Biomass Pretreatment with the Szego Mill™ for Bioethanol and Biogas Production

processes

Article Biomass Pretreatment with the Szego Mill™ for Bioethanol and Biogas Production

Merlin Raud 1,* , Kaja Orupõld 2 , Lisandra Rocha-Meneses 1 , Vahur Rooni 1, Olev Träss 3 and Timo Kikas 1

1 Institute of Technology, Estonian University of Life Sciences, 56 Kreutzwaldi Str., 51014 Tartu, Estonia; [email protected] (L.R.-M.); [email protected] (V.R.); [email protected] (T.K.) 2 Institute of Agricultural and Environmental Sciences, Estonian University of Life Sciences, 5 Kreutzwaldi Str., 51006 Tartu, Estonia; [email protected] 3 Department of Chemical Engineering and Applied Chemistry, University of Toronto, Toronto, ON M5S 3ES, Canada; [email protected] * Correspondence: [email protected]

Received: 18 September 2020; Accepted: 19 October 2020; Published: 21 October 2020

Abstract: Results from an investigation of the mechanical size reduction with the Szego Mill™ as a pretreatment method for lignocellulosic biomass are presented. Pretreatment is a highly expensive and energy-consuming step in lignocellulosic biomass processing. Therefore, it is vital to study and optimize different pretreatment methods to find a most efficient production process. The biomass was milled with the Szego Mill™ using three different approaches: dry milling, wet milling and for the first time nitrogen assisted wet milling was tested. Bioethanol and biogas production were studied, but also fibre analysis and SEM (scanning electron microscope) analysis were carried out to characterize the effect of different milling approaches. In addition, two different process flows were used to evaluate the efficiency of downstream processing steps. The results show that pretreatment 1 of barely straw with the Szego Mill™ enabled obtaining glucose concentrations of up to 7 g L− in the hydrolysis mixture, which yields at hydrolysis efficiency of 18%. The final ethanol concentrations from 1 3.4 to 6.7 g L− were obtained. The lowest glucose and ethanol concentrations were measured when the biomass was dry milled, the highest when nitrogen assisted wet milling was used. Milling also resulted in an 6–11% of increase in methane production rate during anaerobic digestion of straw.

Keywords: Szego Mill™; wet milling; nitrogen assisted milling; bioethanol; biogas

1. Introduction According to revised Renewable Energy Directive published in 2018, the European Union (EU) set a goal to become a global leader in the field of renewable energy. To gain this target, the directive establishes a new and binding target for renewable energy for the EU for 2030 of at least 32%. In addition, the energy in the transport sector should increase to at least 14% by 2030 and the limit of 7% for conventional first generation biofuels in transport sector will be reduced to 3.5% by 2030 [1]. As the goal is to shift the transport sector from fossil fuel use to the use of different fuel mixtures with a larger fraction of different biofuels, such as bioethanol and biodiesel. In addition, biofuel target in transport sector is planned to be covered with biomethane produced by anaerobic digestion of local biomass. Although the production of liquid and sustainable biofuels from lignocellulosic biomass like ethanol, butanol etc. shows potential in laboratory conditions, the large-scale and cost-effective production of cellulosic biofuels is still hindered by several technical barriers. One of the critical issues is the high capital and processing costs of which the most expensive processing step is pretreatment [2]. Therefore, there is an urgent need for research and development of new and

Processes 2020, 8, 1327; doi:10.3390/pr8101327 www.mdpi.com/journal/processes Processes 2020, 8, 1327 2 of 14 more effective methods and processes for biomass pretreatment to overcome major hindrances in lignocellulosic bioethanol production. Over the time, various methods for pretreatment of lignocellulosic biomass have been proposed and many papers studying various methods have been published [2–5]. According to working principles, the pretreatment methods can be divided into biological, chemical, and physical methods and, additionally, combined methods that apply characteristics of multiple methods [5–9]. Amongst them physical or mechanical methods involve size reduction of biomass by means of grinding chipping, or milling [10,11]. The biomass chip size is usually reduced to 10–30 mm after chipping and 0.2–2 mm after grinding or milling [4,12]. The size reduction pretreatments are very often used in combination with other pretreatment methods as a first step in the whole pretreatment process to make the subsequent processes easier and more effective [2,13]. The physical pretreatment methods are environmentally friendly and usually do not produce any toxic substances. On the other hand, they have high energy consumption and the high costs associated with large-scale implementation, and safety hazards. Different types of mills and milling processes like colloid milling, ball milling, hammer milling, vibro-energy milling, and two-roll milling have been proposed [14,15]. In this work The Szego Mill™ was used. The Szego Mill™ is a planetary ring-roller mill consisting of a stationary, cylindrical grinding surface inside which a number of helically grooved rollers rotate. The biomass is fed by gravity or pumped into the mill from top, and then discharged at the bottom of the mill. The biomass is crushed and sheared inside the mill between cylindrical surface and rotating rollers. The radial acceleration of the rollers create the crushing force while the high velocity gradients generated in the mill causes the shearing action. The biomass is transported through the mill by roller grooves and exits from the bottom of the mill [16,17]. Szego Mills™ have been used to process various woody materials like chips, sawdust, and wood wastes, such as pelletized wood waste and hog fue, as well as minerals and other materials [18]. However, the effect of this mill has not been tested for biomass milling as a pretreatment method in the second generation lignocellulosic biofuel and biogas production process. In addition, the efficiency of nitrogen gas-assisted milling process by the Szego Mill™ has not been reported for milling any biomass nor material. Previous research has shown that when using NED (nitrogen explosive decompression pretreatment), a higher efficiencies can be obtained in further biofuel production process when pressurized nitrogen gas was added during pretreatment. The biomass destruction is achieved on sudden pressure release where the gas volume inside the biomass is expanding and thereby breaking the structure of biomass [19,20]. The paper proposes that similar effect could be obtained using a Szego Mill™. By adding nitrogen gas to the milling cylinder, high pressure micro-areas arise in the cylinder during milling process. Due to rapid change in pressure, similar effects can occur as in NED pretreatment. During milling, the sudden pressure changes in between high and low pressure areas causes the moisture and gas inside the biomass to increase and decrease volume and this breaks the biomass structure. As a result, the biomass structure is opened for the next processing steps. The aim of the paper was to investigate the mechanical size reduction of barely straw with a Szego Mill™ as a pretreatment method in biofuels production. The biomass was milled with a Szego Mill™ using three different techniques: dry milling, wet milling and nitrogen assisted wet milling. The milled biomass was used in bioethanol and biogas production processes using different process schemes to evaluate the effect of different pretreatment approaches.

2. Materials and Methods

2.1. Biomass Barley straw (Hordeum vulgare) used in the experimental procedure was collected in September 2013 from the plant ﬁeld collection of Estonian University of Life Sciences, near Tartu (Estonia). Processes 2020, 8, 1327 3 of 14 Processes 2020, 8, x FOR PEER REVIEW 3 of 14

1 Thesamples samples were were dried dried to gain to gain a mo aisture moisture content content less than less 100 than g kg 100−1 and g kg ground− and with ground a SM with 100 Cutting a SM 100 CuttingMill comfort Mill comfort (Retsch (RetschGmbH, GmbH,Haan, Germany) Haan, Germany) to a particle-size to a particle-size 4 mm or less 4 mm (referred or less to (referredas “original to as “originalmaterial”). material”). Particle size Particle distributi sizeon distribution of milled straw of milled was analysed straw wasby the analysed vibratory by sieve the shaker vibratory AS 200 sieve shaker(Retsch AS GmbH) 200 (Retsch passing GmbH) the straw passing through the straw a series through of sieves a series(2.0, 1.0, of 0.5, sieves 0.25, (2.0, 0.125, 1.0, 0.05, 0.5, 0.02 0.25, mm) 0.125, and 0.05, 0.02weighing mm) and the weighing amount of the material amount on ofeach material sieve. on each sieve.

2.2.2.2. Pretreatment Pretreatment with with Szego Szego Mill Mill TheThe Szego Szego Mill Mill™™ (SM-220, (SM-220, GeneralGeneral ComminutionComminution Inc., Inc., Toronto, Toronto, ON, ON, Canada) Canada) was was used used for for biomassbiomass pretreatment. pretreatment. The The details details of mill of constructionmill construction and working and working principles principles are outlined are outlined in published in paperspublished [18] and papers in Figure [18] and1. Three in Figure modes 1. Three of milling modes were of milling used—dry were milling, used—dry wet milling, milling, wet and milling, nitrogen assistedand nitrogen wet milling. assisted In wet all modes,milling. 50In Hzall modes, and 1100 50 RPMHz and milling 1100 RPM parameters milling wereparameters used andwere 3.6 used kg of dryand biomass 3.6 kg wasof dry grounded. biomass Inwas case grounded. of dry mill, In case the of biomass dry mill, was the fed biomass through was the fed mill through twice. Afterthe mill each pass,twice. the After biomass each was pass, collected the biomass and anwas averaged collectedsample and an wasaveraged taken. sample In case was of wettaken. milling, In case the of wet 3.6 kg ofmilling, dry biomass the 3.6 was kg mixedof dry withbiomass 18 kg was of watermixed (biomass-waterwith 18 kg of water ratio (biomass-water 1:5) and fed through ratio 1:5) the and mill fed once. Forthrough the third the mode,mill once. the For biomass the third was mode, moistened the biomass with water was moistened similarly towith the water previous similarly method to the and additionally,previous method nitrogen and was additionally, fed through nitrogen the mill was and fed the through collection the mill system and with the collection biomass duringsystem milling.with biomass during milling. After milling, samples were used in the enzymatic hydrolysis or stored after After milling, samples were used in the enzymatic hydrolysis or stored after drying for later analysis. drying for later analysis.

Figure 1. A schematic describing the working principle of the Szego Mill™. Reproduced with Figure 1. A schematic describing the working principle of the Szego Mill™. Reproduced with permission permission from [Gravelsins, R. J.; Trass, O.], [Powder Technology]; published by [Elsevier], [2013]. from [Gravelsins, R. J.; Trass, O.], [Powder Technology]; published by [Elsevier], [2013].

Processes 2020, 8, x FOR PEER REVIEW 4 of 14 Processes 2020, 8, 1327 4 of 14 2.3. Route Design 2.3. Route Design Bioethanol production from biomass was evaluated using two different routes (Figure 2). In both routes the biomassBioethanol was production used in fromthe traditional biomass was thre evaluatede step using bioethanol two diﬀerent producti routeson (Figure process.2). In The both biomass was pretreatedroutes the using biomass the was Szego used Mill™. in the traditional Enzymatic three hy stepdrolysis bioethanol was production applied process.to convert The biomassthe cellulose to was pretreated using the Szego Mill™. Enzymatic hydrolysis was applied to convert the cellulose to sugars andsugars finally and ﬁnallyyeast yeastwas wasused used to toferment ferment the the sugarssugars to to ethanol. ethanol. In routeIn rout 2 thee 2 solid the andsolid liquid and liquid fractionsfractions were separated were separated after after enzy enzymaticmatic hydrolysis hydrolysis andand only only the the liquid liquid part part was usedwas inused the ethanol in the ethanol productionproduction process. process. In route In route 1, the 1, thehydrolyzed hydrolyzed bi biomassomass slurry slurry was was used used in a fermentationin a fermentation process process without withoutadditional additional processing processing and and phase phase sepa separationration waswas applied applied after after the fermentation.the fermentation.

Figure 2.Figure A flow 2. chartA flow of chart the of studied the studied routes routes of of biomass biomass forfor biofuel biofuel and and biogas biogas production. production. Reproduced Reproduced with permissionwith permission from from[Merlin [Merlin Raud Raud and and Timo Timo Ki Kikas],kas], [Environmental[Environmental and and Climate Climate Technologies]; Technologies]; publishedpublished by [Sciedo], by [Sciedo], [2020]. [2020]. 2.4. Enzymatic Hydrolysis and Fermentation 2.4. Enzymatic Hydrolysis and Fermentation Enzymatic hydrolysis was used to convert cellulose to glucose. Distilled water and enzyme Enzymaticwere added hydrolysis to the 100 was g of used pretreated to convert biomass cellulose in order to to glucose. gain the Distilled final volume water of the and mixture enzyme were 1000 mL. An enzyme mixture (Accellerase 1500 from DuPont®, city, USA; 30 FPU g 1 cellulose) was added to the 100 g of pretreated biomass in order to gain the final volume of− the mixture 1000 mL. added to biomass suspension at a ratio of 0.3 mL per g of biomass [21]. Hydrolysis lasted for 24 h at An enzyme mixture (Accellerase 1500 from DuPont®, city, USA; 30 FPU g−1 cellulose)1 was added to a temperature of 50 ◦C under constant stirring in a rotating shaker/incubator (250 min− ) (IKA 4000 i biomass Controlsuspension IKA®-Werke at a ratio GmbH of & Co.0.3 KG,mL Mindelheim, per g of biomass Germany). [21]. Hydrolysis lasted for 24 h at a temperature Afterof 50 enzymatic °C under hydrolysis, constant two stirring approaches in a were rotating used (Figure shaker/incubator1). In the first option, (250 themin biomass−1) (IKA 4000 i Control IKAslurry®-Werke was used GmbH in fermentation & Co. KG, process Mindelheim, without separation Germany). and 2.5 g of dry yeast Saccharomyces cerevisiae per 100 g of biomass was added to all samples to start the fermentation process. For second After enzymatic hydrolysis, two approaches were used (Figure 1). In the first option, the biomass option, centrifugation (Heraeus Megafuge 40, Thermo, USA, 3400 rpm) was used to separate the liquid slurry wasphase used of the in suspension fermentation from theprocess solid phase without and only separation liquid was and used 2.5 in fermentation.g of dry yeast In this Saccharomyces case, cerevisiae1 per g of 100 yeast gSaccharomyces of biomass was cerevisiae addedwas to added all samples to 200 mL to of start liquid the to startfermentation the fermentation process. process. For second option, centrifugationIn both options, the(Heraeus fermentation Megafuge process was40, carriedThermo, out atUSA, room 3400 temperature rpm) (22was 1used◦C) for to 7 separate days the ± liquid phaseunder of low the oxygen suspension conditions from in 250 the or solid 1000 mLphase glass and bottles only sealed liquid with was a fermentation used in fermentation. tube. In this case, 1 g of yeast Saccharomyces cerevisiae was added to 200 mL of liquid to start the fermentation process. In both options, the fermentation process was carried out at room temperature (22 ± 1 °C) for 7 days under low oxygen conditions in 250 or 1000 mL glass bottles sealed with a fermentation tube.

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2.5. Biogas Analysis The BMP (biochemical methane potential) measurements were performed according to the guideline of Angelidaki et al. [22]. Barley straw, milled straw (dry, wet and N2 assisted milling) as well as biomass samples from bioethanol production after hydrolysis and fermentation (Figure1) were analysed for BMP. The inoculum was collected from the anaerobic reactor of the municipal wastewater treatment plant in Tartu (Estonia). Before use the inoculum was sieved through 2 mm mesh and preincubated at 36 ◦C to ensure depletion of degradable organic material. The experiments were carried out in 575 mL plasma bottles filled with 200 g of inoculum and substrate mixture with the substrate to inoculum VS (volatile solids) ratio of 0.25. The test bottles were flushed with nitrogen to assure anaerobic conditions and incubated at 36 ◦C in a Mermet isothermal thermo-chamber. All tests were performed in triplicates with each sample. Additional nutrients were not added to the BMP test. The blanks with only inoculum were included to measure the background biogas and methane production from the inoculum, which were later subtracted from that of the tests with substrate. Biogas production was determined by measuring pressure in the headspace of bottles with pressure meter BMP-Testsystem WAL (WAL Mess- und Regelsysteme GmbH, Oldenburg, Germany). The quantification of methane in the produced biogas was done by gas chromatography (CP-4900 Micro-GC, Varian Inc., Palo Alto, CA, USA) at conditions described by Luna-del Risco et al. [23]. Biogas and biomethane potential, cumulative yield achieved in the BMP test, were calculated as liters of cumulative gas produced per kg of TS or VS for normal conditions (0 ◦C, 1 atm). To characterize the methane production rate during anaerobic digestion, the first-order rate constant k was calculated from Equation (1): Bt = Bmax (1 exp( k t)) (1) × − − × where Bt is the cumulative biomethane yield at time t, Bmax is the maximum biomethane yield, k is the first-order rate constant and t is time.

2.6. Biomass Analysis The solid and liquid parts of the biomass mixture were weighed before and after pretreatment and enzymatic hydrolysis and corresponding samples were taken for analysis. The initial biomass, but also samples of solid biomass gained after pretreatment and enzymatic hydrolysis, were dried for later analysis. Dry matter content was analyzed with a Kern MLS-D moisture analyzer (KERN & SOHN GmbH, Balingen, Germany) and ash content according to NREL Technical Report NREL/TP-510-42622 [24]. The biomass was subjected to ADF (acid detergent fiber), NDF (neutral detergent fiber) ADL (acid detergent lignin) tests to determine the contents of cellulose, hemicellulose and lignin using ANKOM 2000 analyzer (ANKOM Technology, Macedon, NY, USA). The glucose and ethanol concentrations were measured using a Prominence-i LC-2030 3D Plus HPLC system (Shimadzu, Japan) and a Shimadzu RID-20A detector. The analytical separation of glucose was accomplished with the RPM-Monosaccharide Pb+2 and RHM-Monosaccharide H+ columns TM which were arranged in sequence (Rezex ). The column oven temperature was 85 ◦C and the detector temperature 60 ◦C. Autoclaved and degassed distilled water was used as a mobile phase at a flow rate of 0.8 mL/min. For the sugar quantification, a calibration range from 50 mg/L to 2000 mg/L was chosen. The injection volume of the sugar samples was 20 µL. A RezexTM ROA-Organic Acid H+ column was used for the detection of ethanol. The column oven and detector temperature were set to 50 ◦C. Autoclaved and degassed 5 mM H2SO4 was used as mobile phase at a flow rate of 0.5 mL/min. The calibration ranged from 200 mg/L to 20 g/L for the Processes 2020, 8, 1327 6 of 14

acetic acid and ethanol quantification. The sample injection volume was 10 µL. All the measurements were conducted in triplicate and average and standard deviations were calculated. Processes 2020, 8, x FOR PEER REVIEW 6 of 14 SEM at the University of Tartu at the Institute of Geology was used to take pictures of the biomass. SEM imaging of uncoated samples was carried out using an EVO MA15 SEM (ZEISS) in variable 3. Resultspressure mode. The images were captured in backscattered electronand variable pressure secondary electron modes. 3.1. Effect of Milling 3. Results Size distribution of straw samples ground with a cutting mill and the Szego MillTM are given in Figure 2.3.1. It can Effect be of Millingseen that there is a much higher mass of smaller particles in the sample ground with the SzegoSize Mill distributionTM. Grinding of straw with samples the ground cutting with mill a cutting provided mill and most the of Szego the Mill particlesTM are given with in size 1–2 mm whereasFigure with2. It can the be Szego seen that Mill thereTM size is a much fractions higher 0.5–1 mass ofmm smaller and particles 0.25–0.5 in themm sample were ground dominant with (Figure the Szego MillTM. Grinding with the cutting mill provided most of the particles with size 1–2 mm 3). The Szego MillTM was more efficient for particle size reduction than the cutting mill, which can be whereas with the Szego MillTM size fractions 0.5–1 mm and 0.25–0.5 mm were dominant (Figure3). seen alsoThe from Szego Figure MillTM 4 wherewas more SEM efficient pictures for particle of biomass size reduction milled than in different the cutting ways mill, whichhave canbeen be shown. In additionseen to also a decrease from Figure in average4 where SEM particle pictures size, of the biomass particle milled structure in di fferent has waysalso changed have been due shown. to milling with the InSzego addition Mill toTM a decrease (Figure in 5). average While particle particles size, the from particle the structure cutting has mill also have changed smooth due to millingsurfaces and TM straight edges,with the the Szego surface Mill structur(Figuree5 ).of Whileparticles particles from from the theSzego cutting Mill millTM have have smoothrough surfaces and and pieces straight edges, the surface structure of particles from the Szego MillTM have rough surfaces and pieces have sharp edges. Furthermore, the particles from wet milling show a number of loose fibres and have sharp edges. Furthermore, the particles from wet milling show a number of loose fibres and ragged endsragged (Figure ends (Figure 4B–D).4B–D). Dry Dry milling milling (Figure (Figure 44B)B) has has done done visibly visibly less damageless damage to the biomassto the biomass comparedcompared to wet tomilling wet milling (Figure (Figure 4C,D).4C,D). Moreover, Moreover the, nitrogenthe nitrogen assisted wetassisted milling wet shows milling even smaller shows even smaller particlesparticles andand more more visible visible damage damage to the to biomass the biomass through fibrethrough separation. fibre separation.

50 Original Szego, dry milling 40

10 Fractionation by size, % by Fractionation

Size fractions

Figure 3. Size distribution of barley straw after grinding with cutting mill and Szego mill. Figure 3. Size distribution of barley straw after grinding with cutting mill and Szego mill.

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Processes 2020, 8, 1327 7 of 14 Processes 2020, 8, x FOR PEER REVIEW 7 of 14

Figure 4.FigureFigure SEM 4. 4.(scanning SEM (scanning electron electron microscope) microscope) pictures pictures ofof of milledmilled milled biomassbiomass biomass samples samples by cutting by cutting mill (A)) mill (A) and the andSzegoand thethe MillSzegoSzego (( MillMillB)—dry ((((BB)—dry)—dry milling, milling,milling, (C ((C)—wetC)—wet)—wet milling,milling, ((D D()—nitrogenD)—nitrogen)—nitrogen assistedassisted assisted wetwet milling). milling). wet milling).

10 10 9 9 8 8 7 7 6 6 5 4 5 3 Concentration, g/l Concentration, 4 2 3 Concentration, g/l Concentration, 1 2 0 Glucose Ethanol, with solids Ethanol, liquid 1 Dry milling Wet milling Nitrogen assisted wet milling 0 Glucose Ethanol, with solids Ethanol, liquid Figure 5. Concentration of glucose and ethanol in hydrolysis and fermentation mixtures in case of different milling methodsDry milling and fermentationWet milling approaches.Nitrogen assisted wet milling

The composition of initial biomass and biomass treated using different modes with the Szego FigureMill 5.TMFigure Concentrationwas 5.characterizedConcentration of byglucose of fibre glucose analysis and and et ethanolhanol results. in The hydrolysishydrolysis untreated and and biomass fermentation fermentation contained mixtures mixtures7.8%, in case 40.0% ofin andcase of different27.1%di milling ffoferent lignin, milling methods cellulose, methods and and and fermentation hemicellulose, fermentation approaches. approaches. respectively. The fibre analysis after milling indicated minimal changes in biomass composition after wet milling and no change after dry milling. In case of wetThe milling composition methods, of initial the cellulose biomass and and lignin biomass contents treated of using biomass different decreased modes 1–2% with thewhile Szego the The compositionTM of initial biomass and biomass treated using different modes with the Szego Millhemicellulosewas characterized content did by not fibre chan analysisge. This results. variation The untreated in fibre biomass analysis contained results are 7.8%, in 40.0%line with and TM Mill was27.1%measurement characterized of lignin, variability cellulose, by fibre and hemicellulose,analysisstandard results.deviation respectively. The of parallel untreated The fibremeasurements. biomass analysis after contained Therefore, milling indicatedit7.8%, can be40.0% and 27.1% ofconcluded lignin, cellulose, that milling and did hemicellulose,not cause significant respectively. chemical changes The infibre biomass analysis composition. after milling indicated minimal changes in biomass composition after wet milling and no change after dry milling. In case of wet milling methods, the cellulose and lignin contents of biomass decreased 1–2% while the hemicellulose content did not change. This variation in fibre analysis results are in line with measurement variability and standard deviation of parallel measurements. Therefore, it can be concluded that milling did not cause significant chemical changes in biomass composition.

Processes 2020, 8, 1327 8 of 14 minimal changes in biomass composition after wet milling and no change after dry milling. In case of wet milling methods, the cellulose and lignin contents of biomass decreased 1–2% while the hemicellulose content did not change. This variation in ﬁbre analysis results are in line with measurement variability and standard deviation of parallel measurements. Therefore, it can be concluded that milling did not cause signiﬁcant chemical changes in biomass composition.

3.2. Bioethanol Production Figure5 illustrates the glucose and ethanol yields, and hydrolysis and fermentation e fficiencies when biomass was milled in different ways using the Szego MillTM. The lowest glucose concentration, 1 3.9 g L− in hydrolysis media, was measured when biomass was dry milled. Higher glucose 1 concentrations, 6.0 and 8.0 g L− , were measured when the biomass wet milling and nitrogen assisted wet milling were used, respectively. This can be attributed to the effect of wet milling to biomass. In the SEM pictures, it was also seen that milling gave smaller biomass pieces and wet milling additionally lead to even greater extent of biomass shredding. It has been shown that milling causes the size reduction and rupture of the polysaccharide chains bonds, which increases their exposure at particles surfaces and reduces cellulose crystallinity [25]. In addition, when wet and dry milling were compared with the Pennisetum hybrid, then at the same milling conditions, wet milling gave higher pore volume, surface area, and average pore diameter [26]. This effect helps to increase the accessibility of the substrate to enzymes and, thereby, improve hydrolysis efficiency. 1 Similarly to glucose, the lowest ethanol concentrations, 3.4–4.2 g L− , were measured after fermentation when biomass was dry milled and hydrolyzed. In comparison, wet milling methods enabled a gain of 60–100% higher ethanol concentrations. The higher ethanol yields are partially caused by the higher glucose concentrations obtained when milled moist biomass was used in the hydrolysis step. When wet milled and nitrogen assisted wet milled biomass was used in hydrolysis, 54% and 104% higher glucose concentrations were gained compared to glucose yields gained with dry milled straw, respectively. The glucose formed in hydrolysis was directly fermented into ethanol in the fermentation step. Therefore, the proportions that were seen in glucose concentration, when different milling methods were used, remained the same after fermentation in ethanol concentrations. Fermentation was carried out using two different approaches to find the most effective process in terms of ethanol and biogas yields. In the first approach, the hydrolyzed biomass slurry was used in fermentation, and in the second approach, the biomass and liquid fractions were separated using filtration and only the liquid fraction was used in fermentation (Figure1). Applying the phase separation ensured that the sugars available in the fermentation step were only from the hydrolysis step. As expected, the higher ethanol concentrations were measured in samples where biomass slurry was used in fermentation. The measured ethanol concentrations were 5–19% higher compared to the fermentation of only liquid fraction. Without solid and liquid separation, the residual cellulose remaining in the solid fraction is continuously hydrolyzed by enzymes to glucose during the fermentation step as well and then fermented to ethanol. This yielded higher concentrations of ethanol in samples, where biomass slurry was fully used for fermentation compared to samples where only the liquid fraction was used. Fermentation efficiencies exceeding 100% were caused by sugars available in forms other than glucose. The hydrolysis mixture also contains sugars in oligomeric forms and in short polymeric chains. During fermentation, the enzymes that remained in the fermentation broth continued to hydrolyze these polymers into sugars. These sugars were fermented into ethanol, which increased the final ethanol yield. Similar results have been reported in earlier studies [19,21]. Compared to the results obtained with NED pretreatment when the same biomass was used, the glucose and ethanol concentrations gained with Szego milling as a pretreatment were lower. 1 When the same biomass was used with NED pretreatment up to 20 g L− of ethanol was gained with a fermentation efficiency of 46% depending on pretreatment temperature. The ethanol concentrations 1 obtained with NED pretreatment method were up to 12 g L− [21]. In addition to pressure release, Processes 2020, 8, x FOR PEER REVIEW 9 of 14

3.3. Biogas Figure 6 shows the biomethane potential (BMP) of samples that were pretreated with dry milling, wet milling and nitrogen assisted wet milling. Samples that were pretreated using dry milling had the highest methane yields (292 L CH4 kg−1 original material), followed by samples that were pretreated with wet milling (285 L CH4 kg−1 original material), N2 assisted milling (278 L CH4 kg−1 original material) and untreated barley straw (269 L CH4 kg−1 original material). It can be seen from the Figure 5 that all materials that were Szego milled had higher BMP than the one milled using conventional cutting mill. The differences in BMP results between cutting milling and Szego milling were statistically significant (p < 0.05). Although the highest BMP was obtained using the dry milling mode,Processes the2020 highest, 8, 1327 reaction rate was achieved with nitrogen assisted wet milled material9 of(Figure 14 5). Figure 5 illustrates that the Szego milling process allows for better access to the biomass than the regular cutting mill as the kinetic rates (slope of the curve) increased up to 133% (see also Figure 7). the NED pretreatment also includes use of elevated temperatures which initiate the dissolution of Furthermore, maximum biogas output is also increased with Szego milling, which implies that some hemicellulose. Therefore, after NED pretreatment the cellulose is more opened to enzymatic treatment crystallineand higher cellulose efficiencies areas resulted have [also19]. been On the disrupted. other hand, when the Szego Mill was used in different modes,Figure neither 7 shows the hemicellulose the biomethane nor ligninand biogas were removed potentials from for biomass. original Due material to this, and the glucosesamples and that were dryethanol milled, concentrations wet milled were and lower.nitrogen assisted wet milled and subjected to bioethanol production, calculated per kg of dry matter of the original sample used in bioethanol production. The biomethane 3.3. Biogas yields varied between 210 L CH4 kg−1 original material (samples from the fermentation stage that were pretreatedFigure6 shows with the wet biomethane milling) potential and 289 (BMP) L CH of4 sampleskg−1 original that were material pretreated (samples with dry from milling, the milling stagewet millingthat were and pretreated nitrogen assisted with wet wet milling). milling. Samples A similar that trend were pretreatedwas obtained using for dry biogas milling yields. had the Samples 1 fromhighest the methanefermentation yields (292stage L CHthat4 kgwere− original wet milled material), had followed the lowest by samples yields that(385 were L CH pretreated4 kg−1 original 1 1 material),with wet while milling samples (285 L CH from4 kg the− original milling material), stage that N 2wereassisted dry millingmilled (278had Lthe CH highest4 kg− original biogas yields 1 material) and untreated barley straw (269 L CH kg− original material). It can be seen from the Figure5 (550 L CH4 kg−1 original material). As can be4 seen from the results, dry milling has higher biogas and that all materials that were Szego milled had higher BMP than the one milled using conventional biomethane yields. For different milling methods, the highest biogas and methane yields were cutting mill. The differences in BMP results between cutting milling and Szego milling were statistically achieved in trials with samples only milled compared to samples from bioethanol production. significant (p < 0.05). Although the highest BMP was obtained using the dry milling mode, the highest However,reaction ratethe wasfact achievedthat even with with nitrogen higher assisted ethanol wet production, milled material the (Figuresolid residue5). Figure from5 illustrates hydrolysis and fermentationthat the Szego steps milling of process the nitrogen allows for assisted better access wet tomilled the biomass material than thegenera regularted cuttingconsiderably mill as more biogas/biomethanethe kinetic rates (slope than of from the curve) other increased materials up implies to 133% that (see the also structure Figure7). of Furthermore, the biomass maximum was more open forbiogas the biological output isalso processes. increased with Szego milling, which implies that some crystalline cellulose areas have also been disrupted.

Figure 6. Biomethane production in time during experiments and corresponding best-ﬁt curves Figure(according 6. Biomethane to Equation (1))production for material in milledtime during with cutting experiments mill (untreated) and corresponding and samples that best-fit were curves (accordingdry milled, to wet Equation milled and (1)) nitrogen for material assisted milled wet milled with using cutting the mill Szego (untreated) Mill. Inset shows and samples the starting that were dryslope milled, of the wet respective milled anaerobicand nitrogen digestion assisted processes. wet milled using the Szego Mill. Inset shows the starting slope of the respective anaerobic digestion processes. Figure7 shows the biomethane and biogas potentials for original material and samples that were dry milled, wet milled and nitrogen assisted wet milled and subjected to bioethanol production, calculated per kg of dry matter of the original sample used in bioethanol production. The biomethane 1 yields varied between 210 L CH4 kg− original material (samples from the fermentation stage that were 1 pretreated with wet milling) and 289 L CH4 kg− original material (samples from the milling stage that were pretreated with wet milling). A similar trend was obtained for biogas yields. Samples from 1 the fermentation stage that were wet milled had the lowest yields (385 L CH4 kg− original material), while samples from the milling stage that were dry milled had the highest biogas yields (550 L CH4 1 kg− original material). As can be seen from the results, dry milling has higher biogas and biomethane Processes 2020, 8, 1327 10 of 14

yields. For diﬀerent milling methods, the highest biogas and methane yields were achieved in trials with samples only milled compared to samples from bioethanol production. However, the fact that even with higher ethanol production, the solid residue from hydrolysis and fermentation steps of the Processesnitrogen 2020, 8, x assisted FOR PEER wet REVIEW milled material generated considerably more biogas/biomethane than from other 10 of 14 materials implies that the structure of the biomass was more open for the biological processes.

Figure 7. Maximum biogas and biomethane yields for untreated material and samples from the Figuredi 7.ﬀerent Maximum stages of biogas bioethanol and production biomethane pretreated yields with for dry untreated milling, wet material milling and and nitrogen samples assisted from the differentwet stages milling. of bioethanol production pretreated with dry milling, wet milling and nitrogen assisted wet milling. The kinetic rate constants are shown in Figure8. As can be seen from the ﬁgure, conventionally 1 milled barley straw has the lowest kinetic rate constant (0.074 d− ). Samples that were N2 assisted wet The kinetic rate constants are shown in Figure1 8. As can be1 seen from the figure, conventionally milled had the highest kinetic rate (between 0.16 d− and 0.17 d− ),−1 followed by samples that were wet milled barley straw has the1 lowest kinetic1 rate constant (0.074 d ). Samples1 that were1 N2 assisted wet milled (between 0.12 d− and 0.15 d− ), and dry milled (being between 0.10 d− and 0.14 d− ) with the −1 −1 milledSzego had the Mill. highest The results kinetic are inrate accordance (between with 0.16 SEM d pictures and 0.17 in d Figure), followed3, where itby can samples be seen thatthat the were wet millednitrogen (between assisted 0.12 wetd−1 and milling 0.15 resulted d−1), and in the dry smallest milled particle (being size between and the most0.10 visibled−1 and damage 0.14 d to−1) the with the Szego straw.Mill. The For samplesresults thatare werein accordance N2 assisted with wet milled, SEM thepict kineticures in rate Figure constant 3, where was 106% it can to 133% be seen higher that the nitrogenthan assisted that for conventionallywet milling result milleded barley in the straw; smallest for samples particle that size were and wet the milled, most the visible kinetic damage rate was to the straw. 68%For tosamples 111% higher that than were original N2 assisted material; wet and formilled, samples the that kinetic were dryrate milled, constant the kinetic was 106% rate was to 133% higher39% than to that 87% for higher conventionally than that of original milled biomass. barley Forstraw; all the for milling samples methods, that were the highest wet milled, kinetic ratesthe kinetic were achieved for the samples that were just milled compared to further hydrolyzed and fermented rate was 68% to 111% higher than original material; and for samples that were dry milled, the kinetic samples. It can be explained by the presence of more easily hydrolyzable substances in the milled rate wassamples 39% thatto 87% were higher used in thethan enzymatic that of hydrolysisoriginal biomass. step of bioethanol For all production.the milling Overall, methods, fermented the highest kineticsamples rates were had higherachieved kinetic for ratesthe samples than hydrolyzed that were materials. just milled compared to further hydrolyzed and fermented samples. It can be explained by the presence of more easily hydrolyzable substances in the milled samples that were used in the enzymatic hydrolysis step of bioethanol production. Overall, fermented samples had higher kinetic rates than hydrolyzed materials.

Processes 2020, 8, 1327 11 of 14 Processes 2020, 8, x FOR PEER REVIEW 11 of 14

Figure 8. Kinetic rate constant (k) for untreated material and samples from the different stages of Figurebioethanol 8. Kinetic production rate constant pretreated (k) withfor untreated dry milling, materi wet millingal and and samples nitrogen from assisted the wetdifferent milling. stages of bioethanol production pretreated with dry milling, wet milling and nitrogen assisted wet milling. 3.4. Mass Balances 3.4. Mass TheBalances liquid and solid phases from all process stages were weighed, fiber analysis results of all biomassesThe liquid and and solid solid residues, phases and from glucose all process and ethanol stages concentrations, were weighed, as well fiber as methane analysis production, results of all measured after hydrolysis and fermentation stages, respectively, were recorded. Based on these data, biomasses and solid residues, and glucose and ethanol concentrations, as well as methane mass balances were calculated, and mass flow charts prepared for ethanol and biogas production production, measured after hydrolysis and fermentation stages, respectively, were recorded. Based processesProcesses 2020 using, 8, x FOR diff PEERerent REVIEW milling methods (Figures9 and 10). 12 of 14 on these data, mass balances were calculated, and mass flow charts prepared for ethanol and biogas production processes using different milling methods (Figures 9 and 10). The results show that during bioethanol production a small mass loss was recorded after the hydrolysis and fermentation steps. During hydrolysis, the cellulose in the biomass is partially hydrolyzed and dissolved by the enzymes. From an initial 100 g as low as 85.6 g of solid fraction was left after hydrolysis. The hydrolysis process and dissolution of cellulose partially continues during fermentation and causes continuous mass loss in the solid fraction. Therefore 89.9–79.7 g of solid fraction was left after fermentation. After hydrolysis, 272–391 mL of liquid fraction was separated and used for fermentation. When the liquid fraction was separated after fermentation, 339–380 mL of it was obtained. In both fermentation approaches used, the least amount of liquid fraction was obtained after wet milling with nitrogen addition.

Two different strategies were used for fermentation. Depending on the milling method, 1.33– Figure 9.9. The mass balance of bioethanol and anaerobicanaerobic digestion process using didifferentﬀerent millingmilling 1.70 g of ethanol could be producedTM when solid and liquid fractions were separated, and only the methods with thethe SzegoSzego MillMillTM when liquid and solid fractions (dry matter) are separatedseparated after liquid fractionhydrolysis was andand used beforebefore in fermentation. fermentation.fermentation. When the biomass broth was fully used in fermentation, 1.45–2.26 g of ethanol could be produced with different milling methods. Depending on the fermentationThe resultsstrategy, show wet that milling during gives bioethanol 34–45% production more ethanol a small compared mass loss to wasdry recordedmilling using after the samethe fermentation hydrolysis and strate fermentationgy. When nitrogen steps. During was hydrolysis,added to wet the milling, cellulose the in theethanol biomass yield is partiallywas 27–55% hydrolyzed and dissolved by the enzymes. From an initial 100 g as low as 85.6 g of solid fraction was higher than with dry milling using the same fermentation strategy. left after hydrolysis. The hydrolysis process and dissolution of cellulose partially continues during fermentation and causes continuous mass loss in the solid fraction. Therefore 89.9–79.7 g of solid

Figure 10. The mass balance of bioethanol and anaerobic digestion process using different milling methods with the Szego MillTM when liquid and solid fractions (dry matter) are separated after

fermentation.

Biogas production experiments revealed that the highest amount of biomethane could be produced from milled biomass—28.7–29.8 L methane from 100 g of biomass. After processing the biomass in hydrolysis and fermentation steps the biomethane yield decreases with each step since some of the volatile matter has been removed. If the solid would be removed after hydrolysis and solid residue would be used for biomethane production, then additionally 23.3–28.1 L biomethane could be produced. If the solids would be separated after fermentation, the biomethane yield would be 21.0–25.2 L. Overall, more biomethane was produced in case of nitrogen assisted wet milling in case of combined bioethanol and biomethane production. As the biogas and biomethane potentials were similar in all milling methods the difference in yields was most likely caused by the mass difference of solid residues. When nitrogen assisted wet milling was used as a pretreatment, the larger amount of solid residues after each process step remained and, therefore, more biomethane could be produced. Although nitrogen assisted wet milling produced almost double the glucose compared to dry milling, all milling methods are too ineffective as a single pretreatment. The same biomass was used with steam explosion pretreatment and explosive decompression pretreatment at similar conditions and up to 9.0 g of ethanol from 100 g of biomass was obtained [19,21]. Pretreatment by milling enables a breakdown of the biomass structure but as hemicellulose is not removed with pretreatment, the enzyme accessibility to cellulose is not sufficient for efficient bioethanol production. Compared to earlier studies, less liquid fraction was removed and therefore, the final ethanol yield is smaller although the ethanol concentration in media was comparable.

Processes 2020, 8, x FOR PEER REVIEW 12 of 14

Processes 2020, 8, 1327 12 of 14 fraction was left after fermentation. After hydrolysis, 272–391 mL of liquid fraction was separated and usedFigure for fermentation. 9. The mass balance When theof bioethanol liquid fraction and anaerobi was separatedc digestion after process fermentation, using different 339–380 milling mL of it was obtained.methods with In both the fermentationSzego MillTM when approaches liquid and used, solid the leastfractions amount (dry ofmatter) liquid are fraction separated was after obtained afterhydrolysis wet milling and with before nitrogen fermentation. addition.

Figure 10. The mass balance of bioethanol and anaerobic digestion process using different Figure 10. The mass balance of bioethanol and anaerobic digestion process using different milling milling methods with the Szego MillTM when liquid and solid fractions (dry matter) are separated methods with the Szego MillTM when liquid and solid fractions (dry matter) are separated after after fermentation. fermentation. Two different strategies were used for fermentation. Depending on the milling method, 1.33–1.70 g Biogas production experiments revealed that the highest amount of biomethane could be of ethanol could be produced when solid and liquid fractions were separated, and only the liquid produced from milled biomass—28.7–29.8 L methane from 100 g of biomass. After processing the fraction was used in fermentation. When the biomass broth was fully used in fermentation, 1.45–2.26 g biomass in hydrolysis and fermentation steps the biomethane yield decreases with each step since of ethanol could be produced with different milling methods. Depending on the fermentation strategy, some of the volatile matter has been removed. If the solid would be removed after hydrolysis and wet milling gives 34–45% more ethanol compared to dry milling using the same fermentation strategy. solid residue would be used for biomethane production, then additionally 23.3–28.1 L biomethane When nitrogen was added to wet milling, the ethanol yield was 27–55% higher than with dry milling could be produced. If the solids would be separated after fermentation, the biomethane yield would using the same fermentation strategy. be 21.0–25.2 L. Overall, more biomethane was produced in case of nitrogen assisted wet milling in Biogas production experiments revealed that the highest amount of biomethane could be produced case of combined bioethanol and biomethane production. As the biogas and biomethane potentials from milled biomass—28.7–29.8 L methane from 100 g of biomass. After processing the biomass in were similar in all milling methods the difference in yields was most likely caused by the mass hydrolysis and fermentation steps the biomethane yield decreases with each step since some of the difference of solid residues. When nitrogen assisted wet milling was used as a pretreatment, the volatile matter has been removed. If the solid would be removed after hydrolysis and solid residue larger amount of solid residues after each process step remained and, therefore, more biomethane would be used for biomethane production, then additionally 23.3–28.1 L biomethane could be produced. could be produced. If the solids would be separated after fermentation, the biomethane yield would be 21.0–25.2 L. Overall, Although nitrogen assisted wet milling produced almost double the glucose compared to dry more biomethane was produced in case of nitrogen assisted wet milling in case of combined bioethanol milling, all milling methods are too ineffective as a single pretreatment. The same biomass was used and biomethane production. As the biogas and biomethane potentials were similar in all milling with steam explosion pretreatment and explosive decompression pretreatment at similar conditions methods the difference in yields was most likely caused by the mass difference of solid residues. and up to 9.0 g of ethanol from 100 g of biomass was obtained [19,21]. Pretreatment by milling enables When nitrogen assisted wet milling was used as a pretreatment, the larger amount of solid residues a breakdown of the biomass structure but as hemicellulose is not removed with pretreatment, the after each process step remained and, therefore, more biomethane could be produced. enzyme accessibility to cellulose is not sufficient for efficient bioethanol production. Compared to Although nitrogen assisted wet milling produced almost double the glucose compared to dry earlier studies, less liquid fraction was removed and therefore, the final ethanol yield is smaller milling, all milling methods are too ineffective as a single pretreatment. The same biomass was used although the ethanol concentration in media was comparable. with steam explosion pretreatment and explosive decompression pretreatment at similar conditions and up to 9.0 g of ethanol from 100 g of biomass was obtained [19,21]. Pretreatment by milling enables a breakdown of the biomass structure but as hemicellulose is not removed with pretreatment, the enzyme accessibility to cellulose is not sufficient for efficient bioethanol production. Compared to earlier studies, less liquid fraction was removed and therefore, the final ethanol yield is smaller although the ethanol concentration in media was comparable.

4. Conclusions Milling of biomass with the Szego MillTM as a pretreatment method in the bioethanol and biogas production process was investigated. The biomass was milled with the Szego MillTM using three different approaches to evaluate the effect of dry milling, wet milling and nitrogen assisted wet milling. The results showed that the wet milling methods are more effective than dry milling. Wet milling Processes 2020, 8, 1327 13 of 14 gave higher bioethanol yields and biogas from process residues than dry milling. The comparison of different fermentation strategies showed that in terms of ethanol yield the separation of solid and liquid phase should be done after the fermentation step. Although ethanol yields were lower than obtained with similar biomass in previous studies using NED pretreatment, the method is suitable as a pretreatment step for size reduction. One of the benefits of using the Szego MillTM is the increased rate of anaerobic digestion. Straw pretreatment with a Szego MillTM resulted in a higher methane production rate than grinding with the cutting mill. The rate was increased the most in case of N2 assisted wet milling.

Author Contributions: Conceptualization, M.R. and T.K.; experimental work, M.R., K.O., V.R.; data analysis M.R., K.O., L.R.-M.; writing—original draft preparation, M.R.; writing—review and editing, M.R., K.O., L.R.-M., O.T., T.K.; funding acquisition, M.R. All authors have read and agreed to the published version of the manuscript. Funding: We gratefully acknowledge the financial support of the European Regional Development Fund via the Mobilitas Pluss (project MOBERA2) of the Estonian Research Council and the Doctoral School of Energy and Geotechnology III, supported by the European Union, European Regional Development Fund (Estonian University of Life Sciences ASTRA project “Value-chain based bio-economy”). Acknowledgments: Anti Perkson for introducing and helping with Szego mill and Kalle Kirisimäe for helping with SEM pictures are gratefully acknowledged. Conflicts of Interest: The authors declare no conflict of interest.

Nomenclature

NED nitrogen explosive decompression pretreatment BMP biochemical methane potential VS volatile solids TS total solids ADF acid detergent ﬁber NDF neutral detergent ﬁber ADL acid detergent lignin SEM scanning electron microscope

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