The increase in consumption of fossil fuels, the environmental pollution and the threat of greenhouse effect have forced a dynamic development of alternative fuel markets.
A promising lignocellulosic raw material for bioethanol (second generation biofuel) is hemp (Cannabis sativa L.) biomass. In recent years, there has been a dynamic increase in the area of hemp cultivation in Poland (over 1000 ha). The cultivation of hemp for seed purposes is currently intensively developed, therefore hemp biomass remains unused in the field, which can be a suitable raw material for the production of second generation bioethanol. The hemp dry matter yield is 10–15 t·ha−1. It is an environmentally friendly plant, with a short vegetation period of 3–4 months and a rapid growth of up to 4 m in height, which improves soil quality and is useful for the reclamation of degraded areas (post-mining heaps). Based on data from 2016, it is estimated that in Poland, the area of devastated and degraded land requiring remediation, constituting a potential area for growing hemp for energy purposes, is about 65,000 ha.
Hemp is also extremely resistant, perfectly adapt to different climatic conditions. They grow on almost any soil and can improve its quality, are not susceptible to various pests and do not require the use of plant protection products, and 1 ha of hemp binds around 2.5 t CO2
Hemp biomass contains in its structure a polymeric complex called lignocellulose, which is relatively difficult to degrade. The lignocellulosic complex found in cell walls of hemp is composed of the cellulose, hemicellulose and lignin. Cellulose and hemicellulose, after efficient deconstruction, can become productive substrates in the fermentation process. Lignin, consisting of phenolic alcohol derivates, is an effective obstacle in bioethanol production from plant biomass. The production of biofuel from lignocellulosic material requires deconstruction of the cell wall into individual polymers and the hydrolysis of carbohydrates into monomeric sugars [3
Bioethanol production from lignocellulosic material can be divided into three steps: physical treatment, followed by chemical pretreatment, enzymatic hydrolysis and ethanol fermentation.
In order to disintegrate the biomass and remove lignin, several pretreatment methods often used including - physical, chemical and biological methods [6
]. The physical methods of the pretreatment of lignocellulosic biomass, whose aim is to reduce the size of the substrate as well as to facilitate the access of bioactive substances to the surface, reduction of polymerization and crystallization degree of lignocellulose, include: milling, an extrusion method, and an ultrasound pretreatment. The chemical processes include treatment with acid (typically sulfuric or hydrochloric acid), alkali (sodium hydroxide, calcium carbonate, ammonia) or neutral (ionic liquids, liquid hot water LHW), the organosolv process, steam explosion, SO2
or ammonia (AFEX), the ammonia recycle percolation (ARP), ozonolysis [6
]. Depending on the method used, different changes occur within the lignocellulosic complex. The alkali pretreatment’s function is mainly delignification, while the acid pretreatment process degrades most hemicellulose. A neutral solvent mainly depolymerises lignin, while the application of hot liquid or steam induce the degradation of hemicellulose and a small quantity of lignin. The non-specificity of the acidic treatment leads to the formation of complex sugars and compounds inhibitory to the functioning of microorganisms utilized for ethanol production [9
]. An effective pretreatment process should solves the following issues: decrystallize the cellulose without causing its hydrolysis; depolymerize hemicellulose; restrict the formation of inhibitors which impede the hydrolysis of carbohydrates; require low energy input; allow the value added products such as lignin to recover; and, finally, it should be cost-effective [10
]. Unfortunately, none of the lignocellulosic biomass pretreatment methods described above meets all these criteria at the same time.
It is therefore necessary to subject the lignocelullosic biomass to pretreatment, which significantly affects the course of the further stages of bioethanol production i.e., enzymatic hydrolysis and fermentation process [11
The alkaline pretreatment removes the acetyl and uronic acid groups present on the hemicellulose, thus increasing access for hemicellulases. This process can significantly improve in solubilizing lignin, showing less solubility of cellulose and hemicellulose. This pretreatment allows to increase the internal surface of cellulose, reduce the degree of polymerization and crystallinity and disrupt the structure of lignin [12
The next stage is enzymatic hydrolysis, which determines the amount of simple sugars metabolized by yeast in the fermentation process. The decomposition of cellulose to simple sugars requires synergistic action of three types of cellulases: endoglucanases, cellobiohydrolases and ß-glucosidase. The action of enzymes involves the attack on the cellulose by bonding with cellulose fibres in amorphous places, the cleavage of cellulosic chains, cutting off their considerable fragments, and then degrading them until the glucose polymer is obtained [13
The last stage of bioethanol production is an ethanol fermentation. The ethanol fermentation process can be carried out in two ways; the first is separated hydrolysis and fermentation (SHF) while the other is simultaneous saccharification and fermentation (SSF). In SHF process the enzymes obtain the optimum at 50–60 °C and the pretreated biomass is first converted into fermentable sugar by cellulase. In SSF process the enzymes must be adapted to the temperature of the fermentation process 30–40 °C (the optimum temperature for Saccharomyces cerevisiae
) and the pretreated biomass is converted into bioethanol in the presence of both enzymes and yeast in one bioreactor [14
The aim of the study was to evaluate the pretreatment, enzymatic hydrolysis and fermentation process of hemp biomass during the preparation of the material for the production of bioethanol. The main goal is to increase the digestibility of maximum available sugars. Each chemical pretreatment and enzymatic hydrolysis has a specific effect on the cellulose, hemicellulose and lignin fraction. Thus, the most efficient methods and conditions should be chosen, allowing maximum cost reduction of the entire process.
2. Materials and Methods
The Saccharomyces cerevisiae strain yeast Ethanol Red was obtained from the French company Lesaffre. This strains are resistant to elevated concentrations of ethanol (12–14%) and temperatures above 35 °C. The microorganisms was stored on YPD medium with the addition of 2% w/v agar-agar kept at temperature 4–8 °C.
2.2. Biomass Preparation
The raw material used in the study was Tygra biomass hemp (Cannabis sativa L.) from the Experimental Farm of INF & MP in Pętkowo, Poland. The raw material was subjected to preliminary crushing to particles of size 20–40 mm and then dried in 50–55 °C for 24 h. Next, the material was disintegrated on knife mill (Retsch SM-200, Haan, Germany) with a sieve of the mesh size of 2 mm.
2.3. Chemical (Alkaline) Pretreatment of Hemp Biomass
The evaluation of pretreatment conditions for hemp biomass was carried out at 5 h treatment with 1.5–3% sodium hydroxide in 90 °C. The alkali effect on the content of the released reducing sugars was determined by Miller’s method with 3,5-dinitrosalicylic acid (DNS) in the enzymatic test [17
]. The test was performed with the use of Celluclast 1.5 L (Novozymes) enzymatic preparation at the dose of 10 FPU·g−1
. The raw material was incubated in 55 °C in 0.05 M citrate buffer of pH 4.8 for 24 h. Then, after the enzymatic test supernatant was respectively diluted, DNS reagent was added and the mixture was incubated in a boiling water bath for 10 min. After cooling to room temperature, the absorbance of the supernatant at 530 nm was measured (UV-VIS Spectrophotometer, Jasco V-630, Pfungstadt, Germany).
2.4. Enzymatic Hydrolysis Process
The selection of the enzyme complex for the enzymatic hydrolysis process was made by conducting enzymatic tests using selected enzymes, their mixtures (compositions — 30/70, 50/50 and 70/30%) and by supplementing them with glucosidase, xylanase and their mixture (50/50%). The enzyme test was carried out under the following conditions: substrate concentration 5%, enzyme dose 10 FPU/g, temperature 55 °C (SHF process)/38 °C (SSF process), pH 4.8, time 24 h. The selection parameter was the content of released reducing sugars determined by the Miller method with 3,5-dinitrosalicylic acid (DNS).
The optimization of the enzymatic hydrolysis of hemp biomass as the SHF process was carried out acc. to the Response Surface Methodology (RSM) using the parameters: biomass content 5–10% w/v, temp. 50–70 °C, time 24–72 h, pH 4.2–5.4, dose of Flashzyme (AB Enzymes) 10–30 FPU·g−1. Also, the Flashzyme enzyme supplementation by using glucosidase 20 CBU·g−1 and xylanase 500 XU·g−1 (Sigma-Aldrich, St. Louis, MI, USA) was tested. Next, the fermentation process was carried out in 100 mL Erlenmeyer flasks containing 40 mL of medium with added S. cerevisiae (1 g dry matter/L) in the following process conditions: 37 °C, pH 4.8, 120 h.
To optimize the SSF process according to the RSM, the ranges of process parameters were selected: substrate content 5–7% w/v, dose of Flashzyme/Celluclast 1.5 L enzymes 10–30 FPU g−1 using S. cerevisiae yeast (1 g dry matter/L) at 37 °C, pH 4.8 and 120 h.
2.5. Ethanol Fermentation
The ethanol fermentation was carried out in bioreactor Biostat B Plus (Sartorius) with 2 L vessel equipped with pH, temperature and agitation controls. The temperature was maintained at 37 °C and agitation at 900 rpm, pH was controlled at 4.8 by adding 1 N NaOH or 1 N HCl. The fermentation process was used not hydrated freeze-dried yeast S. cerevisiae at a dose of 1 g/L, which corresponded to cell concentration after inoculation of about 1 × 107 cfu/mL. After inoculation, a 96 h-fermentation was carried out and samples were taken every 24 h.
2.6. Analytical Methods
The chemical composition of hemp biomass before the pretreatment was determined, i.e., cellulose acc. to TAPPI T17 m-55 [18
], hemicellulose as the difference holocellulose according to TAPPI T9 m-54 and cellulose [19
], and lignin acc. to TAPPI T13 m-54 [20
In order to provide a more complete picture of the molecular structure of hemp biomass before and after the chemical pretreatment the analysis of FTIR spectroscopy was performed using a Fourier Transform Infrared Spectrometer (FTIR, Bruker ISS 66v/S, Karlsruhe, Germany) at infrared wavenumbers of 400–4000 cm−1
The physical morphologies of hemp biomass before and after the chemical treatment were performed by using Scanning Electron Microscope (SEM, S-3400N, Hitachi, Tokyo, Japan) in high vacuum conditions. The samples were covered with gold dust.
The content of glucose and ethanol was determined by High Performance Liquid Chromatography on Elite LaChrom by VWR-Hitachi using an RI L-2490 detector, Rezex ROA 300 × 7.80 mm column from Phenomenex, at a flow rate of 0.6 mL/min, at 40 °C.
2.7. Statistical Analysis
The experiments of ethanol fermentation were carried out in triplicates. Standard deviation was calculated using the analysis of variance ANOVA, Statistica 13.0 software (p < 0.05).
In this study it is suggested that the hemp biomass is a proper source for second-generation bioethanol, as an alternative to petroleum-oil based fossil fuels. Optimal pretreatment, enzymatic hydrolysis and ethanol fermentation were showed. The use of sodium hydroxide is an efficient pretreatment method of hemp biomass which allows to obtain an increase in cellulose content and partial degradation of hemicellulose. Optimization of enzymatic hydrolysis by the RSM method allowed to achieve glucose yield at the level 36.9 g/L. The ethanol fermentation using S. cerevisiae in the present work resulted in the production of ethanol at 10.51 g/L.
Further research needs to focus on achieving a cost-effective process for the production of bioethanol, e.g., by using the genome shuffling technique, which improves the phenotypic traits of S. cerevisiae yeast, i.e., an increase in fermentation activity and resistance to temperature as well as acidic and osmotic stress.