Comparison of pretreatments and cost-optimization of enzymatic hydrolysis for production of single cell protein from grass silage fibre

Journal Pre-proof Comparison of pretreatments and cost-optimization of enzymatic hydrolysis for production of single cell protein from grass silage fibre

Ville Pihlajaniemi, Simo Ellilä, Sakari Poikkimäki, Marja Nappa, Marketta Rinne, Raija Lantto, Matti Siika-aho PII:

S2589-014X(19)30247-6

DOI:

https://doi.org/10.1016/j.biteb.2019.100357

Reference:

BITEB 100357

To appear in:

Bioresource Technology Reports

Received date:

29 August 2019

Revised date:

8 November 2019

Accepted date:

29 November 2019

Please cite this article as: V. Pihlajaniemi, S. Ellilä, S. Poikkimäki, et al., Comparison of pretreatments and cost-optimization of enzymatic hydrolysis for production of single cell protein from grass silage fibre, Bioresource Technology Reports(2019), https://doi.org/ 10.1016/j.biteb.2019.100357

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Comparison of Pretreatments and Cost-Optimization of Enzymatic Hydrolysis for Production of Single Cell Protein from Grass Silage Fibre Ville Pihlajaniemi,a,* Simo Ellilä,a Sakari Poikkimäki a,c Marja Nappa,a Marketta Rinne,b

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Natural Resources Institute Finland (Luke), FI-31600, Jokioinen, Finland

Present address: Rinheat Oy, Kutojantie 11, 02630 Espoo, Finland

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c

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*Corresponding author, Email: [email protected]

Declarations of interest: None

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b

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Finland

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VTT Technical Research Centre of Finland Ltd., P.O. Box 1000, FI-02044 VTT, Espoo,

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a

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Raija Lantto,a Matti Siika-ahoa

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ABSTRACT

Grass silage is a promising biorefinery feedstock with surplus production potential, providing a source of readily soluble protein and lignocellulosic fibre. This study presents a concept combining protein extraction with production of single cell protein from enzymatically saccharified grass silage fibre by fermentation of the filamentous fungus

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Paecilomyces variotii. Steam explosion and ammonia soaking were compared as

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pretreatments, leading to 81.2% and 88.1% carbohydrate hydrolysability, respectively.

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Microbial biomass yields of 51% from hydrolysate sugars were reached with a protein

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content of 51% of cell dry weight. A single-reactor ammonia pretreatment and hydrolysis process was demonstrated, including ammonia recovery of up to 66%, while the residual

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ammonia was synergistically utilized as a nitrogen source for protein production. The effect

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of cellulase dosage, hydrolysis time and solids concentration was empirically modelled and the model was applied for cost optimization of enzymatic hydrolysis as a part of a techno-

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economic assessment of the process.

KEYWORDS: single cell protein, grass silage, green biorefinery, ammonia recycling, lignocellulose hydrolysis

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1. INTRODUCTION Population growth and increased living standards are driving the search for new sustainable sources of protein for feed and food production (Boland et al., 2013). In recent years, the concept of green biorefineries has emerged with the aim of producing high value products from grass. Grasses are highly productive crops with a high protein content and a

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lignocellulosic fibre fraction comparable to agricultural residues (Mandl, 2010). Grass crops exhibit several environmental advantages from soil quality improvement to

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groundwater protection (Grass, 2004; Hermansen et al., 2017). However, due to decreasing

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milk and beef production (Grass, 2004), a surplus grass production capacity is emerging

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with an annual estimate of 20 million tons in Europe (Mandl, 2010), providing a significant

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new feedstock source for biorefining. Green biorefineries have so far mainly focused on extraction of the soluble protein of grass for feed applications, as 30–50% of grass protein

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can be recovered by pressing and water extraction (Kamm et al., 2016; Mandl, 2010).

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Further, the yield can be optimized by manipulating the raw material quality and separation methodology (Franco et al., 2019). While direct protein extraction has been shown to be

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technically feasible, the fibre fraction of grass remains as a residue with alternative suggested uses ranging from biogas and biochemicals to insulation materials (Corona et al., 2018; Grass, 2004; Kamm et al., 2016). Simultaneously, second generation lignocellulosic biorefineries are looking for higher value product alternatives to biofuels. Biotechnological production of single cell protein from the lignocellulosic fraction of grass silage could provide a novel and synergistic alternative. Single cell protein (SCP) refers to biomass produced by fermentation with a microbe with a high protein content and it is applied for both food and feed applications, using a wide

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range of microbial species (Bajpai, 2017; Ritala et al., 2017). In 1970–1980, feed protein was produced in Finland on a commercial scale by fermenting sulphite pulping effluent with the filamentous fungus Paecilomyces variotii (Romantschuk and Lehtomäki, 1978). P. variotii showed robustness towards inhibitory compounds as well as the ability to utilize pentose sugars and organic acids, and the produced SCP was successfully used as feed for

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pigs and poultry under the trade name ―Pekilo‖ (Alaviuhkola et al., 1975; Ojala, 1979). Within the current biorefinery concepts, single cell production from lignocellulosic sugars

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has received little attention, and the reported studies have almost exclusively focused on

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hemicellulose hydrolysates or direct cultivation on pretreated materials (Bajpai, 2017).

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Ensiling is the prevailing method for preserving grass for a year-round supply (Kamm et

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al., 2016; McDonald et al., 1991). In our recent study we optimized steam explosion and ammonia soaking pretreatments for maximizing enzymatic hydrolysability of grass silage

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fibre (Niemi et al., 2017). Steam explosion is one of the most common pretreatments in

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industrial scale lignocellulosic biorefineries, (Balan et al., 2013; Humbird et al., 2011; Larsen et al., 2012) with the major effect of hemicellulose dissolution and reformation of

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lignin structure. Ammonia pretreatments are applicable for alkaline delignification (da Costa Sousa et al., 2016) or lignin degradation without lignin removal (Lee et al., 2010; Sendich et al., 2008), and ammonia is considered to be easily recyclable by evaporation. Ammonia soaking can be performed with simple equipment at low pressures and temperatures and could therefore be considered for smaller scale localized processes. Besides evaporation and distillation, steam stripping has been suggested in ammonia fibre explosion (AFEX) process modelling studies (Sendich et al., 2008) with a proposed ammonia recovery of 99.5% (Laser et al., 2009), although it is also known that an ammonia

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amount of up to 3% per biomass is reactively consumed or neutralized by organic acids released from biomass (Balan et al., 2013). Nevertheless, the recovery of ammonia from pretreated biomass has rarely been studied in practice, and clarification of the potential is particularly important for grass silage, after its storage in acidic conditions. Enzymatic hydrolysis is a key cost factor for the 2nd generation biorefineries and the

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maximum hydrolysis yield is not likely the economical optimum. Instead, the optimum depends on the costs of the hydrolysis reaction and feedstock, as well as the value of the

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product. Modelling hydrolysis response to enzyme dosage, time and solids concentration

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could allow optimization of hydrolysis yield against the costs of enzyme and reactor

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capacity (Pihlajaniemi et al., 2015). Nevertheless, techno-economic assessment of the 2nd

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generation biorefineries is frequently based on fixed assumptions of enzyme consumption and hydrolysis yield (Eggeman and Elander, 2005; Humbird et al., 2011; Klein-

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Marcuschamer et al., 2012; Sendich et al., 2008), and in our knowledge, cost optimization

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of hydrolysis has so far not been reported. In this article we present a novel concept of producing SCP from grass fibre in synergy

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with extraction of soluble silage protein, both of which are applicable as non-ruminant feed. SCP was produced from grass silage fibre hydrolysate after pretreatment and enzymatic hydrolysis by fermentation of P. variotii. Steam explosion and ammonia pretreatments were compared and the efficiency of ammonia recovery and synergy of ammonia utilization as a nitrogen source for biomass production was experimentally evaluated. The dependence of hydrolysis yield on enzyme dosage, time and solids concentration was determined and empirically modelled. Finally, techno-economic assessment (TEA) of the process was

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carried out. For the first time, hydrolysis modelling was applied for integrating cost optimization of hydrolysis in the TEA.

2. MATERIALS AND METHODS 2.1 Grass silage fibre

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Three batches of grass silage were prepared for this study at farm scale by Natural Resources Institute Finland at Jokioinen, Finland (60°48’N, 23°29’E). The ensiled grass

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was a mixture of timothy (Phleum pratense) and meadow fescue (Festuca pratensis),

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harvested with a precision chopper, simultaneously applying formic acid based additive at a

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rate of 5 kg per ton (AIV2 Plus, Eastman Chemical Company, Oulu, Finland). The grass

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was ensiled in a vertical silo after being moderately wilted in the field. The silage was mechanically separated into liquid and solid fractions using either a single screw press or a

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twin screw press and washed with excess water. The carbohydrate content of washed silage

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(as polysaccharides) was 51.8% ± 4% (standard deviation, SD), including 38.1% ± 1% glucose, 15.1% ± 2% xylose, 3.0% ± 0.3% arabinose and 1.0% ± 0.1% galactose on dry

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matter (DM) basis. Other components included 13.2% ± 0.2% lignin, 10.2% ± 2% protein, 4.3% ± 1% extractives and 4.8% ± 1% ash. 2.2 Steam explosion Steam explosion was carried out in a 10 L pressure reactor as described previously (Niemi et al., 2017). Silage fibre impregnated with 1% H2SO4 per DM at 20–30% DM overnight at 4 oC was heated with direct steam injection to 190 oC for 15 min, and rapidly

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discharged into the adjacent flash tank. Three batches of steam-exploded silage were produced, each combining the material from 2–3 reactions.

2.3 Laboratory scale ammonia soaking and ammonia recovery Laboratory scale ammonia soaking of grass silage fibre was carried out in closed 500 ml Schott bottles with a reaction volume of 300 ml at 20 % DM and an ammonia loading of 0-

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10% per DM. Closed bottles were incubated at 90 oC overnight. Ammonia recovery after

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soaking was studied in a laboratory distillation setup with an electric heater, distillation

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flask and a condenser. The ammonia-soaked silage was heated to boiling point in normal

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pressure and the distillate from the condenser was directed into 100 ml of 1 M H2SO4

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solution in a weighed Erlenmeyer flask. The progress of distillation was sampled five times by switching the receiver flask to a fresh one. The sample mass was weighed and the

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ammonia content of the distillate was determined by back-titration of the remaining free

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sulphuric acid with NaOH. The remaining free ammonia in the bottom product was determined by titration with H2SO4.

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The theoretical distillation yield curves for aqueous ammonia were calculated based on literature data on the liquid-vapour phase equilibria of aqueous ammonia (Jennings and Shannon, 1938). The tabulated mass-concentrations of ammonia in liquid corresponding vapour

and the

were fitted to a 3rd degree polynomial (R2 = 1.00) giving a

continuous representation of

( ). The progress of a theoretical distillation was then

calculated by numerically integrating Eq. 1, where

and

represent the mass of total

liquid and ammonia in liquid, respectively, from the initial ammonia concentration of 2.5% w/w using the ordinary different equation solver ode15s in Matlab R2015a (Mathworks).

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( )

2.4 Enzymatic hydrolysis Hydrolysis reactions were performed with the commercial cellulase Flashzyme Plus (Roal Oy, Finland) at 45 oC, pH 5.0. Laboratory scale hydrolysis reactions were performed

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with an enzyme loading of 5-20 mg enzyme protein per g DM at 50 ml reaction volume

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buffered with 100 mM Na-acetate, with an addition of 0.2 mg/mL Na-azide to prevent

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microbial growth, in 250 ml Erlenmeyer flasks with shaking at 150 rpm (5–10% DM) or in closed 200 ml plastic jars placed in a rotating tumbler (20% DM). Weighed samples of 1–2

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g were withdrawn at different time points, diluted 1:5 with water if needed and incubated in

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boiling water for 10 min to inactivate enzymes. The samples were centrifuged and the supernatant was analyzed colorimetrically for reducing sugars using the dinitrosalisylic

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acid method (Miller, 1959).

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High consistency bench scale hydrolysis reactions were performed with 4 kg total

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reaction mass in a reactor with a horizontal mixer and oil jacket heating (Druvatherm DVT 5, Lödige Industries GmbH, Germany) at a mixing rate of 56 rpm for 144 h, with an enzyme dosage of 20 mg/g DM. Prior to hydrolysis, steam-exploded silage was heated to 100 oC for 15 min for preventing microbial growth. Steam-exploded silage was hydrolyzed first at 20% DM. For a second hydrolysis, the free liquid from steam explosion was included, leading to dilution to 16% DM, in order to include all released sugars from the original silage fibre in the hydrolysate. The second hydrolysate was then applied for SCP fermentation (2.7). The average yields of the two hydrolysis reactions and the

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corresponding steam explosion pretreatments are reported. Monomeric sugars were determined by HPAEC with pulse amperometric detection (Dionex ICS 3000 equipped with CarboPac PA1 column). Total sugars were determined after hydrolysis of oligomeric sugars with 4% H2SO4 at 121 oC for 1 h. A correction accounting for dissolved and liquid density

was applied for the liquid volume

of the hydrolysates and steam in each (Eq. 2).

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explosion liquids, based on the known mass of water

( )

)

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(

(%)

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The composition of the solids during the process was determined according to the NRELprotocol (Sluiter et al., 2011), mass balances were determined from DM analysis at 105 oC

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overnight and all carbohydrate yields were calculated as polysaccharides. Ash contents

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were measured gravimetrically after combustion of the samples at 550 oC.

2.5 Single reactor ammonia fractionation process

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Bench scale ammonia pretreatment, ammonia recovery and hydrolysis of grass silage

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fibre was performed with a 4 kg reaction mass in the same bench scale reactor described in section 2.4. The process was then repeated at a larger scale in order to provide enough hydrolysate for the continuous fermentation described below (section 2.7), with a reaction mass of 63.3 kg in a 130 L horizontal stirred tank reactor (Lödige DVT 130, Gebrüder Lödige Maschinenbau GmBH). The reported yields represent the average of the fractionation yields of the bench scale and scale-up reaction. In each case the reactor was loaded with washed silage fibre, water and 25% aqueous ammonia to 20% DM with an ammonia loading of 10% per DM, and incubated at 90 oC for 24 h with mixing. After

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incubation, the reaction slurry was boiled at normal pressure and the released ammoniavapour was directed into cooled water-trap. The headspace of the water-trap was connected to a safety bottle followed by a secondary trap with 1 M H2SO4 in order to determine any escaping ammonia. The whole recovery system was placed on a scale and the boiling was stopped after evaporation of 12% (bench scale) or 16% (scale-up) of the total liquid.

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Recovered ammonia was determined by titration, as described above. At pilot scale, the ammonia recovery could not be accurately determined due to a leakage, and only the bench

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scale recovery is reported. The slurry was cooled to 45 oC, a sample was withdrawn for

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analysis and the pH was adjusted to 5.0 with H3PO4 (bench scale) or H2SO4 (scale-up).

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incubation time of 96 h at pilot scale).

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Hydrolysis was then carried out at 20% DM as described above (except for reduced

2.6 Determination of protein and ammonia nitrogen

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The protein content of solids, liquids and microbial cell samples was determined as the

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nitrogen content multiplied by 6.25, analyzed with a FLASH 2000 series elemental analyzer (Thermo Scientific). In order to distinguish between nitrogen from protein and ammonia, separate liquid samples were analyzed as oven dried solids after trapping ammonia with H2SO4, representing total nitrogen, or after releasing ammonia with NaOH, representing the protein content. The ammonia determined this way correlated well with titration results. The method was tested for standard samples with 1% aqueous ammonia with or without 1% w/v bovine serum albumin (BSA), and compared to direct nitrogen determination of dried BSA solution of equal concentration. An underestimation of 24%

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was observed in protein determination, possibly due to partial degradation of amino acids during oven drying in alkaline conditions, and this was applied as a correction factor for the protein in the actual samples.

2.7 Fermentation

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Paecilomyces variotii strain VTT-D-75018 was obtained from the VTT Culture

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Collection and maintained on Potato Dextrose Agar (PDA) plates at 37 °C. Liquid cultures

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were performed with an initial pH of 5.0, with a defined mineral medium, excluding diammoniumphosphate (DAP) from cultivations on hydrolysates from the ammonia

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process. The silage fibre hydrolysates were diluted to a total sugar concentration of 25 g/L

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(glucose + xylose + arabinose) in the media. A reference solution of pure sugars (Sigma)

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was prepared to mimic the composition of the hydrolysate of steam-exploded grass, containing 17.50 g/L glucose, 6.42 g/L xylose and 1.08 g/L arabinose. Pre-cultures

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inoculated with mycelial fragments from plates were grown on this pure sugar solution.

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After 48 h the cells were harvested by centrifugation, washed twice with sterile 0.9% NaCl, homogenized and diluted to a concentration of 1 g/L. Cultivations were inoculated with a 1:10 volume of this suspension. Shake flask cultures were performed in 50 mL volume in 250 mL Erlenmeyer flasks at 37 °C with 200 rpm agitation. Continuous bioreactor culture was performed in a glass jacketed 2 L Biostat B (Sartorius) reactor at 37 °C in 1 L volume supplemented with 1 mL/L Struktol J647 antifoam with 1 VVM aeration, maintaining pH > 4.0 with 10 M KOH, and dissolved oxygen > 30% with an agitation cascade (400–1400 rpm). After 50 h of batch

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cultivation, media feed was started at a rate of 100 mL/h (50–120 h) and continued at 200 mL/h (120–330 h). Culture broth was removed from the liquid surface level through a 1 cm tube in 15-second pulses every 15 minutes. Cell dry-weight was quantified after filtering culture broth with Whatman GF/B discs and residual sugars were determined from the filtrates using HPLC (Bio-Rad Aminex HPX-

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87H column, 5 mM H2SO4 eluent at 0.5 mL/min, 55 °C). For analyzing the protein content of cells, a separate 2 mL sample was centrifuged, the cells were washed twice with 0.9%

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NaCl and once with demineralized water, and finally freeze-dried prior to analysis.

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2.8 Techno-economic assessment and hydrolysis cost optimization

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Conceptual level TEA was performed for the whole process starting from silage and

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ending with silage juice, dried P. variotii biomass and hydrolysis residue as the products. Capital expenses were calculated using factorial method (Peters et al., 2004). Purchased

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equipment costs were obtained from literature or as in-house information and scaled

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appropriately. All costs were converted to 2017 EUR using chemical engineering plant cost index and exchange rate 0.9 EUR/USD. Economic lifetime of the plant was set to 20 years and rate of return was set to 5%, and used to calculate annual capital charge. To determine the operational expenses the process was evaluated using process simulation software Balas® (VTT, Finland). Variable costs were calculated based on simulated mass and energy balances including feedstock, enzymes, electric and thermal energy, process water, cooling water, ammonia, and diammoniumphosphate, excluding other minor chemical and nutrient consumption. Other fixed costs and maintenance were

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estimated to be 1.5% of the total capital investment, and a workforce of ten persons was assumed with total annual salary costs of 70000 € per person. Further details are presented in supplementary material, including process flowsheet (Fig. S2), investment cost breakdown (Table S1) and unit costs (Table S2). Hydrolysis yield was optimized regarding enzyme dosage at a constant reaction time of

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72 h and consistency of 15% by determining the hydrolysis yield from the response model (Eq. 3) and iteratively maximizing the profit for different values of silage, enzyme and

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protein, assuming 50% biomass yield from released sugars and 50% protein content in

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biomass. Capital expenses and operation expenses other than enzyme or feedstock cost

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were assumed constant. Profit margin was calculated as percentage of total costs.

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3. RESULTS AND DISCUSSION

3.1 Fractionation of grass silage fibre

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Grass silage was pressed and water-extracted, removing 30% ± 3% of DM, 46% ± 6% of

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protein and 32% ± 3% of carbohydrates, in accordance with the soluble carbohydrate content of grasses (McDonald et al., 1991) and previous reports of protein pressing yields of 45–60% from grass (Corona et al., 2018; Hermansen et al., 2017). The protein yield of extraction was 5.2% ± 0.6% of unwashed silage DM. Grass silage fibre was subjected to steam explosion and ammonia soaking pretreatments, followed by enzymatic hydrolysis. The pretreatment conditions were selected for maximal carbohydrate yields according to our recent study (Niemi et al., 2017), including steam explosion with 15 min holding time at 190 oC and 1% H2SO4 per DM, and

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ammonia soaking at 90 oC for 24 h with 10% ammonia loading per DM, followed by hydrolysis with a cellulase loading of 20 mg/g (enzyme protein per DM). In the current work we determined at bench scale the complete carbohydrate, protein and lignin balances for the processes at industrially relevant high consistency hydrolysis conditions (16–20% DM).

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An overall sugar yield of 70% from grass silage fibre was obtained with steam explosion and enzymatic hydrolysis, with a 21.3% yield directly from steam explosion and 48.3%

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from enzymatic hydrolysis, leaving 13% of carbohydrates in the solid residue (Table 1).

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Steam explosion dissolved mainly arabinoxylan, most of which was in oligomeric form and

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an arabinoxylan loss of 24.8% was observed, indicating pentose degradation. The overall

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glucose yield corresponded to 85% hydrolysis of the original cellulose, and the overall arabinoxylan yield was 63.3%. The oligomeric sugars dissolved during steam explosion

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were completely monomerized during hydrolysis. A slightly lower total sugar yield of

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66.8% was obtained with ammonia soaking and hydrolysis, with a lower glucose yield of 59% and a higher arabinoxylan yield of 77%. The ammonia soaking process was gentler for

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carbohydrates, averting the degradation losses observed in steam explosion. As a trade-off, it was less efficient for facilitating cellulose hydrolysis, leaving 30% of the original carbohydrates in the residue. Additionally, unlike with steam exploded silage, nonhydrolysable oligomeric arabinoxylan was observed in the hydrolysate, corresponding to 30% of the original arabinoxylan. Carbohydrate dissolution during ammonia soaking was low, showing 10% dissolution of arabinoxylan in completely oligomeric form. On the other hand, 65% of lignin was dissolved, and precipitated back to solids upon neutralization, showing an overall lignin

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yield of 106%, the excess likely resulting from lignin condensation reactions and binding of cellulases on the solid residue lignin. The observed sugar yields are in the range of previous reports for agricultural biomasses. The yields were higher compared to high consistency hydrolysis (20% DM) of hydrothermally treated wheat straw (Jørgensen et al., 2007) and lower compared to our

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previous pretreatment optimization study for grass silage where a low consistency (1% DM) hydrolysis was applied (Niemi et al., 2017). Glucan conversion up to 69% has been

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previously reported for hydrothermally treated grass silage (Ambye-Jensen et al., 2014).

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Previous ammonia soaking treatments at room temperature have required four days for

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reaching comparable hydrolysability for corn stover (Kim and Lee, 2005), whereas

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comparable overall dissolution of forages have been reached with AFEX (Savoie et al., 1998) which has also been efficient for pretreating Bermuda grass (Lee et al., 2010) and

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swithcgrass (Bals et al., 2010). However, the ammonia loadings for AFEX are at a 10-fold

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level compared to the current study, and require special equipment. Similar extent of protein dissolution occurred during steam explosion (30%) and

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ammonia soaking (33%), and a similar extent of nitrogen loss was observed (23% and 20%, respectively), possible due to formation of volatile protein degradation products (Warner and Cannan, 1942). Further protein dissolution during enzymatic hydrolysis of steam exploded silage was minimal as the soluble protein content in the hydrolysate corresponded to no more than 2.4% of original protein in grass silage fibre. This also indicates that the majority of enzymes were adsorbed on the residual solids. The protein content of the hydrolysis residue was therefore corrected for enzymes assuming complete enzyme binding on solids, showing that at least 72% of the silage fibre protein remained in the hydrolysis

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residue. Similar protein behavior in steam explosion was previously reported for brewers spent grain (Kemppainen et al., 2016). For the ammonia soaked silage, the non-ammonia nitrogen content of the hydrolysate could not be reliably determined. However, according to analysis of the solid residue, no further protein dissolution occurred during hydrolysis of

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ammonia treated silage fibre either.

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Table 1. Yields and balances of total sugars, glucose, arabinoxylan, protein, DM and lignin in pretreatment and enzymatic hydrolysis of grass silage fibre. Values in brackets represent standard deviation of duplicate reactions. Glucose %

Arabinoxylan %

Protein** %

DM %

Steam explosion Pretreatment liquid Oligomeric

21.3 (3.5) 11.9 (2.8)

5 (1) 2 (0)

44 (10) 30.8 (4)

30.2 (1.1)

23.4 (0)

Pretreated solids

62.2 (5)

82.1 (1.4)

31.3 (15.5)

47.1 (0.1)

64.5 (3.4)

Balance

83.5 (1.5)

87.2 (0.4)

75.2 (5.5)

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Total carbohydrates %

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Hydrolysis yields from steam exploded silage

77.3 (1.2)

88.3 (2.3)

87.9 (3.4)

Hydrolysate

78.5 (4.9)

85.8 (1)

68 (25.6)

5 (1.1)

49.2 (0.5)

Solid residue

21.3 (1)

18.1 (1.7)

32.2 (10.8)

83.9 (12.1)

52.9 (1.2)

Balance

99.8 (3.9)

103.9 (0.7)

100.2 (36.3)

88.9 (11.1)

102 (0.7)

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Lignin %

85.8 (23.6)

Combined yield of steam explosion and hydrolysis 70 (2.6)

75.5 (1)

63.3 (7.4)

32.6 (0.6)

Hydrolysate

48.7 (0.9)

70.4 (2)

19.3 (2.6)

2.4 (0.5)

31.7 (1.4)

Solid residue

13.3 (1.7)

14.9 (1.2)

9.2 (1.6)

39.5 (5.8)

34.1 (2.6)

Balance

83.3 (1)

90.4 (0.2)

72.5 (5.8)

72.1 (6.4)

65.8 (4)

0.8 (0.9)

10.3 (8)

33.5 (6.1)

65.5 (0.8)

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Ammonia pretreatment Pretreatment liquid* 5.6 (4.8)

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Total yield

82.3 (1.2)

88.9 (4.3)

72.8 (5.8)

46.5 (3.9)

12.1 (2.2)

Balance

87.9 (6)

89.7 (5.1)

83.1 (2.2)

80 (2.1)

77.7 (1.4)

n.d.

50.7 (3.5)

81.3 (8.6)

54.6 (13.1) 105.3 (16.6)

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Pretreated solids

75.5 (18.9)

55.5 (9.3)

Combined yield of ammonia pretreatment and hydrolysis Total yield

66.8 (2.3)

58.9 (1.6)

76.9 (0.6)

11.8 (3.8)

-2.5 (0.8)

30.1 (7.3)

Solid residue

29.9 (11.8)

35.8 (15.9)

17 (7.8)

Balance

96.7 (9.5)

94.6 (14.3)

94 (7.1)

Oligomeric

105.8 (0)*** 0 (0)

*Soluble fraction prior to neutralization (only oligomeric carbohydrates detected), ** Calculated as nitrogen balance excluding ammonia and solid-bound enzymes, assuming complete binding of enzymes on solid residue, *** Presented for the scale-up reaction alone, since the bench scale lignin yield was highly overestimated, possibly due to a high phosphate content interfering with analysis.

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3.2 Single reactor ammonia process and ammonia recovery Ammonia soaking pretreatment and hydrolysis was demonstrated as a single-reactor process in bench scale (4 L) and pilot scale (68 L), comprising the sequence of ammonia soaking, ammonia recovery, neutralization and enzymatic hydrolysis. The efficiency of ammonia recovery by evaporation was first studied by laboratory distillation of ammonia-

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soaked silage and compared to distillation of aqueous ammonia and to theoretical distillation yield according to known phase equilibria (Jennings and Shannon, 1938).

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Theoretically, 80% of ammonia could be recycled from an aqueous solution by

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evaporation of 12.6% of the liquid and the initial concentration has only a minimal effect

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on the proportional recovery (Fig. 1). However, actual ammonia recovery in laboratory

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distillation was lower, reaching 56.4% and 46.8% recovery with 10% and 5% ammonia loadings, respectively, after evaporation of 18–23% of the total liquid. This resulted from

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partial neutralization of ammonia by organic acids released from biomass. According to

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titration, neutralization at 5% and 10% ammonia loadings was 1.8% ± 0.2% and 2.2% ± 0.67% of ammonia per biomass DM, respectively, in accordance with previously reported

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ammonia neutralization of up to 3% per DM for straw (Balan et al., 2013). This sets the upper limit for ammonia recycling at 78% for the 10% loading and at 64% for 5% loading. Ammonia recovery at bench scale was slightly more efficient, showing a 66% recovery after evaporation of 12.3% of total liquid. Using a cooled water trap was an efficient way for recovering evaporated ammonia, as virtually no ammonia escaped. The mass of the trapped aqueous ammonia solution at bench scale was 88% of the total water and ammonia mass loaded to the reactor initially, confirming that the recovered ammonia could be recycled to a subsequent reaction as such.

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Figure 1. Actual and theoretical ammonia recovery from ammonia soaked grass silage

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fibre by evaporation as a function of total liquid evaporated (mass-%).

3.3 Production of single cell protein from silage hydrolysates

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The original industrially applied strain of Paecilomyces variotii VTT-D-75018 (Forss et al., 1974) was redeployed in this study for producing single cell protein from hydrolysates

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of steam-exploded and ammonia-soaked grass silage fibre. The cultivation conditions were

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chosen to mimic those originally applied in industry (Romantschuk and Lehtomäki, 1978), including a relatively low feed concentration (25 g/L), and a high dilution rate (≥ 0.2) during continuous fermentation, while maintaining relatively low cell densities (10–15 g/L) to ensure sufficient oxygen transfer in the viscous mycelial cultivation broth. The filamentous biomass could be efficiently separated by filtration. Flask scale cultivation showed that both hydrolysates were suitable carbon sources for P. variotii with similar growth curves (Fig. 2A), reaching biomass yields of 51% and 46% from consumed sugars, and protein contents of 51% and 50% of DM with steam explosion

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and ammonia treatment hydrolysates, respectively (Fig. 2B). The biomass yields and protein contents were close to the 56% yield and 52–57% protein content previously reported for continuous fermentation of P. variotii (Forss et al., 1974; Romantschuk and Lehtomäki, 1978). Biomass yield was higher compared to the 39% yield achieved using the reference sugar solution, indicating that carbon sources other than the quantified sugars

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(glucose, xylose and arabinose) likely contributed to biomass growth. P. variotii is known

et al., 1995; Romantschuk and Lehtomäki, 1978).

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to able to consume organic acids as well furaldehydes as carbon sources (Almeida e Silva

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The residual ammonium from the ammonia process was synergistically utilized as the

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nitrogen source by P. variotii, substituting the diammoniumphosphate feed that was

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required for the reference sugars and steam-exploded silage hydrolysate. However, not all of the residual ammonia could be used. The nitrogen requirement for protein production

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would have been fulfilled by an ammonia amount corresponding to 1.7% of grass silage

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fibre DM, indicating that ammonia neutralization during pretreatment would already lead to an excess of ammonia (2.2% per silage fibre DM) in the hydrolysate, even if the free

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ammonia was completely recycled. With the obtained ammonia recovery of 66% in bench scale, 1.7% of ammonia per silage DM travels through the process as excess ammonium salts. Nevertheless, the excess is relatively small and could be reduced by further optimization of the ammonia loading in pretreatment or by introducing an additional carbon source to fermentation. A continuous fermentation was carried out with the hydrolysate produced in the scale-up ammonia process and sustained for 330 h (Figure S1). Continuous operation was commenced at 48 hours at a dilution rate of 0.1 h-1, and the rate was increased to 0.2 h-1 at

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120 h. During operation at 0.1 h-1 dilution rate, glucose, xylose and arabinose were all consumed, and dissolved oxygen could mostly be maintained above the set point of 30%. During cultivation at 0.2 h-1 dilution rate the fungus was no longer able to utilize arabinose and utilized xylose only partially, likely due to the limitations in oxygen transfer. A biomass yield of 50% from consumed sugars was reached at steady state with a biomass protein content of 46%. The average biomass concentration during continuous growth at

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0.2 h-1 dilution rate was 13.2 g/L, corresponding to a volumetric productivity of 2.64 g/L/h,

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in accordance with previous reports (Forss et al., 1974; Romantschuk and Lehtomäki,

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1978).

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P. variotii SCP provides a higher value alternative compared to the benchmark

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biorefinery product ethanol, showing biomass yields similar to the theoretical yield of sugars to ethanol (0.51 g/g), while the value of protein is more than double compared to

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ethanol. The SCP yields correspond up to 140 g biomass and 70 g protein per kg of

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unwashed silage DM, leading to a combined protein yield of 122 g together with the extracted silage protein. A previous study reported ethanol yields of 91 g/kg grass silage by

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simultaneous saccharification and fermentation (Sieker et al., 2011), which was 30% higher compared to the SCP protein yield. However, the combined protein yield surpasses the ethanol yield by 34%.

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Figure 2. Single cell protein production from silage hydrolysates by fermentation of P. variotii. A) Flask scale growth curves, B) biomass yield from consumed sugars and protein

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content of biomass per DM.

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3.4 Modelling enzymatic hydrolysis Hydrolysis of steam-exploded and ammonia-soaked silage was studied (Fig. 3) as a function of enzyme dosage (E = 5–20 mg/g DM), time (t = 6–144 h) and solids concentration (c = 5–20%). Hydrolysis yields were fitted to an empirical model previously reported by the authors (Pihlajaniemi et al., 2015) with modifications (Eq. 3). (𝑌𝑚𝑎𝑥

𝛽 𝑐)

𝛽2 𝐸

𝛽3 𝑡

𝛽4 𝐸𝑡

𝛽2 𝐸+

𝛽3 𝑡+

𝛽4 𝐸𝑡+

(3)

of

𝑌

), known as the ―dry matter effect‖ (Jørgensen

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function of solids concentration (slope =

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The model was further improved by including a linear decrease in hydrolysis yield as a

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et al., 2007). The model parameters are presented in Table 2, showing a good fit (R2 >

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0.972) for both processes. At a solids concentration of 5%, the maximum hydrolysis yield was higher with ammonia-soaked silage (88.1%) compared to steam exploded silage

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(81.2%), whereas the contrary was observed at 20% solids concentration, with a maximum yield of 58.9% and 68.5% for ammonia and steam explosion processes, respectively. The

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dry matter effect was therefore larger for the ammonia process. Hydrolysis yield showed an

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asymptotic response to enzyme dosage and time, as frequently presented in the literature (Ghose, 1987; Pihlajaniemi et al., 2015; Taneda et al., 2012). Accordingly, the hydrolysis yields at an enzyme dosage of 10 mg/g DM reached in average 82% of those at 20 mg/g DM, and the yields after 48 h hydrolysis were 86% of those after 144 h.

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Figure 3. Hydrolysis sugar yields of steam exploded (panel A) and NH3-treated (panel B) grass silage fibre as a function of hydrolysis time, enzyme loading and solids concentration.

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response surface.

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Dots represent averages of duplicates, black bars represent vertical distance from the

Table 2. Fitting parameters of the empirical hydrolysis model (Eq. 3).

Steam explosion

1.38 0.89 928.7 0.0119 97.0

0.972

Ammonia treatment 2.14 4.04 702.0 0.0115 101.5 0.979

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3.5 Techno-economic assessment integrated with hydrolysis cost optimization The techno-economic viability was evaluated for a protein production process combining water extraction of silage protein with SCP-production from grass silage fibre. The annual processing capacity of the plant was 60 000 ton silage DM. Silage and enzymes were the major components of the operational expenses, underlining the importance of optimizing

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the costs of enzymatic hydrolysis. Therefore, the hydrolysis model (Eq. 3) was applied for determining optimal enzyme consumption as a function of feedstock and enzyme costs and

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product value. The operational expenses other than enzyme and silage costs were estimated

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to be 71 €/ton and 65 €/ton of silage DM for steam explosion and ammonia processes,

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while the corresponding annual capital charge was 74 €/ton and 51 €/ton, respectively. The

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total investment cost was considerably lower for the ammonia process (38.8 M€) compared to steam explosion (55.8 M€), suggesting that it may be particularly suitable for localized

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small scale processes (Table S1, supplementary material). The hydrolysis model was

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applied for iteratively maximizing profit against enzyme costs as a function of the value of silage, enzymes and protein. Costs per enzyme protein of 2.8 €/kg (Ellilä et al., 2018) 3.8

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€/kg (Humbird et al., 2011) and 9.0 €/kg (Klein-Marcuschamer et al., 2012) have been reported for on-site enzyme production, whereas the cost of commercial cellulases is expected to reside at 10–20 €/kg. The value of the produced protein required for the investment to break even was determined as a function of silage cost (Fig. 4A), while optimizing enzyme consumption at each point (Fig. 4B). At the low end of the silage cost range, 80 €/ton (McEniry et al., 2011; Seppälä et al., 2014), the required protein value was in the range of 2000–2700 €/ton depending on enzyme price, whereas the highest silage costs of 200 €/ton DM required protein values of 3200–4000 €/ton. The corresponding

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optimum enzyme dosages ranged from 2 mg/g to 8 mg/g in the commercial enzyme price range, and up to 16 mg/g and 11 mg/g at the lowest on-site enzyme production cost for steam explosion and ammonia processes, respectively. Due to mass reduction in steam explosion, the optimum enzyme dosage per steam-exploded silage DM was higher compared to ammonia-treated silage. In the range of optimal enzyme dosages, the

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corresponding SCP protein yields were 37–59 kg and 48–71 kg per ton of silage DM for ammonia and steam explosion processes, respectively. Direct extraction yield of silage

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protein by pressing and washing was assumed to be 46 kg (90% of theoretical).

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While feed protein is considered to be the primary target of the process, it is

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apparent that values similar to soy meal feed (ca. 1000 €/ton protein) are not sufficient for

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economically feasible operation of the process and instead, values at least double should be obtained for the produced protein. This seems unlikely in the near future, regardless of the

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environmental benefits related to grass utilization (Grass, 2004). On the other hand, such

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values could easily be reached if the protein could be applied directly as food protein. This possibility exists, as single cell protein is considered a sustainable alternative for meat

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products (Ritala et al., 2017) and the Pekilo protein has already been successfully tested in food products (Koivurinta et al., 1979). Grass itself has also been suggested as a source of food protein (Kamm et al., 2016). For further perspective of optimal hydrolysis in an economically viable process, the effect of enzyme cost on the optimum yield and the corresponding optimum enzyme dosage was determined for a scenario assuming silage cost of 80 €/ton and protein value of 2500 €/ton (Fig. 4C). Increasing enzyme cost from 2 to 20 €/kg led to a decrease of the optimum yield from 60–66% to 46%, whereas the corresponding optimum enzyme dosage shows

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more than three-fold asymptotic decrease. This signifies that the yield optimum of lignocellulose hydrolysis is far from 100% even at favorable conditions. In this scenario, the profit margin falls below break-even at an enzyme cost of 12–14 €/kg. As described, it is relatively simple to integrate hydrolysis cost optimization into the TEA of a biorefinery process using basic hydrolysis data, which is abundant in the literature for

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all common lignocellulosic biomasses. A future improvement could include optimization of reaction time and DM in a similar way through their effect on reactor size and the

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corresponding costs.

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Figure 4. A) The dependence of break-even protein value on silage cost with optimized enzyme consumption at different enzyme costs (€/kg enzyme protein) for steam explosion (SE) and ammonia processes. B) Corresponding optimal enzyme dosage per pretreated

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silage DM at break-even protein value (same legend as in A). C) Dependence of maximum profit margin and the corresponding optimum hydrolysis yield and optimum enzyme dosage on enzyme cost, in a favourable scenario with a protein value of 2500 €/ton and a silage cost of 80 €/ton per DM. 4. CONCLUSIONS

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The concept of SCP production from grass silage fibre in synergy with direct extraction

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of silage protein was demonstrated as an extension to traditional green biorefining. While

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SCP provides a high-value product alternative for lignocellulosic biorefineries, the economic success of the process would require protein values corresponding to food,

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instead of feed applications. Ammonia pretreatment had synergy with SCP production and

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ammonia recycling was efficient with simple equipment, suggesting particular suitability

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for smaller scale operation compared to steam explosion. It was concluded that cost optimization of enzymatic hydrolysis can be an essential part of the techno-economic

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analysis of lignocellulosic biorefinery processes.

ACKNOWLEDGMENTS We gratefully acknowledge Business Finland (The Innofeed project) for funding of this work. We thank Erika Winquist and Heimo Kanerva for their expertise and Riitta Alander, Mariitta Svanberg, Jari Leino, Jenni Lehtonen, Atte Mikkelson, Marita Ikonen, Ulla Vornamo and Taru Koitto for excellent technical assistance.

REFERENCES

29

Journal Pre-proof

Alaviuhkola, T., Korhonen, I., Partanen, J., Lampila, M., 1975. Pekilo Protein in the Nutrition of Growing-finishing Pigs. Acta Agric. Scand. 25, 301–305. Almeida e Silva, J.B., de Mancilha, I.M., Vannetti, M.C.D., Teixeira, M.A., 1995. Microbial protein production by Paecilomyces variotii cultivated in eucalyptus hemicellulosic hydrolyzate. Bioresour. Technol. 52, 197–200.

of

Ambye-Jensen, M., Johansen, K.S., Didion, T., Kádár, Z., Meyer, A.S., 2014. Ensiling and hydrothermal pretreatment of grass: Consequences for enzymatic biomass conversion monosaccharide

yields.

https://doi.org/10.1186/1754-6834-7-95

Biotechnol.

ro

total

Biofuels

7,

1–12.

-p

and

re

Bajpai, P., 2017. Single-Cell Protein from Lignocellulosic Wastes, in: Single Cell Protein

lP

Production from Lignocellulosic Biomass. pp. 41–58. Balan, V., Chiaramonti, D., Kumar, S., 2013. Review of US and EU initiatives toward

na

development, demonstration, and commercialization of lignocellulosic biofuels.

ur

Biofuels, Bioprod. Biorefining 7, 732–759. https://doi.org/10.1002/bbb Bals, B., Rogers, C., Jin, M., Balan, V., Dale, B., 2010. Evaluation of ammonia fibre

Jo

expansion (AFEX) pretreatment for enzymatic hydrolysis of switchgrass harvested in different seasons and locations. Biotechnol. Biofuels 3, 1. Boland, M.J., Rae, A.N., Vereijken, J.M., Meuwissen, M.P.M., Fischer, A.R.H., van Boekel, M.A.J.S., Rutherfurd, S.M., Gruppen, H., Moughan, P.J., Hendriks, W.H., 2013. The future supply of animal-derived protein for human consumption. Trends Food Sci. Technol. 29, 62–73. https://doi.org/10.1016/j.tifs.2012.07.002 Corona, A., Ambye-Jensen, M., Vega, G.C., Hauschild, M.Z., Birkved, M., 2018. Technoenvironmental assessment of the green biorefinery concept: Combining process

30

Journal Pre-proof

simulation and life cycle assessment at an early design stage. Sci. Total Environ. 635, 100–111. https://doi.org/10.1016/j.scitotenv.2018.03.357 da Costa Sousa, L., Jin, M., Chundawat, S.P.S., Bokade, V., Tang, X., Azarpira, A., Lu, F., Avci, U., Humpula, J., Uppugundla, N., Gunawan, C., Pattathil, S., Cheh, A.M., Kothari, N., Kumar, R., Ralph, J., Hahn, M.G., Wyman, C.E., Singh, S., Simmons,

cellulosic

biofuel

production.

of

B.A., Dale, B.E., Balan, V., 2016. Next-generation ammonia pretreatment enhances Energy

Sci.

9,

1215–1223.

ro

https://doi.org/10.1039/C5EE03051J

Environ.

Bioresour.

Technol.

96,

2019–2025.

re

technologies.

-p

Eggeman, T., Elander, R.T., 2005. Process and economic analysis of pretreatment

lP

https://doi.org/10.1016/j.biortech.2005.01.017

Ellilä, S., Kujanpää, L., Marjamaa, K., Paasikallio, T., Saloheimo, M., Aro, N., 2018. Low-

na

cost glucose-based cellulase production, in: Hytönen, E., Vepsäläinen, J. (Eds.),

ur

NWBC 2018: Proceedings of the 8th Nordic Wood Biorefinery Conference. VTT, Helsinki, pp. 1–6.

Jo

Forss, K.G., Gadd, G.O., Lundell, R.O., Williamson, H.W., 1974. Process for the manufacture of protein-containing substances for fodder, foodstuffs and technical applications, Patent US3809614A. US3809614A. Franco, M., Hurme, T., Winquist, E., Rinne, M., 2019. Grass silage for biorefinery—A meta-analysis of silage factors affecting liquid–solid separation. Grass Forage Sci. 74, 218–230. https://doi.org/10.1111/gfs.12421 Ghose, T.K., 1987. Measurement of cellulase activities. Pure Appl. Chem. 59, 257–268. Grass, S., 2004. Utilisation of grass for production of fibres,Protein and energy, in:

31

Journal Pre-proof

Biomass and Agriculture: Sustainability, Markets and Policies. OECD Publication services, Paris, pp. 169–178. Hermansen, J.E., Jørgensen, U., Lærke, P.E., Manevski, K., Boelt, B., Jensen, S.K., Weisbjerg, M.R., Dalsgaard, T.K., Danielsen, M., Asp, T., Amby-jensen, M., Aage, C., Sørensen, G., Jensen, M.V., Gylling, M., Lindedam, J., Lübeck, M., Fog, E., 2017.

of

Green Biomass - Protein Production Through Bio-Refining. Humbird, D., Davis, R., Tao, L., Kinchin, C., Hsu, D., Aden, A., Schoen, P., Lukas, J.,

ro

Olthof, B., Worley, D., Sexton, D., Dudgeon, D., 2011. Process Design and

-p

Economics for Biochemical Conversion of Lignocellulosic Biomass to Ethanol,

re

Dilute-Acid Pretreatment and Enzymatic Hydrolysis of Corn Stover. Technical Report

lP

NREL/TP-5100-47764. National Renewable Energy Laboratory, U.S. Department of Energy.

na

Jennings, B.H., Shannon, F.P., 1938. Tables of the Properties of Aqua-Ammonia Solutions,

ur

in: The Thermodynamics of Absorption Refrigeration. Bethlehem, Pa., Lehigh University, pp. 1–11.

at

Jo

Jørgensen, H., Vibe-Pedersen, J., Larsen, J., Felby, C., 2007. Liquefaction of lignocellulose high-solids

concentrations.

Biotechnol.

Bioeng.

96,

862–870.

https://doi.org/10.1002/bit.21115 Kamm, B., Schönicke, P., Hille, C., 2016. Green biorefinery - Industrial implementation. Food Chem. 197, 1341–1345. https://doi.org/10.1016/j.foodchem.2015.11.088 Kemppainen, K., Rommi, K., Holopainen, U., Kruus, K., 2016. Steam explosion of Brewer’s spent grain improves enzymatic digestibility of carbohydrates and affects solubility and stability of proteins. Appl. Biochem. Biotechnol. 180, 94–108.

32

Journal Pre-proof

https://doi.org/10.1007/s12010-016-2085-9 Kim, T.H., Lee, Y.Y., 2005. Pretreatment of Corn Stover by Soaking in Aqueous Ammonia. Appl. Biochem. Biotechnol. 121–124, 1119–1131. Klein-Marcuschamer, D., Oleskowicz-Popiel, P., Simmons, B. a., Blanch, H.W., 2012. The challenge of enzyme cost in the production of lignocellulosic biofuels. Biotechnol.

of

Bioeng. 109, 1083–1087. https://doi.org/10.1002/bit.24370 Koivurinta, J., Kurkela, R., Koivis-, P., 1979. Uses of Pekilo, a microfungus biomass from

ro

Paecilomyces varioti in sausage and meat balls 561–570.

reality.

Biomass

and

Bioenergy

46,

36–45.

re

commercial

-p

Larsen, J., Haven, M.Ø., Thirup, L., 2012. Inbicon makes lignocellulosic ethanol a

lP

https://doi.org/10.1016/j.biombioe.2012.03.033 Laser, M., Jin, H., Jayawardhana, K., Lynd, L.R., 2009. Coproduction of ethanol and power

na

from switchgrass Mark. Biofuels, Bioprod. Biorefining 3, 195–218.

ur

Lee, J.M., Jameel, H., Venditti, R.A., 2010. A comparison of the autohydrolysis and ammonia fiber explosion (AFEX) pretreatments on the subsequent enzymatic

Jo

hydrolysis of coastal Bermuda grass. Bioresour. Technol. 101, 5449–5458. https://doi.org/10.1016/j.biortech.2010.02.055 Mandl, M.G., 2010. Status of green biorefining in Europe. Biofuels, Bioprod. Biorefining 4, 268–274. https://doi.org/10.1002/bbb.219; McDonald, P., Henderson, N., Heron, S., 1991. The Biochemistry of Silage, 2nd ed. Chalcombe Publications, United Kingdom. McEniry, J., O’Kiely, P., Crosson, P., Groom, E., Murphy, J.D., 2011. The effect of feedstock cost on biofuel cost as examplified by biomethane production from grass

33

Journal Pre-proof

silage. Biofuels, Bioprod. Biorefining 5, 670–682. Miller, G.L., 1959. Use of Dinitrosalicylic Acid Reagent for Determination of Reducing Sugar. Anal. Chem. 31, 426–428. https://doi.org/10.1021/ac60147a030 Niemi, P., Pihlajaniemi, V., Rinne, M., Siika-aho, M., 2017. Production of sugars from grass silage after steam explosion or soaking in aqueous ammonia. Ind. Crops Prod.

of

98, 93–99. https://doi.org/10.1016/j.indcrop.2017.01.022 Ojala, J., 1979. Pekilo in Finnish feed industry, in: Kiisikinen, T. (Ed.), Research Reports

ro

of Pekilo-Symposium, Tampere 12-15.9.1978. Tampere, pp. 27–32.

-p

Peters, M.S., Timmerhaus, K.D., West, R.E., 2004. Plant Design and Economics for

re

Chemical Engineers, 5th Ed. ed. McGraw-Hill, Singapore.

lP

Pihlajaniemi, V., Sipponen, M.H., Pastinen, O., Lehtomäki, I., Laakso, S., 2015. Yield optimization and rational function modelling of enzymatic hydrolysis of wheat straw

na

pretreated by NaOH-delignification, autohydrolysis and their combination. Green

ur

Chem. 17, 1683–1691. https://doi.org/10.1039/C4GC02218A Ritala, A., Häkkinen, S.T., Toivari, M., Wiebe, M.G., 2017. Single cell protein-state-of-theindustrial

landscape

Jo

art,

and

patents

2001-2016.

Front.

Microbiol.

8.

https://doi.org/10.3389/fmicb.2017.02009 Romantschuk, H., Lehtomäki, M., 1978. Operational Experiences of First Full Scale Pekilo SCP-Mill Application. Process Biochem. 13, 16–29. Savoie, P., de Halleux, D., Belkacemi, K., Turcotte, G., Nadeau, P., Chiquette, J., 1998. Enzymatic hydrolysis of forage and straw pretreated with ammonia. Can. Agric. Eng. 40, 207–212. Sendich, E. (Newton), Laser, M., Kim, S., Alizadeh, H., Laureano-Perez, L., Dale, B.,

34

Journal Pre-proof

Lynd, L., 2008. Recent process improvements for the ammonia fiber expansion (AFEX) process and resulting reductions in minimum ethanol selling price. Bioresour. Technol. 99, 8429–8435. https://doi.org/10.1016/j.biortech.2008.02.059 Seppälä, A., Kässi, P., Lehtonen, H., Aro-Heinilä, E., Niemeläinen, O., Lehtonen, E., Höhn, J., Salo, T., Keskitalo, M., Nysand, M., Winquist, E., Luostarinen, S., Paavola, T.,

of

2014. Nurmesta biokaasua liikennepolttoaineeksi, MTT Report 151. Sieker, T., Neuner, A., Dimitrova, D., Tippkötter, N., Muffler, K., Bart, H.J., Heinzle, E.,

ro

Ulber, R., 2011. Ethanol production from grass silage by simultaneous pretreatment,

-p

saccharification and fermentation: First steps in the process development. Eng. Life

re

Sci. 11, 436–442. https://doi.org/10.1002/elsc.201000160

lP

Sluiter, A., Hames, B., Ruiz, R.O., Scarlata, C., Sluiter, J., Templeton, D., Crocker, D., 2011. Determination of Structural Carbohydrates and Lignin in Biomass: Laboratory

na

Analytical Procedure (LAP). U.S. Department of Energy.

cellulose

ur

Taneda, D., Ueno, Y., Ikeo, M., Okino, S., 2012. Characteristics of enzyme hydrolysis of under

static

condition.

Bioresour.

Technol.

121,

154–60.

Jo

https://doi.org/10.1016/j.biortech.2012.06.104 Warner, R.., Cannan, K., 1942. The formation of ammonia from proteins in alkaline solution. J. Biol. Chem. 142, 725–739.

35

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Author Contribution Statement

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Ville Pihlajaniemi: Conceptualization, Investigation, Formal analysis, methodology, Writing - original draft; Simo Ellilä: Investigation, Conceptualization, Writing - Review & editing; Sakari Poikkimäki: Investigation, formal analysis, Validation; Marja Nappa: Methodology, Formal analysis, Writing - Review & editing; Marketta Rinne: Conceptualization, resources, Supervision; Raija Lantto: Conceptualization, Funding acquisition, Supervision; Matti Siika-aho: Conceptualization, Supervision, Project administration.

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Conflict of interest

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The authors declare that no competing financial interests exist regarding this work.

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Graphical abstract

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HIGHLIGHTS

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Single cell protein was efficiently produced from grass silage hydrolysates Unrecyclable ammonia was utilized as a nitrogen source in fermentation Enzymatic hydrolysis of pretreated silage fibre was empirically modelled Hydrolysis cost optimization was integrated in techno-economic analysis

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1. 2. 3. 4.

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