Accepted Manuscript

Biogas potential of green biomass after protein extraction in an organic biorefinery

concept for feed, fuel and fertilizer production

M. Santamaría-Fernández, B. Molinuevo-Salces, M. Lübeck, H. Uellendahl

PII: S0960-1481(17)30192-1
DOI: 10.1016/j.renene.2017.03.012
Reference: RENE 8605

To appear in: Renewable Energy

Received Date: 5 January 2017

Revised Date: 27 February 2017
Accepted Date: 4 March 2017

Please cite this article as: Santamaría-Fernández M, Molinuevo-Salces B, Lübeck M, Uellendahl H,

Biogas potential of green biomass after protein extraction in an organic biorefinery concept for feed, fuel
and fertilizer production, Renewable Energy (2017), doi: 10.1016/j.renene.2017.03.012.

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 ACCEPTED MANUSCRIPT

1 Biogas potential of green biomass after protein extraction in an organic biorefinery

2 concept for feed, fuel and fertilizer production

4 M. Santamaría-Fernández1*, B. Molinuevo-Salces1, M. Lübeck, H. Uellendahl.

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6 Section for Sustainable Biotechnology, Department of Chemistry and Bioscience,

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7 Aalborg University Copenhagen, A C Meyers Vaenge 15, 2450 Copenhagen SV,

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8 Denmark

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10 *Corresponding author.
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11 Tel. +45 5270 7443

12 E-mail address: msf@bio.aau.dk

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14 Authors contributed equally to this work.

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16 Abstract

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18 The biogas potential of the residual fractions of four organically grown green crops after

19 protein extraction was studied. The protein extraction method involved screw pressing

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20 of freshly harvested biomass to obtain a plant juice, followed by precipitation of the

21 proteins. After protein extraction, 95% of organic matter was still present in the residual

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22 press cake and juice. Methane yields in the range of 219-375 and 429-539 ml-CH4 g-

VS-1 were obtained for the mono-digestion of press cake and the residual juice,

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24 respectively, and up to 81% of the methane potential of the fresh crops was recovered in

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25 the two residual fractions when evaluated separately. Co-digestion of the press cake and
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26 the residual juice at the organic matter ratio at which those fractions leave the

27 biorefinery, resulted in a methane yield of 400 ml-CH4 g-VS-1 according to the

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28 regression model equation developed for red clover. Consequently, 65% of the methane
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29 potential from fresh red clover could be recovered by co-digestion of the residual

fractions from the green biorefinery after extraction of proteins.

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30

31
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32 Keywords: Green biorefinery; biogas potential; organic biomass; central composite

33 design; press cake; brown juice.

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34
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35 Abbreviations

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AD Anaerobic Digestion
BJ Brown juice
BMP Biomethane Potential
CCD Central Composite Design

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FS Fixed Solids
GJ Green Juice
PC Press Cake
PCN Protein Concentrate
S0/X0 Substrate/Inoculum Ratio
TKN Total Kjeldahl Nitrogen

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TS Total Solids
VFAs Volatile Fatty Acids

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VS Volatile Solids
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38 Introduction

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40 As the development of a bio-based economy is one of the priorities in Europe, biomass

41 will become a precious resource in the future as it will serve not only for the production

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42 of food and feed, but also for fuel and biochemicals to substitute fossil resources and

43 cope with an increasing demand of energy (Höltinger et al. 2014). Thus, the key is to

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44 develop biorefinery systems that efficiently convert biomass into a variety of products

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45 by means of flexible, efficient and zero-waste processes (Clark et al. 2012). More

46 specifically, the green biorefinery aims at utilizing green biomass to produce valuable

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47 products such as fibers, proteins, amino acids, lactic acid or energy (Kamm et al. 2010).
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48

49 On the other hand, a green biorefinery may also be the solution for current challenges in
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50 organic farming with monogastric animals by: 1) problems with supply of organic
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51 protein feed with the right amino acid profile, 2) low crop yields of non-leguminous

crops and low value of leguminous forage crops in organic crop rotations in areas with
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53 little milk production, and 3) the lack of sufficient supply of organic fertilizer. In
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54 Denmark for example, organic farmers are forced to import organic feed and are in need

55 of utilizing manure from conventional farming with a maximum limitation of 70 kg

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56 N/ha (Ingvorsen and Bertelsen, 2010).

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58 The scope of the current green biorefinery concept is that, organic crops are utilized for

59 animal feed, fertilizer and energy production in the form of methane. The freshly

60 harvested green biomass is firstly separated into a solid fraction (press cake, PC) and a

61 liquid fraction (green juice, GJ). Then, the green juice is processed through a lactic acid

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62 fermentation obtaining two fractions, namely protein concentrate (PCN) and a residual

63 brown juice (BJ). The protein concentrate is the primary product within this biorefinery

64 concept. This protein concentrate is aimed at substituting imported soybeans that is

65 currently the main protein source for animal feeding in organic farming. Indeed,

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66 soybean protein currently accounts for 67% of the global animal feed market (Dozier

67 and Hess, 2011), and there are difficulties for the supply in an organic context due to the

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68 increasing demand for non-gmo organic soybeans.

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69

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71
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72 Figure 1. Scheme of the green biorefinery process.

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73
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74 Beside this protein feed product, two sub-products are obtained during this organic

75 protein production, namely the press cake (PC) and the brown juice (BJ) (Fig. 1), from

76 which nutrient and energy value should be exploited. The press cake (PC) and the

77 brown juice (BJ) could be processed through anaerobic digestion (AD) to produce

78 bioenergy in the form of methane. The liquid effluent from the AD process, which

79 contains residual organic matter and nutrients, could be valuable as an organic fertilizer.

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80 The conversion efficiency as well as the stability of the AD process is dependent on the

81 biomass composition. Previous studies have reported a nutrient deficiency in mono-

82 digestion of energy crops (Weiland, 2010, Nges and Björnsson 2012). Co-digestion

83 with other substrates has often been used to overcome this deficiency. In this context,

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84 Molinuevo-Salces et al., (2015) obtained up to 1.8-fold higher biogas yields in co-

85 digestion of green biomass together with manure compared to green biomass alone.

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86 However, manure from organic farming is a limited resource, so that the development

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87 of a suitable AD process with no or low addition of manure is necessary in this case.

88 Therefore, adjusting the protein separation process and optimizing the ratio PC and BJ

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89 in the biomass input are proposed to overcome possible nutrient deficiencies and adjust
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90 dry matter concentration.

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92 The main objective of the present study is to evaluate the potential of PC and BJ as
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93 feedstock for biogas production. PC and BJ from four different crops, namely red

clover, clover grass, alfalfa and oilseed radish, were studied in mono-digestion. A
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95 central composite design was used to evaluate the co-digestion of PC and BJ in red
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96 clover and optimize the ratio between both substrates. Finally, the biomethane potential

97 (BMP) of these two sub-products alone as well as in co-digestion was related to the
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98 BMP from the corresponding fresh biomass.

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100 Material and Methods

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102 2.1. Agricultural practices, green biorefinery and sampling

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104 The following crops or crop mixtures were studied: red clover (Trifolium pratense),

105 clover grass (Trifolium pretense and Lolium multiflorum), alfalfa (Medicago sativa) and

106 oilseed radish (Raphanus sativus var. Oleiferus). The crops were organically grown and

107 no fertilizer was used in any case. Red clover, clover grass and alfalfa were processed

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108 immediately after the harvest. In the case of oilseed radish, it was spread outdoors after

109 the harvest at relatively cold conditions (bellow 10°C) overnight to reduce the water

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110 content before mechanical separation.

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112 The first step of the green biorefinery was a mechanical separation of the fresh crops

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113 (FC), obtaining a green juice (GJ) and a press cake (PC). The method developed by Kiel
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114 et al. (2015) for extraction of functional proteins from plant materials was applied. The

115 green juice was lactic acid fermented until the pH was in the range of 4.0-4.7, whereby
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116 the proteins precipitate. Afterwards, two fractions were obtained by centrifugation: a
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117 protein concentrate (PCN) and a residual brown juice (BJ). The different fractions

obtained were kept at -20⁰C for composition analysis and further experiments.
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119
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120 2.2. Analyses and mass balances

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122 Biomass samples were analyzed for total solids (TS), volatile solids (VS), fixed solids
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123 (FS) and total Kjeldahl nitrogen (TKN) according to APHA Standard Methods (2005).

124 Free sugars (i.e. sum of glucose, xylose, cellobiose and arabinose) and lactic acid were

125 determined by HPLC on a Dionex Ultimate 3000-LC system with an Aminex® HPX-

126 87H column coupled to a refractive index detector. As mobile phase H2SO4 (4 mmol L-
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127 ) was used. The concentration of Total Volatile Fatty Acids (TVFA, i.e. sum of acetic,

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128 propionic, butyric, iso-butyric, valeric and iso-valeric acids) was determined using a gas

129 chromatograph (PerkinElmer, Clarus 400) equipped with an Agilent HP-FFAP capillary

130 column of 30 m length and 0.53 mm i.d. followed by a flame ionization detector (FID).

131 The carrier gas was nitrogen (13 ml min-1). The temperatures of the detector and the

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132 injector were 230 and 240⁰C, respectively.

133

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134 Process yields in terms of wet weight (WW), organic matter content (volatile solids,

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135 VS) and inorganic matter content (fixed solids, FS) were calculated for the process

136 fractions i.e. PC, BJ and PCN and for the process running at steady state.

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137
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138 
 ,  ℎ % =  / !"# $ % 100 (1)

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140 
 , 
( ) % =

* %  / * % !"# $ % 100

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141 (2)
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143 
 , 

( ) % =
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144 + %  / + % !"# $ % 100 (3)

145
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146 2.3. Biomass composition

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148 Table 1 presents the composition of the fresh crops, PC and BJ of the four crops in

149 terms of dry matter (TS) and organic matter (VS), total nitrogen (TKN), free sugars (as

150 sum of glucose, xylose, cellobiose and galactose), organic acids (lactic acid and TVFA)

151 as well as their pH. The screw press separation resulted in a press cake fraction with

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152 higher dry matter concentration compared to the respective fresh crops, containing only

153 little amounts of free sugars and mainly fiber-bound proteins and lignocellulose. On the

154 other hand, the brown juice obtained after lactic acid fermentation and separation of the

155 protein concentrate (PCN) from the green juice was rich in free sugars and organic

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156 acids, presenting low pH (4.0-4.7) and containing also some nitrogen (0.8-3.3 g kg-1).

157

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158 2.4 Biomethane potential (BMP) tests

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160 The biomethane potential (BMP) was determined in batch experiments according to the

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161 methodology followed by Biswas et al. (2012). Batch vials (117 cm3 total volume) were
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162 filled in a substrate/inoculum (S0/X0) ratio of 0.5 or 1 (based on VS) due to the

163 characteristics of the substrates and in order to compare different organic loadings.
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164 According to Angelidaki et al. (2009), the relative volume of inoculum depends on the
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165 characteristics of the substrate, its biodegradability and on the characteristics of the

inoculum itself which should be enough to avoid VFAs accumulation and acidic
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167 conditions. Mesophilic inoculum (37°C) was obtained from Hashøj biogas plant
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168 (Denmark). In all samples with BJ, the pH was adjusted to 7.5 at the beginning of the

169 experiment by using NaHCO3 (12 g L-1). Batch tests were performed in triplicates.
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170 Blanks containing inoculum were set-up in triplicates to determine the endogenous
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171 methane production of the inoculum. The vials were flushed with a gas mixture of 80%

172 N2 and 20% CO2 and gas tight sealed with rubber stoppers and metal cramps to ensure

173 anaerobic conditions. The vials were incubated at 37 ± 2°C. Methane production was

174 monitored by measuring the methane concentration in the headspace using a gas

175 chromatograph (SRI GC model 310), equipped with a Porapak Q column of 182.88 cm

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176 length and 2.1 mm i.d. Nitrogen was used as carrier gas. As standard gas, a mixture of

177 30% CH4 and 70% N2 was utilized. Methane production was measured until no

178 significant increase in gas production was observed. Methane yield, expressed as mL-

179 CH4 per g-VS added, was calculated according to equation 4 where V is the headspace

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180 volume in the vials and indexes S and B correspond with added substrate and blank

181 vials, respectively (Biswas et al. 2012).

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,-.   = %,-.,/ % *#!0"$!,/ − %,-.,2 % *#!0"$!,2 /*00!0

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183 (4)

184

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185 2.5. Central composite design (CCD)
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187 A central composite design (CCD) was carried out in order to study the anaerobic co-
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188 digestion of BJ and PC from red clover in batch tests. In order to identify both the
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189 optimum mixture of BJ and PC and possible inhibition effects of the substrates on the
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190 inoculum, the proportion of BJ (%BJ) added as co-substrate (based on VS) and the

191 substrate/inoculum ratio (S0/X0, based on VS) were the selected factors for the
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192 experimental design. The range chosen for the %BJ was from 0 to 100% while the

193 S0/X0 ratio was between 0.26 and 1.83. Table 2 shows the 9 different treatments
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194 selected for the experimental design where the analyzed factors are presented as coded
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195 factor levels and as actual factor levels. All treatments were carried out in triplicates

196 except for the central point (T9) which was repeated 6 times in order to estimate the

197 experimental error. Batch vials were prepared as previously explained in Section 2.3,

198 and methane production was monitored periodically until no significant gas production

199 was observed.

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200

201 2.6. Data analysis

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203 Response surface methodology was utilized to fit the experimental data into a second-

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204 order polynomial model equation, which describes the influence of the two selected

205 factors over the response:

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206

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207 3 = 45 + 47 87 +49 89 + 477 879 + 499 899 + 479 87 89 (5)

208

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where Y is the predicted response, namely the methane yield (ml CH4 g-1 VS). β0, β1, β2,
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210 β11, β22 and β12 are the regression coefficients, which describe the influence of each
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211 factor X1 (BJ in mixture, %) and X2 (substrate/inoculum ratio, S0/X0) over the response

212 value.
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213
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214 The software RStudio (2015) was used to develop a multiple linear regression (MLR)

215 model to obtain the regression coefficients from the data set. The quality of the fit of the
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216 polynomial model equation, namely how much variability of the data can be explained
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217 by the model, was evaluated by the determination coefficient R2. Validation and
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218 statistical significance of the model were assessed by analysis of variance (ANOVA).

219 The impact of the regression coefficients on the predicted response was determined by

220 p-values and significant model terms were indicated by p-values lower than 0.05. The

221 optimal response was identified by derivation of the regression model equation in order

222 to find a stationary point in the response surface.

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224 Results and discussion

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226 3.1. Mass balances

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229 Table 3 presents the mass balances for the separation of the fresh crops into PC, BJ and

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230 PCN in the green biorefinery process scheme. Data reveal that between 92 and 97% of

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231 the fresh crop wet weight was transferred to the PC and BJ, for the four green crops

232 tested. Indeed, the PC accounted for 39-50 % of the wet weight while between 46 and

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233 57% of the wet weight was recovered in the BJ. The PCN represented 7%, 4%, 8%, and
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234 3% of the wet weight from red clover, clover grass, alfalfa, and oilseed radish fresh

235 crops, respectively. With regards to the organic matter (VS), 74-82% and 12-14% of the
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236 VS was recovered in the PC and in the BJ, respectively. As most of the organic matter
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237 was left in the green biorefinery by-products (PC and BJ), these fractions contained

most of the energy potential of the fresh crop to be used for biogas production.
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239 Significant nitrogen content in the PC and BJ, on the other hand, indicated that the
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240 fractions also contained enough nitrogen for microbial growth.

241
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242 BJ and PC contained between 89 and 96% of inorganics of the fresh crop, which may
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243 relate to sufficient micro-nutrients for the AD process on the one hand, but may also

244 lead to an accumulation of inorganic solids (sand) in large-scale reactors, resulting in

245 technical problems as for example decreased mixing efficiency (Burke, 2000).

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247 3.2. Anaerobic mono-digestion of fresh crop, press cake and brown juice

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248

249 The feasibility of the press cake and the brown juice as sole substrates for anaerobic

250 digestion was studied in batch tests. For the brown juice, two different

251 substrate/inoculum (S0/X0) ratios were tested, namely 0.5 and 1, based on VS. Table 4

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252 presents the obtained methane yields as mL-CH4 per g-VS added after 15 days and 55

253 days of incubation.

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254

Methane yields of the fresh crops after 55 days of AD were between 331 ml-CH4 g-VS-1

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256 for red clover and 452 ml-CH4 g-VS-1 for oilseed radish, being in the range of previous

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257 studies (Molinuevo-Salces et al. 2013). The methane yield of the press cakes was
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258 around 1.5-fold lower compared to the fresh crops for red clover and alfalfa and about

259 1.2 fold lower in the case of clover grass and oilseed radish. The final methane yield
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260 (after 55 days) of the brown juice was in the range of 429-539 ml-CH4 g-VS-1,
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261 indicating a high content of degradable organic matter.

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263 A significant increase of the methane yield from day 15 to day 55 especially for the
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264 fresh crop and the press cake of red clover and clover grass indicate that these crops

265 have a higher content of slowly degradable organic matter. For the brown juice, a higher
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266 conversion rate when using a lower S0/X0 ratio (0.5) indicated some substrate inhibition
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267 at a higher organic loading, similar to previous studies (Baier et al., 2005). A relatively

268 high yield after 55 days, also for the high S0/X0 ratio (1.0) could indicate, however, that

269 the inhibition was overcome during the experiments. Similar results were obtained for

270 all four crops studied.

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272 3.3. Co-digestion of brown juice and press cake: The case of red clover

273

274 Since red clover was identified as the most valuable crop in the green biorefinery

275 concept due to its high biomass yield and high protein content, anaerobic digestion of

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276 the residual fractions of red clover was studied in more detail. The optimal BJ/PC

277 mixture (BJ%) and substrate/inoculum ratio (So/Xo) in the co-digestion were

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278 determined by a central composite design (CCD). The experimental response from the

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279 batch tests in terms of methane yield (mL-CH4 per g-VS added) after 42 days of

280 anaerobic digestion is presented in Table 2. The regression analysis for the co-digestion

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281 of BJ and PC resulted in Eq. 6 for the methane yield (YCH4), expressed as ml-CH4 g-VS-
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282 :

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284 3:;< = 341.93 + 7.24CD − 113.495 /85  − 0.05CD9 + 63.56 GIH J − 1.40CD 5 /85  (6)
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285
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286 This model equation is presented with actual levels for the regression coefficients. The

287 response model presented an adjusted R2 coefficient of 0.71, meaning that the assessed
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288 factors (% BJ and S0/X0) and their interactions are able to explain 71% of the data

289 variability found in the response, i.e. methane yield. In addition, statistical significance
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290 of the model terms was assumed for p-value < 0.05. As it can be observed in Table 5,
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291 the %BJ (β1) and the quadratic term for the %BJ (β11) presented the most significant

292 effect on the methane yield followed by the interaction term between the two studied

293 factors (β12). Accordingly, the changes in the methane yield are mainly caused by the

294 different proportions of brown juice (%BJ) in the substrate mixture. The

295 substrate/inoculum ratio (S0/X0) did not have a significant influence on the methane

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296 yield by itself but influenced slightly the effect that the proportion of brown juice (%BJ)

297 had on the methane yield. This is according to the fact that the organic matter (VS) of

298 the BJ has a considerably higher specific methane yield than the organic matter (VS) of

299 the PC. The resulting surface plot (Fig. 2) indicates the presence of a stationary point at

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300 51.88 %BJ and S0/X0 ratio of 1.47. However, this stationary point is a local maximum

301 of the methane yield only for the %BJ, while it is a minimum for the S0/X0 ratio with

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302 increasing methane yields towards lower S0/X0 ratio.

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303

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304
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306 Figure 2. Surface response plot and contour plot for the methane yield response (ml-
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307 CH4 g-VS-1). The red point in the contour plot represents a stationary plot in the
308 response surface.
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310 As illustrated by the surface graph and contour plot, the highest methane yields were
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311 achieved for a mixture of 50-70% of brown juice with increasing yields for lower S0/X0

312 ratios. For lower S0/X0 ratios the methane yield was highest for the higher percentage of

313 BJ, indicating that the inhibiting effect of the BJ was overcome in the 70%BJ ratio if

314 sufficient inoculum was added. Anaerobic digestion of either press cake or brown juice

315 alone (T7 and T8) resulted in the lowest methane yields. Addition of 15% BJ in the

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316 mixture with PC (T1 and T2) increased slightly the methane yield due to a higher

317 content of easily degradable organic matter. AD of brown juice alone (T8) resulted in

318 significantly lower methane yields than in the previous batch (Section 3.2), probably

319 due to the fact that the pH – in contrast to the previous experiments – was not controlled

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320 and the low pH of the brown juice (pH 4.7) was far below the optimum for the AD

321 process. Indeed, the pH was between 6.7 and 7.7 at the commencement of the co-

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322 digestion batch tests for the nine different treatments. However, higher pH values (8.0-

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323 8.3) were measured in all treatments at the end of the experiments indicating thus, a pH

324 increase along with the time reaching the proper value for AD. Besides, the

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325 concentration of TVFAs at the end of the experiments was low (between 0.04-0.09 g/L),
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326 indicating that initial inhibition of methanogens was overcome over the time of the

327 experiment.
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329 3.4. Methane yield of the by-product fractions of the biorefinery process compared to

the fresh crops

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331
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332 Based on the specific methane yields of the different by-product fractions (Table 4) and

333 their mass balance along the Organofinery concept (Table 3) the resulting methane
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334 yields per ton of fresh crop input are presented in Table 6.
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336 The results reveal that 50%, 81%, 69% and 79% of the potential methane yield of

337 freshly harvested red clover, clover grass, alfalfa, and oilseed radish, respectively could

338 be recovered by the mono-digestion of press cake and brown juice. The recovery of the

339 biogas potential in the by-product fractions of clover grass and oilseed radish is

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340 significantly higher than for red clover and alfalfa, mainly because the specific methane

341 yield of the press cake is much higher of these two crops.

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343 On the other hand, a substrate mixture containing 14% of VS of BJ and 86% of VS of

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344 PC, which is the ratio of the two fractions leaving the biorefinery (Table 3), could result

345 in a methane yields of 400 m3- ton-VS-1(for substrate/inoculum ratio of 1.8) based on

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346 the model equation. Accordingly, 31 m3-CH4 could be recovered from 1 ton of fresh red

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347 clover by co-digestion of the residual fractions of the green biorefinery. Co-digestion of

348 the press cake and brown juice implies a 1.3-fold increase in the methane recovery in

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349 comparison with mono-digestion in the case of a red clover-based green biorefinery.
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351 Conclusions
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353 Up to 95% of the organic matter of four different green crops remained in the by-

products of the green biorefinery, press cake (PC) and brown juice (BJ), after the
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355 extraction of the protein concentrate. Methane yields of 218-375 ml-CH4 g-VS-1 and
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356 429-539 ml-CH4 g-VS-1 were obtained for PC and BJ, respectively. Initial inhibiting

357 effects of the BJ were overcome over time and no nutrient deficiencies were observed
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358 during the anaerobic mono-digestion batch tests of these by-products of the green crops.
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359 Results from the central composite design of co-digestion of BJ and PC indicated that

360 the highest methane yields were achieved for BJ/PC mixtures containing between 50-

361 85% of BJ (in terms of VS). The need of pH control during the AD of BJ alone might

362 be avoided when utilizing PC as co-substrate. The recovery of methane after the

363 extraction of proteins from red clover accounted for around 65% when the PC and BJ

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364 are co-digested in the same ratio as they are leaving the biorefinery. Therefore, the

365 combination of protein refining and biogas production could be more economically

366 favorable compared with solely biogas production from the crops. Further research is

367 needed to optimize the biogas production from the PC and BJ in a continuous reactor as

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368 well as to evaluate the potential of the digestate to be used as organic fertilizer.

369

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370 Acknowledgements

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372 Authors are grateful to the "Green Development and Demonstration Program (GUDP)

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373 under the Danish Ministry of Food and to the Organic RDD-2 program, coordinated by
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374 ICROFS, for financial support.

375
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376 References
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377

Angelidaki, I., Alves, M., Bolzonella, D., Borzacconi, L., Campos, L., Guwy, A.J.,
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379 Kalyuzhnyi, S., Jenicek, P., van Lier, J.B. 2009. Defining the biomethane potential
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380 (BMP) of solid organic wastes and energy crops: a proposed protocol for batch assays.

381 Water Science and Technology 59: 927-934.

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382
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383 APHA, AWWA, WEF. 2005. Standard Methods for the Examination of Water and

384 Wastewaster. 21st ed. American Public Health Association, Washington, D.C.

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386 Baier U., Delavy P. 2005. UASB treatment of liquid residues from grass bioraffination.

387 Water Science and Technololgy 52: 405-11.

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388

389 Biswas R., Ahring B.K., Uellendahl H. 2012. Improving biogas yields using an

390 innovative concept for conversion of the fiber fraction of manure. Water Science and

391 Technology 66: 1751-58.

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392

393 Burke, D. A. 2000. Anaerobic treatment process with removal of inorganic material.

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394 U.S. Patent No. 6,113,786. Washington, DC: U.S. Patent and Trademark Office.

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395

396 Clark J.H., Luque R., Matharu A.S. 2012. Green Chemistry, Biofuels, and Biorefinery.

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397 Annual Review of Chemical and Biomolecular Engineering 3: 183-207.
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398

399 Dozier W.A., Hess J.B. 2011. Soybean meal quality and analytical techniques. In: El-
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400 Shemy H. (ed.) Soybean and nutrition pp. 111-124. InTech, available from:
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401 http://www.intechopen.com/books/soybean-and-nutrition/soybean-meal-quality-and-

analytical-techniques.
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402

403
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404 Höltinger S., Schmidt J., Schönhart M., Schmid E. 2014. A spatially explicit techno-

405 economic assessment of green biorefinery concepts. Biofuels, Bioproducts and

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406 Biorefining 8: 325-34.

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407

408 Ingvorsen, B., Bertelsen, I. 2010. Regler for økologisk planteavl. Videncentret for

409 Landbrug, available from: https://www.landbrugsinfo.dk/oekologi/regler-for-

410 produktion/sider/startside.aspx.

411

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412 Kamm B., Hille C., Schönicke P. 2010. Green biorefinery demonstration plant in

413 Havelland (Germany). Biofuels, Bioproducts and Biorefining 4: 253-262.

414

415 Kiel, P., Andersen, M., Lübeck, M. 2015."A method of providing functional proteins

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416 from a plant material". PCT DK2015 050185. WO2015197078.

417

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418 Molinuevo-Salces B., Larsem S.U., Ahring B.K., Uellendahl H. 2013. Biogas

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419 production from catch crops: Evaluation of biomass yield and methane potential of

420 catch crops in organic crop rotations. Biomass and Bioenergy 59: 285-292.

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421
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422 Molinuevo-Salces, B., Fernández-Varela, R., Uellendahl, H. 2014. Key factors

423 influencing the potential of catch crops for methane production. Environmental
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424 Technology 35: 1685-1694.

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425

Molinuevo-Salce, B., Ahring, B.K., Uellendahl, H. 2015. Optimization of the co-

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426

427 digestion of catch crops with manure using a central composite design and reactor
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428 operation. Applied Biochemistry and Biotechnology 175: 1710-1723.

429
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430 Nges, I.A., Björnsson, L. 2012. High methane yields and stable operation during
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431 anaerobic digestion of nutrient-supplemented energy crop mixtures. Biomass and

432 Bioenergy 47: 62-70.

433

434 RStudio Team (2015). RStudio: Integrated Development for R. RStudio, Inc., Boston,

435 MA. http://www.rstudio.com/.

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436

437 Weiland P. 2010. Biogas production: current state and perspectives. Applied

438 Microbiology and Bio-technology 85: 849-60.

439

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440 Table 1. Biomass composition of the four crops used. Standard deviation in brackets;

441 n.d.: not determined.

442

RED CLOVER Fresh crop Press cake Brown juice

TS g/kg 163.6 (9.7) 236.4 (3.4) 24.6 (0.1)

PT
VS g/kg 144.5 (9.2) 217.7 (2.8) 18.8 (0.2)
TKN g/kg 5.3 (0.2) 7.0 (0.2) 0.8 (0.1)

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Free sugars* g/kg 23.0 (2.7) 26.3 (1.0) 7.8 (0.2)
Lactic acid g/kg 0.0 0.0 6.8 (0.2)
TVFA** g/kg 0.5 (0.0) 0.8 (0.2) 1.0 (0.0)

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pH 5.7 5.5 4.7
CLOVER GRASS Fresh crop Press cake Brown juice

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TS g/kg 186.8 (6.8) 355.6 (25.1) 43.8 (0.4)
VS g/kg 170.6 (6.0) 337.8 (26.0) 32.9 (0.3)
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TKN g/kg 3.1 (0.1) 4.9 (0.9) 0.8 (0.0)
Free sugars* g/kg 25.0 (2.0) 7.8 (5.0) 27.5 (2.4)
Lactic acid g/kg 0.0 0.0 13.5 (1.3)
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TVFA** g/kg 0.5 (0.1) 1.0 (0.3) 2.5 (0.1)

pH 5.6 5.6 4.2
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ALFALFA Fresh crop Press cake Brown juice

TS g/kg 153.8 (0.2) 255.7 (0.6) 52.6 (0.0)
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VS g/kg 136.4 (0.7) 233.8 (2.1) 39.6 (1.9)

TKN g/kg 5.0 (0.1) 8.6 (0.8) 3.1 (0.0)
Free sugars* g/kg 0.0 (0.0) 4.0 (0.3) 6.8 (0.1)
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Lactic acid g/kg 0.0 (0.0) 0.0 (0.0) 11.8 (0.1)

TVFA** g/kg 0.5 (0.1) 0.8 (0.1) 2.3 (0.0)
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pH 6.03 5.6 4.4

OILSEED RADISH Fresh crop Press cake Brown juice
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TS g/kg 86.3 (9.8) 134.0 (7.2) 32.2 (0.1)

VS g/kg 72.1 (8.6) 114.8 (6.6) 20.5 (0.4)
TKN g/kg 3.4 (0.6) 5.3 (0.3) 1.1 (0.0)
Free sugars* g/kg 0.0 (0.0) 4.5 (0.4) 3.3 (0.0)
Lactic acid g/kg 0.0 (0.0) 3.3 (0.1) 14.6 (0.0)
TVFA** g/kg 0.2 (0.0) 0.8 (0.1) 1.0 (0.0)
pH 6.0 5.7 4.0
* Sum of glucose, xylose, cellobiose and galactose.
**Sum of acetic, propionic, butyric, iso-butyric, valeric and iso-valeric acids.

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443 Table 2. Codified values, real values and response values for the co-digestion of BJ and
444 PC from red clover. Standard deviation is shown in brackets.
445

Coded levels Actual levels Experimental response

BJ (%) So/Xo BJ (%) So/Xo CH4 yield (ml-CH4 g-VS-1)

PT
T1 -1 1 14.64 1.60 346 (51)
T2 -1 -1 14.64 0.49 364 (32)

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T3 1 1 85.36 1.60 420 (28)
T4 1 -1 85.36 0.49 534 (11)

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T5 0 1.4142 50.00 1.83 450 (32)
T6 0 -1.4142 50.00 0.26 528 (45)
T7 -1.4142 0 0.00 1.05 330 (19)

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T8 1.4142 0 100.00 1.05 321 (37)
T9 0 0 50.00 1.05 467 (11)
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448

449

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450 Table 3. Mass balances for the separation of the fresh biomass into PC, BJ and PCN in the Organofinery process for red clover, clover
451 grass, alfalfa and oilseed radish.

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452
453
RED CLOVER PC BJ PCN CLOVER GRASS PC BJ PCN

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Wet weight yield (%) 40.1 53.2 6.7 Wet weight yield (%) 39.5 56.8 3.7

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Organic matter yield (%) 74.1 11.8 14.1 Organic matter yield (%) 82.4 12.8 4.8

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Inorganic matter yield (%) 53.0 36.5 10.5 Inorganic matter yield (%) 49.2 46.6 4.2

ALFALFA PC BJ PCN OILSEED RADISH PC BJ PCN

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Wet weight yield (%) 45.2 46.4 8.4 Wet weight yield (%) 49.7 47.7 2.8

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Organic matter yield (%) 77.6 13.8 8.7 Organic matter yield (%) 80.1 13.9 6.0

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Inorganic matter yield (%) 53.7 37.9
EP 8.4 Inorganic matter yield (%) 61.1 32.7 6.2
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456 Table 4. Methane yields of fresh crop (FC), press cake (PC) and brown juice (BJ) of the
457 different crops tested after 15 days and 55 days of anaerobic digestion. Standard
458 deviation is shown in brackets.
459

RED CLOVER
Ratio pH Methane yield 15d. Methane yield 55d.

PT
So/Xo control ml-CH4 g-VS-1 ml-CH4 g-VS-1
FC 1 no 57.1 (5.5) 330.6 (33.2)
PC 1 no 24.1 (5.3) 218.6 (19.3)

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1 yes 131.1 (11.9) 428.7 (26.9)
BJ
0.5 yes 319.1 (4.1) 506.4 (20.7)

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CLOVER GRASS
Ratio pH Methane yield 15d. Methane yield 55d.
ml-CH4 g-VS-1 ml-CH4 g-VS-1

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So/Xo control
FC 1 no 97.9 (4.8) 343.6 (47.9)
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PC 1 no 46.0 (11.6) 295.6 (13.0)
1 yes 193.7 (3.7) 464.4 (49.7)
BJ
0.5 yes 353.0 (29.5) 539.2 (19.1)
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ALFALFA
Ratio pH Methane yield 15d. Methane yield 55d.
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So/Xo control ml-CH4 g-VS-1 ml-CH4 g-VS-1

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FC 1 no 221.1 (9.0) 361.4 (22.1)

PC 1 no 176.6 (18.3) 239.9 (21.5)
1 yes 153.4 (6.5) 456.7 (10.2)
BJ
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0.5 yes 278.2 (12.3) 478.1 (14.4)

OILSEED RADISH
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Ratio pH Methane yield 15d. Methane yield 55d.

So/Xo control ml-CH4 g-VS-1 ml-CH4 g-VS-1
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FC 1 no 152.5 (7.3) 452.2 (39.9)

PC 1 no 278.6 (10.7) 374.7 (12.6)
1 yes 80.2 (5.4) 475.0 (6.9)
BJ
0.5 yes 178.9 (6.1) 489.5 (42.5)
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462 Table 5. Regression results for co-digestion of BJ and PC. Regression coefficients are
463 expressed as actual levels.
464

Methane yield (ml-CH4 g-VS-1)

Coefficient p-Value
β0 341.93 <0.001

PT
β1 7.24 <0.001
β2 -113.49 0.210
β11 -0.05 <0.001
β22 63.56 0.119

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β12 -1.40 0.047
R2 = 0.77, Adj. R2 =0.71, F = 14.4, Sres = 43.0

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465 R2, determination coefficient; Adj. R2, adjusted determination coefficient, F value, from F-test and Sres,
466 residual standard error.
467

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468 Table 6. Methane yields per t-VS and per t-fresh crop of the PC and BJ fractions of the
469 Organofinery concept and compared to the yields of the fresh crops. Standard deviation
470 is shown in brackets.

471

RED CLOVER CLOVER GRASS

Fresh crop 1000 Fresh crop 1000

PT
(kg) (kg)
3 3
Methane yield (m /t VS) 331 Methane yield (m /t VS) 344
(m3/t fresh crop) 48 (m3/t fresh crop) 59

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Press cake (kg) 401 Press cake (kg) 395
3 3
Methane yield (m /t VS) 219 Methane yield (m /t VS) 296

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3 3
(m /t fresh crop) 19 (m /t fresh crop) 39
Brown juice (kg) 532 Brown juice (kg) 568
Methane yield* (m3/t VS) 468 Methane yield* (m3/t VS) 502

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3 3
(m /t fresh crop) 5 (m /t fresh crop) 9
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Methane yield (PC+BJ)/FC 50% Methane yield (PC+BJ)/FC 81%
ALFALFA OILSEED RADISH
M

Fresh crop (kg) 1000 Fresh crop (kg) 1000

3 3
Methane yield (m /t VS) 361 Methane yield (m /t VS) 452
(m3/t fresh crop) 49 (m3/t fresh crop) 33
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Press cake (kg) 452 Press cake (kg) 497

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3 3
Methane yield (m /t VS) 240 Methane yield (m /t VS) 375
3 3
(m /t fresh crop) 25 (m /t fresh crop) 21
Brown juice (kg) 464 Brown juice (kg) 477
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Methane yield* (m3/t VS) 467 Methane yield* (m3/t VS) 482
3 3
(m /t fresh crop) 9 (m /t fresh crop) 5
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Methane yield (PC+BJ)/FC 69% Methane yield (PC+BJ)/FC 79%

*Average methane yield of the two methane yields presented in Table 4 for each brown juice.
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