generated from renewable raw materials and used as renewable source of energy. Agricultural waste is a promising alternative biomass to renewable energy production once different kind can be utilized as feedstock for the biological ethanol, hydrogen and biogas productions [3,4]. The relative abundance, the world-wide distribution of these cellulosic materials is attractive factors for generating biotechnological products. The bioconversion of lignocellulosic compounds into hydrogen and biogas can occur at ambient temperature and atmospheric pressure, which is other attractive condition to biohydrogen production [5].

Sugarcane Bagasse (SCB) is a residual product of sugarcane processing, which is one of the most important process adopted in Brazil for fuel production. This residue is used as animal feed or burned to energy recovery. Nevertheless, it could be used as substrate for second generation bioethanol, methane [3] and hydrogen productions [6]. The bioproduction of hydrogen and biogas is recognized as a very promising, environmental friendly and feasible strategy [5], although some factors can affect its effectiveness.Among the factors that affect the bioconversion of lignocellulosic wastes into bioenergy, the high lignin content and cellulose crystallinity have been considered the main cause of low digestibility of the substrate. The SCB is basically composed of cellulose (40-45%), hemicellulose (30-55%) and lignin (20-30%) [3]. In this context, pretreatments have been applied to disrupt the biomass components (cellulose and lignin) and improve the enzymatic digestibility. So, the applicability of pretreatments at industrial scale should be considered i.e., economic viability, minimum generation of microbial inhibitory compounds and fewer environmental impacts [7]. Hydrothermal pretreatment is a process applied to liberate sugars from lignocellulosic materials releasing two fractions, a solid fraction, mainly containing cellulose and lignin, and a liquid fraction (hydrolyzed) containing pentose and hexose. This process can be performed without the addition of chemicals, making it a potential solution for the pretreatment of large quantities of lignocellulosic substrates [8].

The origin of inoculum is also an important factor that affects the biogas and hydrogen production from lignocellulosic biomass, because a small microbial variety can produce cellulolytic enzymes responsible for efficient degradation of the crystalline cellulose structure. Moreover, microbial consortium can work synergistically to produce all enzymes needed for complete cellulose bioconversion [9]. In addition, the use of microbial consortium makes the process simpler, from the point of view of operation and control [10], it is a more robust alternative. The great advantage of the microbial consortium application is regarding the ability to convert many substrates due to its metabolic flexibility, when compared to the pure cultures [11]. Therefore, this study evaluated the effect of inoculum origin on the hydrogen and biogas production from hydrothermally pretreated Sugarcane Bagasse (SCB).

Materials and Methods

Raw materials sugarcane bagasse

The sugarcane bagasse used in this study was provided by São Martinho sugarcane mill (Pradópolis, SP, Brazil).

Pretreatment of sugarcane bagasse

The hydrothermal pretreatment of SCB was carried out in a stainless steel-reactor. The reactor was previously filled with 100mL of water, and then, 5.0g of the substrate was introduced. After substrate addition, the reactor was turned on and set to operate at 200°C with a pressure of 15bar. After 10 minutes under these conditions, the reactor was depressurized and shut down. The solid fraction was collected, dried at ambient temperature for 48 hours, and used in the experiments.

Microbial consortium

Two microbial consortium designated MC1 and MC2 were collected from cultivation of anaerobic hydrogen-producing bioreactor with cellulose and from sludge of a facultative pond of a paper and pulp mill Wastewater Treatment Plant (WWTP), respectively.

The bacterial communities from MC1were identified and characterized by 16S rRNA gene sequence analysis. It was mainly composed by Clostridium, Bacillus, Bacteroides and Paenibacillus genus. The sequencing data of MC1 was deposited in NCBI Sequence Read Archive under the accession number of PRJNA383576. The MC1 was cultured in Reinforced Clostridia Medium, and preserved as frozen stocks at -80°C in 50% glycerol. Before each batch test, aliquots (0.2L) of the frozen stocks were cultured in Reinforced Clostridia Medium (1.8L) for 48 hours and then used as inoculums (107CFU/mL).

The MC2 was mainly composed of Clostridium, Raoultella, Klebsiella and Desulfovibrio genus, as reported in previous study [12]. The sequencing data of the microbial consortium were deposited in NCBI Sequence Read Archive under the accession number KP715408, KP715409, KP715412 and KP715410. The MC2 was enriched in 5L Duran®flasks, in which 40% was composed of reaction volume and 60% of headspace (N2 100%). Reaction volume contained 1.8L of the enrichment medium (10g/L of yeast extract, 5g/L of tryptone and 10g/L of glucose) and 0.2L of the sludge. The initial pH was adjusted to 6.8 with HCl (1.0M). The system was incubated at 37°C for 48 hours.

Both microbial consortium were previously subjected to a total anaerobic bacteria count onto Reinforced Clostridia Medium plates (Oxoid, UK) and incubated at 37°C for 48 hours in anaerobic jars for enumeration, in order to maintain the same concentration of bacteria in each reactor (107CFU/mL).

Biohydrogen and biogas production in batch reactors

Hydrothermally pretreated sugarcane bagasse was used as substrate for the biohydrogen and biogas production through dark fermentation. This step was carried out in triplicate using1.0L batch reactors, with a 0.5L working volume constituted by the culture medium (PCS), inoculum (MC1 or MC2) and the substrate (5.0g/L). Nitrogen (N2, 100%) gas was flushed into the reactors to create anaerobic conditions. The reactors were closed with rubber stoppers and incubated at 37°C. A control assay without sugarcane bagasse was also conducted.

Culture medium used in batchreactors

PCS (peptone cellulose solution) was used as a culture medium as previously reported (Haruta et al. 2002). The constitution of the culture medium was: yeast extract (1.0g/L), peptone (5.0g/L), CaCO3 (5.0g/L) and NaCl (5.0g/L).

Analytical procedures

The biogas composition in the headspace was determined by a gas chromatography (Shimadzu GC-2010) equipped with a thermal conductivity detector using argon as the carrier gas. The temperatures of the injector, detector and column were 30°C, 200°C and 300°C, respectively. An aliquot (0.5mL) of gas samples were collected from each pressurized reactor with a pressure-lock gastight syringe. The pH and Volatile Solids Concentration (VSS) were determined in accordance with APHA (2005) [14]. Soluble carbohydrates were determined using the colorimetric phenol-sulfuric acid method [15]. The determination of Volatile Organic Acids (VFA) and alcohols was performed using a High Performance Liquid Chromatography (HPLC Shimadzu) in accordance with Penteado et al. (2013) [16].

Kinetic parameters

The experimental data was fit to the mean values of the triplicate sets of reactors using the Statistica 8.0 software. The average of the hydrogen evolution data was adjusted to the modified Gompertz model [17], which has been described as a suitable model for the adjustment of accumulated biogas production data in batch experiments [4].

In the modified Gompertz equation (Eq. 1), H is the cumulative hydrogen production, t is the time of operation (days), P is the maximum hydrogen production potential (mmol/L or mL/L), Rm is the maximum hydrogen production rate (mmol/ or mL/, ? is the lag-phase period (day) and e is 2.71.

The hydrogen yield (mL H2/g SCB) was calculated as hydrogen production (mL/L) divided by SCB concentration added (g SCB/L).

Results and Discussion

Hydrogen production

The hydrogen production (Figure 1) from MC1 was higher (5.33mmol/L) than hydrogen production from MC2 (2.45mmol/L).