Electronic Journal of Polish Agricultural Universities (EJPAU) founded by all Polish Agriculture Universities presents original papers and review articles relevant to all aspects of agricultural sciences. It is target for persons working both in science and industry,regulatory agencies or teaching in agricultural sector. Covered by IFIS Publishing (Food Science and Technology Abstracts), ELSEVIER Science - Food Science and Technology Program, CAS USA (Chemical Abstracts), CABI Publishing UK and ALPSP (Association of Learned and Professional Society Publisher - full membership). Presented in the Master List of Thomson ISI.
Volume 22
Issue 2
DOI:10.30825/5.ejpau.174.2019.22.2 , EJPAU 22(2), #04.
Available Online: http://www.ejpau.media.pl/volume22/issue2/art-04.html


Piotr ŁysakowskI, Natalia Brona, Włodzimierz Grajek
Department of Biotechnology and Food Microbiology, Poznań University of Life Sciences, Poznań, Poland



Effectiveness and, by extension, profitability of the second-generation bioethanol production process is dependent on pretreatment. The aim is to develop an inexpensive, easy-to-conduct pretreatment that would highly increase hydrolysis efficiency and, at the same time, maximize the fermentable sugar yield and minimize substrate loss. In this work, optimal conditions of low-cost miscanthus and sorghum biomass pretreatment were determined and the Brunauer-Emmett-Teller surface analysis (BET) was used to characterize specific substrate area. Microscopic structure changes of both substrates were demonstrated by Scanning Electron Microscope (SEM) images. Lignin content in pretreated materials was determined according to NREL procedure. While alkali pretreatment had minor effects on the structure, acid pretreatment resulted in the emergence of noticeable pores and fissures in the surface of miscanthus and sorghum fibres. The increase in specific surface area and substrate porosity improved the efficiency of enzymatic hydrolysis of polysaccharides. However, the decrease of lignin content turned out to be a key factor in hydrolysis efficiency enhancement.

Key words: bioethanol, lignocellulose, structure, surface area.


Greenhouse gas emissions, pollution, resource depletion and unbalanced supply demand relations (all connected to fossil fuels) are the main reasons for an increased interest in renewable energy. The long list of disadvantages of petrol and diesel is strongly reduced in the case of transportation biofuels. Biofuels (i.e. bioethanol and biodiesel) are eco-friendly and the feedstock for their production, i.e. plant biomass, is common and inexhaustible [16].

Bioethanol is produced by fermentation of simple sugars, mainly glucose, cellobiose and xylose, obtained from plant biomass. First-generation bioethanol is produced from starch sugar crops such as corn, sugar cane, potatoes, wheat, rye and rice. The technology is well-developed and, in consequence, cost-effective. The biggest drawback of first-generation bioethanol is the fact that the substrates for its production may be used either as food or animal feed [12]. Second-generation bioethanol is produced from lignocellulosic biomass (LCB), i.e. fast-growing energy trees (e.g. poplar, willow), energy crops (e.g. miscanthus, sorghum) and plant waste biomass (e.g. straw, forest industry waste) [4, 10, 15]. All listed substrates are non-food and are found in abundance. Furthermore, energy plants have low soil quality requirements, therefore they may be cultivated on lands not suitable for growing food crops. Although there are numerous demonstration facilities and first commercial scale plants, the technology still needs to be upgraded to be more economical and more popular worldwide [7].

One of key factors determining the profitability of LCB derived biofuel is high fecundity and a short growth time of energy plants. Various reports show that miscanthus and sorghum are exceptionally attractive substrates for bioethanol production. Their yields reach 27 to 44 t/ha [3] and 25–42 t/ha [18], respectively. The main organic fraction of the biomass are cellulose and hemicellulose fibres surrounded by lignin. Cellulose fibres, such as hemicelluloses, require the enzymatic digestion of polysascharides to hexoses and pentoses before ethanol fermentation. Because of the robust structure of plant biomass and low efficiency of enzymatic hydrolysis, pretreatment is essential. The aim of pretreatment is to make the substrate more susceptible to enzyme hydrolysis by loosening the tightly linked structure of LCB and partial hydrolysis of plant polymers by physical-chemical agents.

Cellulose fibres are surrounded by hemicelluloses and lignin chains, which preclude access of hydrolytic enzymes. Generally, most pretreatments damage the crystalline structure of cellulose by spatial spreading of cellulose chains after the breaking of hydrogen bonds between the chains and uncovering cellulose fibres via dissolution of surrounding lignin and hemicellulose. Disruption of the cellulose crystalline structure takes place during the size reduction of biomass and treatment with rapid changes of pressure by explosive ejection of biomass from high pressure reactors or by cavitation. In order to dissolve lignin and hemicellulose, acid and alkali treatments or organic solvent extraction are carried out. Some authors suggest selective elimination of fractions by their enzymatic hydrolysis utilizing specialized enzyme preparations, ion-liquid extraction or treatment with heavy oxidants. A vast majority of processes use dilute acid or alkali to treat comminuted biomass. Pretreatment at alkaline pH causes mainly the removal of lignin, whereas hemicellulose is hydrolyzed at acid pH [2, 20]. Many pretreatment technologies have been developed, e.g. steam-explosion, ammonia fibre explosion (AFEX), CO2 explosion, dilute acid hydrolysis, alkaline hydrolysis, high-pressure water pretreatment and ultrasound pretreatment [1, 9, 14, 17, 19, 22]. Almost all pretreatment technologies exploit high pressure and high temperature, often exceeding 200ºC, which increases the cost of operations and requires specially designed reactors. Basic methods employed to verify pretreatment efficiency are chemical composition analysis of substrate after the pretreatment and determination of substrate susceptibility to enzymatic hydrolysis. Additionally, cellulose crystallinity, specific surface area, pore diameter and volume, as well as wetting angle analyses are made. SEM images are presented to illustrate morphological changes in LCB as a result of substrate pretreatment.

The main aim of any pretreatment is to ensure maximum sugar recovery from biomass at a minimal substrate loss. Most advanced modern technologies are based on this strategy, which requires the use of high energy outlays and expensive equipment. Alternatively, in a more ecological concept of bioethanol production the aim is to obtain the greatest possible amounts of simple sugars without high energy consumption. Produced lignocellulosic residues resistant to enzyme hydrolysis may be used to produce biofuels by different technologies (e.g. methane fermentation, direct combustion). Chemicals, food ingredients and animal feed may also be obtained from the above-mentioned residues. This approach is currently gaining in importance and is being intensively studied and reported by many authors [5, 6, 8, 13, 21].

The main aim of the study was to develop a low-cost method of biomass conversion into mono- and disaccharides, based on dilute acid and alkali pretreatments combined with incubation at moderate temperatures of 90–121ºC. Effectiveness of both pretreatment types was assessed based on surface area (SA) determination, SEM images of materials after chemical pretreatments and susceptibility of treated substrates to enzymatic hydrolysis.


Energy grasses
Miscanthus gighanteus (tetraploid from the Institute breeding program) was provided by the Institute of Plant Genetics, Polish Academy of Sciences, Poznań (Poland). Sorghum bicolor (variety Sucrosorgo 506) was provided by the Institute of Natural Fibers and Medicinal Plants, Poznań. The biomass was harvested in October 2014. All samples were dried at room temperature.

Composition analysis
Glucan, xylan and lignin contents were determined according to the National Renewable Energy Laboratory procedure (Determination of Structural Carbohydrates and Lignin in Biomass, www.nrel.gov/). Results are shown as the average of three repetitions. Prior to the analysis, raw materials were cut into 3–5 cm pieces, dried at room temperature to moisture content below 10%, milled on the Retch SM 100 knife mill equipped with a 2.0 mm sieve and extracted for 8 hours with 99.8% ethanol.

Prior to pretreatments raw materials were milled on the Retsch SM 100 knife mill equipped with a 4.0 mm sieve. Acid pretreatment was carried out by soaking raw material in a sulfuric acid solution at 10% w/v dry mass and autoclaving at 121°C for 1 h. Alkali pretreatment was carried out by soaking raw material in a hydroxide solution at 10% w/v solids and incubation in a water bath at 90°C for 5 h. After the pretreatment materials were chilled and thoroughly rinsed with tap water to neutral pH. Materials were stored at 4°C in zipper bags.

Mass loss
Mass loss was determined as the difference between dry mass before and after the pretreatment and water rinsing. Results are shown as the average of three repetitions.

Enzyme activity
Cellulolytic activity of Flashzyme Plus 200 (AB Enzymes, Germany) was determined according to the NREL procedure (Measurement of Cellulase Activities) and expressed as FPU/ml. 50 mg Whatman No. 1 filter paper strips were used as substrate for assays. Tests were carried out at 50°C in 0.05 M citrate buffer (pH 4.8). After the addition of the enzyme solution, samples were stirred and left in the water bath for 60 minutes. The reaction was stopped by addition of DNS reagent. The amount of liberated glucose was measured via spectroscopy at a 540 nm wave length.

Enzymatic hydrolysis
Enzymatic hydrolysis was run based on the NREL procedure Enzymatic Saccharification of Lignocellulosic Biomass at 50°C, pH 4.8 with light stirring. The applied enzyme loading was 60 FPU/g biomass. The concentration of liberated simple sugars (glucose, xylose) was determined after 120 h on HPLC. Results are shown as the average of three repetitions.

HPLC analysis
The concentration of sugars (glucose, xylose) was determined on the VWR-HITACHI LaChrom Elite system consisting of an autosampler (model L-2200), a pump (model L-2130) and a refractive index detector (model L-2490). Analyses were performed isocratically at a flow rate of 0.6 ml/min at 40°C, on the Rezex ROA- Organic Acid H+, 300 x 7.8 mm column (Phenomenex). 0.005 N sulfuric acid was used as a mobile phase. Standards were used to identify peaks in chromatograms, while peak area was used to determine the sample concentrations. It was performed by computer integration (EzChrom Elite, Version 3.3.2 SP2) operating in the mode of the external standard. Results are shown as the average of three repetitions.

Scanning Electron Microscopy (SEM)
Prior to analysis, pretreated materials were dried at 100°C for 24 h and subsequently coated with gold particles (120 s) using a gold sputter coater. Morphology of samples were characterized by scanning electron microscope (SEM) Quanta FEG 250 (FEI). Before analysis samples were transferred to SEM and characterized in Low Vacuum mode at 70 Pa using beam voltage 10 kV.

Gas porosimetry
About 0.5g samples were weighed for each analysis. Next samples were dried in vacuum at 120°C for 100 h. Complete nitrogen adsorption/desorption isotherms were gathered in liquid nitrogen temperature (77 K). Gas porosimetry was carried out with ASAP 2420 (Micrometrics, USA). Surface area was calculated according to the Brunauer–Emmett–Teller (BET) model. Pore shapes in materials were determined based on isotherm types.


Substrate composition
Miscanthus gighanteus and Sorghum bicolor had similar chemical compositions (Tab. 1). Total polysaccharide percentage was 62.6% dry mass (d.m.) and 60.9% d.m. for miscanthus and sorghum, respectively.

Table 1. Feedstock composition
Substrate Glucan Xylan Lignin
% w/w (dry mass)
Miscanthus gighanteus 40.2
± 3.1
± 1.5
± 0.1
Sorghum bicolor 38.9
± 2.3
± 1.0
± 0.0

Sorghum contained less lignin in comparison to miscanthus biomass. The chemical composition of raw materials presented by other authors varied and depended on genotypic variations and harvest time. [11] reported that the average lignin content in miscanthus is 12.0–12.5% d.m., cellulose 50.3–52.1% d. m. and hemicelluloses 24.8–25.7% d.m. Similar data on the chemical composition of miscanthus are presented by [3]. In our study the holocellulose content was lower, while the content of lignin was higher when compared to the data mentioned above. A slightly higher glucan content in miscanthus might suggest that it is a better substrate for bioethanol production; however, the higher lignin content remains an obstacle. The decision which feedstock is better suited for the process should not be only based on its chemical composition. Composition changes and mass loss during pretreatment are also of high importance.

Substrate composition after pretreatment (lignin content)
Lignin content was determined after pretreatment using various concentrations of NaOH and H2SO4 (Fig. 1). In both plant substrates a similar reaction to the pretreatment was observed. At the lowest NaOH concentration of 0.5% w/v, both treated solid materials showed a slight increase in lignin content when compared to raw materials. Presumably, it is caused by a partial decomposition of hemicellulose and extractives. At NaOH concentration from 1% to 2%, lignin content was lower than in raw materials. At 2.5% NaOH, the lignin content increased again, which might be caused by partial polysaccharide hydrolysis. The results prove the mode of alkali action on lignocellulosic biomass, as described in literature. In alkaline environment lignin is decomposed and, as a result, hemicellulose and cellulose fibres are unveiled. Acid pretreatment, in contrast to alkali, resulted in a gradual relative increase of lignin content in materials with the growing concentration of sulfuric acid. Lignin content ranged between 29–34% and 24–31% for miscanthus and sorghum, respectively. The increase of lignin content is caused by acid hydrolysis of hemicellulose. Both lignin and hemicellulose surround cellulose fibres and inhibit enzyme hydrolysis. Removal of these cell wall components provided access of enzyme molecules to the polysaccharide chains and enhanced their enzymatic digestion.  

Fig. 1. Lignin contents after substrate pretreatment

Mass losses
Dry matter analysis showed significant differences in mass losses between alkali and acid pretreatments (Fig. 2). Alkali pretreatment resulted in 16–36% loss of dry matter for miscanthus and 26–41% for sorghum. A greater mass loss was observed after acid pretreatment, amounting to 33–43% and 34–49% for miscanthus and sorghum, respectively. Generally, the higher the NaOH/H2SO4 concentration, the greater the mass loss. The study showed that miscanthus has a higher glucan content and is less susceptible to mass loss than sorghum, which makes it a better substrate for bioethanol production. Mass loss determination is usually omitted in reports concerning pretreatment even though it is as important in the calculation of efficiency as changes in biomass composition. Pretreatment is almost always connected with partial feedstock wastage, which needs to be taken into account when comparing various pretreatments.

Fig. 2. Mass loss after pretreatment

Enzyme susceptibility after chemical pretreatments
Table 2 presents the concentrations of glucose and xylose liberated from biomass after 120 h enzyme hydrolysis. Generally, a higher concentration of sodium hydroxide/sulfuric acid used for pretreatment causes an increase of glucose concentration in enzyme hydrolysates. Alkali pretreatment caused the greatest increase in glucose concentration when NaOH concentration was increased from 0.5% to 1%, namely 2.58 g/l to 6.01 g/l glucose from miscanthus and 2.91 g/l to 6.10 g/l from sorghum. A further increase of NaOH concentration to 2% caused a slight gain in released glucose. At a 2.5% NaOH the glucose concentration falls as a result of inhibited enzyme activity by products of the reaction. Xylose concentration increased twofold between 0.5% and 1% NaOH and afterwards remained constant. Similar results were obtained for both grasses.

Table 2. Sugar concentrations after enzymatic hydrolysis
of pretreatment catalyst %
Sugar concentration
NaOH treatment H2SO4  treatment
Miscanthus Sorghum Miscanthus Sorghum
Glucose Xylose Glucose Xylose Glucose Xylose Glucose Xylose
0.5 2.58
± 0.26
± 0.01
± 0.05
± 0.09
± 0.12
± 0.04
± 0.23
± 0.03
1 6.01
± 0.52
± 0.26
± 0.70
± 0.15
± 0.43
± 0.07
± 0.27
± 0.09
1.5 7.31
± 0.36
± 0.14
± 0.10
± 0.19
± 0.31
± 0.06
± 0.43
± 0.05
2 8.70
± 0.72
± 0.29
± 0.60
± 0.05
± 0.28
± 0.09
± 0.34
± 0.16
2.5 7.90
± 0.22
± 0.17
± 0.43
± 0.14
± 0.12
± 0.02
± 0.16
± 0.04

Acid pretreatment did not cause such a sharp increase in glucose concentration between 0.5% and 1% H2SO4. The amount of released glucose was lower than in the case of alkali pretreatment at every concentration. What was noticeable, enzyme hydrolysis after acid pretreatment liberated much less xylose, regardless of sulfuric acid concentration. Most likely it was caused by an almost complete decomposition of hemicellulose under experimental conditions at every sulfuric acid concentration.  

When analyzing the data related to mass losses and glucose releases (data not shown), it was found that the most efficient pretreatment conditions were obtained at 1.5% NaOH and 2.5% H2SO4 concentrations. Samples prepared according to the optimized protocols, i.e. alkali pretreatment: incubation in 1.5% NaOH, 90°C for 5 h, and acid pretreatment: autoclaving in 2.5% H2SO4, 121°C for 1 h,  were analyzed by gas porosimetry and SEM.

Table 3 presents the amount of glucose released from 1000 g dry matter of substrates under optimal conditions for pretreatment and enzymatic hydrolysis. Alkali pretreated substrates liberated more glucose than acid pretreated. Miscanthus turned out to be a better feedstock when compared to sorghum. The greatest theoretical calculated amount of ethanol is 168 g from 1000 g of raw miscanthus.

Table 3. A comparison of optimized pretreatments
Substrate/pretreatment Dry mass before pretreatment [g] Mass loss [%] Dry mass after pretreatment [g] Glucose [g] Theoretical ethanol [g]
Miscanthus/alkali 1000 31 690 336 168
Sorghum/alkali 1000 43 570 290 145
Sorghum/acid 1000 49 510 244 122
Miscanthus/acid 1000 43 570 214 107

SEM pictures and gas porosimetry results
Analyses of nitrogen sorption isotherm shape showed that all isotherms have a hysteresis loop, which signals the mesoporous capillary condensation. The hysteresis loop type was matched with the shape of loop H3 (according to the IUPAC classification). The H3 isotherm class is usually observed in materials composed of aggregated lamellar structures. SEM pictures of examined materials (Fig. 3) confirm material topography. The pictures show a similar structure of both feedstocks in their native form. Miscanthus and sorghum are fibrous materials with a high number of aggregated lamellar structures. Grasses after alkali pretreatment exhibit a closed, fibrous structure, whereas acid pretreated materials show an open, macro-porous architecture.

Fig. 3. SEM pictures (1500x magnitude) of untreated (left), alkali pretreated (middle) and acid pretreated (right) miscanthus (top) and sorghum (bottom).

These observations were confirmed by surface area (SA) analyses. SA is significantly greater in acid pretreated feedstocks. Table 4 presents surface area compared for both substrates and both types of pretreatment. SA of native raw materials were 0.46 m2/g and 0.64 m2/g for miscanthus and sorghum, respectively. A high increase in SA is observed in samples after acid pretreatment, amounting to 274% (miscanthus) and 225% (sorghum). Alkali pretreatment resulted in a slight increase in miscanthus SA (28%) and a slight decrease in sorghum SA (-22%).

Table 4. Surface area comparison
  Surface area [m2/g]
Raw material Acid pretreated Alkali pretreated
Miscanthus 0.46 1.72 0.59
Sorghum 0.64 2.08 0.50


In summary, it is possible to efficiently pretreat lignocellulose materials with no effect of rapid material expansion, as it is the case in steam-explosion or AFEX. Analyses of changes in surface area of materials following pretreatment and lignin contents in the material after treatment shows that the removal of lignin is a more significant effect of pretreatment from the point of view of enzymatic hydrolysis efficiency. In the tested materials alkaline treatment resulted in a smaller raw material mass loss and greater amounts of glucose and xylose released in the enzymatic hydrolysis susceptibility test. Despite the lack of increase in substrate porosity, they were degraded to simple sugars with high efficiency. Sulfuric acid pretreatment also had a positive effect on substrate susceptibility to enzymatic hydrolysis; however, the recorded results were less promising.

The considerable increase in surface area of miscanthus and sorghum obtained as a result of acid pretreatment is most probably caused by hemicellulose dissolution. Lignin content in the material increased in relation to control samples. Pores formed as a result of pentose hydrolysis provide the potential greater penetration of the materials by cellulolytic enzymes, which is manifested in the amount of liberated simple sugars. However, better results were obtained for substrates following alkaline treatment. Although the surface area of the materials remained comparable, as a consequence of the decrease in the lignin fraction the material became more susceptible to enzymatic hydrolysis. Moreover, enzymatic hydrolysis of the material following alkaline pretreatment caused the liberation of a considerable amount of xylose, which was not contaminated by toxins, and which may also be used in ethanol fermentation.

Financial support

This work was sponsored by the grant PBS 181111: The development of an innovative technology of second generation bioethanol production from sorghum (Sorghum sp.) and miscanthus (Miscanthus sp.) biomass.

Conflict of interests

The authors declare no conflict of interest.


  1. Balat M., Balat H., Oz C., 2008. Progress in bioethanol processing. Progress Energy Combustion Sci, 34, 551–573.
  2. Bali G., Meng X., Deneff J.I., Sun Q., Ragauskas A.J., 2015. The effect of alkaline pretreatment methods on cellulose structure and accessibility. Chem. Sus. Chem., 8, 275–279.
  3. Brosse N., Dufour A., Meng X., Sun Q., Ragaukas A., 2012.  Miscanthus: a fast-growing crop for biofuel and chemicals production. Biofuels, Bioproducts and Biorefining, 9.
  4. Carriquiry M.A., Du X., Timilsina G.R., 2011. Second generation biofuels: economics and policies. Energy Policy, 4222–4234.
  5. Chen B-Y., Chen S-W., Wang H-T., 2012. Use of different alkaline pretreatments and enzyme models to improve low-cost cellulosic biomass conversion. Biomass and Bioenergy, 39, 182–191.
  6. Dien B.S., Jung H-J. G., Vogel K.P., Casler M.D., Lamb J.F.S., Iten L., Mitchell R.B., Sarath G., 2006. Chemical composition and response to dilute-acid pretreatment and enzymatic saccharification of alfalfa, reed canarygrass, and switchgrass. Biomass and Bioenergy, 30, 880–891.
  7. Geddes C.C., Nieves I.U., Ingram L.O., 2011. Advances in ethanol production. Current Opinion in Biotechnology, 22, 312–319.
  8. Guo G-L., Chen W-H., Chen W-H., Men L-Ch., Hwang W-S., 2008. Characterization of dilute acid pretreatment of silvergrass for ethanol production. Bioresource Technology, 99, 6046–6053.
  9. Haq F., Ali H., Shuaib M., Badshah M., Hassan S.W., Munis M.F.H., Chaudhary H.J., 2016. Recent progress in bioethanol production from lignocellulosic materials: a review. International Journal of Green Energy, 13 (14), 1413–1441.
  10. Hasunuma T., Kondo A., 2012. Consolidated bioprocessing and simultaneous saccharification and fermentation of lignocellulose to ethanol with thermotolerant yeast strains. Process Biochemistry, 47, 1287–1294.
  11. Hodgson E.M., Nowakowski D.J., Shield I., Riche A., Bridgwater A.V., Clifton-Brown J.C., 2011. Variation in Miscanthus chemical composition and implications for conversion by pyrolysis and thermo-chemical bio-refining for fuels and chemicals. Bioresource Technology, 102, 3411–3418.
  12. Joshi B., Bhatt M.R., Sharma D., Joshi J., Malla R., Sreerama L., 2011. Lignocellulosic ethanol production: Current practices and recent developments. Biotechnology and Molecular Biology Review, 6(8), 172–182.
  13. Khullar E., 2012. Miscanthus conversion to ethanol: effect of particle size and pretreatment conditions for hot water. PhD Thesis. Agricultural and Biological Engineering, Graduate College, University of Illinois at Urbana-Champaign.
  14. Kumar R., Tabatabaei M., Karimi K., Sárvári Horváth I., 2016. Recent updates on lignocellulosic biomass derived ethanol - A review. Biofuel Research Journal, 9, 347–356.
  15. Limayem A., Ricke S.C., 2012. Lignocellulosic biomass for bioethanol production: Current perspectives, potential issues and future prospects. Progress in Energy and Combustion Science, 38, 449–467.
  16. Naik S.N., Goud V.V., Rout P.K., Dalai A.K., 2010. Production of first and second generation biofuels: A comprehensive review. Renewable and Sustainable Energy Reviews,  14, 578–597.
  17. Nigam P.S., Singh A., 2010. Production of liquid biofuel from renewable resource. Progress in Energy and Combustion Science, 37, 52–68.
  18. Ratnavathi C.V., Kalyana S., Chakravarthy V.V., Komala U.D., Chavan J., Patil V., 2011. Sweet Sorghum as Feedstock for Biofuel Production: A Review, Sugar Tech., 13(4), 399–407.
  19. Revin V., Atykyan N., Zakharkin D., 2016. Enzymatic hydrolysis and fermentation of ultradispersed wood particles after ultrasonic pretreatment. Electronic Journal of Biotechnology, 20, 14–19.
  20. Saha B.C., Iten L.B., Cotta M.A., Wu Y.V., 2005. Dilute acid pretreatment, enzymatic saccharification and fermentation of wheat straw to ethanol. Process Biochemistry, 40, 3693–3700.
  21. Sun S., Chen W., Tang J., Wang B., Cao X., Sun S., Sun R-C., 2016. Synergetic effect of dilute acid and alkali treatments on fractional application of rice straw. Biotechnology and Biofuels, 9, 217.
  22. Sun Y., Cheng J., 2002. Hydrolysis of lignocellulosic materials for ethanol production: a review. Bioresource Technology, 83, 1–11.

Received: 22.06.2018
Reviewed: 18.03.2019
Accepted: 21.05.2019

Piotr ŁysakowskI
Department of Biotechnology and Food Microbiology, Poznań University of Life Sciences, Poznań, Poland
ul. Wojska Polskiego 48
60-627 Poznań
email: pj.lysakowski@gmail.com

Natalia Brona
Department of Biotechnology and Food Microbiology, Poznań University of Life Sciences, Poznań, Poland
ul. Wojska Polskiego 48
60-627 Poznań

Włodzimierz Grajek
Department of Biotechnology and Food Microbiology, Poznań University of Life Sciences, Poznań, Poland
ul. Wojska Polskiego 48
60-627 Poznań

Responses to this article, comments are invited and should be submitted within three months of the publication of the article. If accepted for publication, they will be published in the chapter headed 'Discussions' and hyperlinked to the article.