Journal Pre-proofs

Food Waste from Restaurant Sector - Characterization for Biorefinery Approach

M. Carmona-Cabello, I.L. García, J. Sáez-Bastante, S. Pinzi, A.A. Koutinas,

M.P. Dorado

PII: S0960-8524(20)30048-1
DOI: https://doi.org/10.1016/j.biortech.2020.122779
Reference: BITE 122779

To appear in: Bioresource Technology

Received Date: 29 September 2019

Revised Date: 6 January 2020
Accepted Date: 7 January 2020

Please cite this article as: Carmona-Cabello, M., García, I.L., Sáez-Bastante, J., Pinzi, S., Koutinas, A.A., Dorado,
M.P., Food Waste from Restaurant Sector - Characterization for Biorefinery Approach, Bioresource Technology
(2020), doi: https://doi.org/10.1016/j.biortech.2020.122779

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 1 FOOD WASTE FROM RESTAURANT SECTOR - CHARACTERIZATION FOR
2 BIOREFINERY APPROACH
3 M. Carmona-Cabello , I.L. García1, J. Sáez-Bastante1, S. Pinzi1, A.A. Koutinas2, M.P.
1

4 Dorado1,*
1
5 Department of Physical Chemistry and Applied Thermodynamics, EPS, Edificio
6 Leonardo da Vinci, Campus de Rabanales, Universidad de Córdoba, Campus de
7 Excelencia Internacional Agroalimentario ceiA3, 14071 Córdoba, Spain
2
8 Department of Food Science and Human Nutrition, Agricultural University of Athens,
9 Iera Odos 75, 118 55 Athens, Greece
10 *Corresponding author: MP Dorado; email: pilar.dorado@uco.es
11
12 Abstract
13 The aim of this study is the analysis of food waste (FW) composition from local
14 catering services to assess potential biorefinery development. Moisture content of
15 different FW samples showed that 27-47% (w/w) was organic material. Main
16 components were lipids (25.7-33.2, w/w), starch (16.2-29.4%, w/w) and proteins (23.5-
17 18.3%, w/w) on a dry basis. A metal profile with Na and Mg as main components,
18 followed by trace elements, i.e. Zn or Fe, was also found in food waste samples.
19 Statistical tests in combination with principal component analysis provides an efficient
20 methodology to establish specific composition variations between FW from different
21 catering services, while relating them to FW typology. The combination of chemical
22 characterization with statistical study constitutes a promising decision-making tool for
23 FW processing and valorization. The innovative methodology presented in this study
24 provides systematic evaluation of FW composition and variability to allow selection of
25 the most appropriate valorization paths.
26 Keywords
27 Restaurant and catering waste; waste classification; statistical analysis; valorization;
28 biorefinery development

1
29 1. Introduction
30 Among organic residues, food waste (FW) is becoming an emerging problem as a potential
31 contaminant of water and land, as well as a source of greenhouse gas (GHG) emissions. The
32 Food and Agricultural Organization (FAO) of the United Nations estimated that more than
33 one third of the total produced foodstuff is lost, either discarded or not consumed (Gustavsson
34 et al., 2013), while other studies move this figure upwards 50% (Lundqvist et al., 2008). FW
35 in the EU represents more than 180 million t (Corrado and Sala, 2018; Stenmarck et al.,
36 2016). Cost associated has been estimated to be around 143 billion € (Scialabba, 2015). The
37 majority of FW is generated once foodstuff reaches the distribution chain, main FW producers
38 belong to household and hospitality sectors, the latter accounting for 21% of total generated
39 FW around the world (Principato et al., 2018). Hospitality FW, originated in restaurants,
40 hospitals, cafeterias or catering services among others, can be defined as discarded foodstuff
41 all over the food chain: from logistics and storage processes (due to transportation damages,
42 expired expiration date or quality losses), through cooking steps (edible, wrong processed and
43 non-edible products, i.e. peels or bones), to not consumed food from plates or catering
44 services leftovers. Hospitality sector FW, together with other residues, are normally collected
45 by municipal or charter services, and accounts for 30-60% of solid urban residue (SUR) (Kaur
46 et al., 2019). In the EU, SUR represents 7 to 10% of total generated wastes, which only in
47 2017 summed 246.94 million t (Eurostat, 2018). Around 29% SUR is recycled and 20% is
48 treated through composting or anaerobic digestion processes, though half of SUR is
49 discharged into landfills or incinerated. However, this situation may change in the future, as
50 European Directives 2008/98/EC (article 11) and 1999/31/EC (article 5) represent the EU
51 commitment to reduce landfills usage to 10%, while increasing recycling routes to 65% by
52 2030.
53 The utilization of FW as feedstock in industrial bioprocesses demands new governmental
54 standards. For instance, the European Waste Framework Directive 2008/98/EC (article 6)
55 presents the required technical specifications of FW for both composting and digestion
56 processes. Moreover, biofuel and bioenergy production is regulated by RSB-STD-01-010
57 standard (RSB standard for certification of biofuels based on end-of-life-products, by-
58 products and residues), that specify levels of hygiene, impurities, odor, pH, particle size,
59 macro/micro matter content, GHG emissions, etc. (Kumar et al., 1998). Therefore, FW-
60 valorization in bioprocesses demands knowledge of its composition and level of degradation
61 (Neves et al., 2008).

2
62 FW can be analyzed from different perspectives, where characterization, variability and bio-
63 stability are critical issues to take into account. FW characterization includes determination of
64 both physicochemical and biochemical properties (Bayard et al., 2018). Physicochemical
65 property determination is focused on total organic content, e.g. C/N ratio, concentration of
66 starch, sugar, protein, lipid, fiber, etc., as well as other analysis, e.g. pH or sample odor.
67 Typological analysis represents the distribution of different foodstuff groups (e.g. fruit, bread,
68 rice, meat or meat-based food, among others) in the collected sample, allowing to express the
69 percentage, in wet basis (w/w) of foodstuff with high fiber (i.e. peels or uneaten fruits and
70 vegetables), starch (e.g. bread, rice, pasta) or protein (meat and fish) content (Bayard et al.,
71 2018). Composition typology plays a key role in the study of FW variability, according to its
72 discarding causes (Fisgativa et al., 2017).
73 Variability also plays an important role in FW analysis. In fact, it is related to sample
74 elementary composition and, consequently, the efficiency of a valorization process (Fisgativa
75 et al., 2017). Main factors of variability in FW samples are related to its geographical origin,
76 collection source (e.g. restaurants, cafeterias, hospitals, patisseries, etc.), FW typology and
77 season, as well as socio-economic factors, such as income level or cultural and religious
78 traditions (Priefer et al., 2013).
79 Biostability defined as resistance to physical, chemical or biochemical decomposition, may be
80 a critical factor to assess the viability of any FW valorization process. Organic matter
81 instability generates odor and GHG emissions, as well as leachate, among other decomposing
82 issues (Abad et al., 2019). To date, two main biodegradation processes have been reported,
83 namely mineralization and humidification. The former takes place when the organic fraction
84 of matter (OF) containing lipids or carbohydrates is degraded by microorganisms, or suffers
85 mechanical abrasion or chemical reactions (e.g. strecker reaction) into its elemental
86 components, such as carbonates, ammonium, nitrate or volatile fatty acids (VFA) (Lü et al.,
87 2018). The second, humidification, consists on the stabilization of organic matter throughout
88 physicochemical interactions between internal components of FW (lignin, melanin,
89 polyphenols, etc.) and external factors, such as soil characteristics (e.g. porosity, soluble
90 organic material in micropores or size of pores), in the presence of microorganisms, resulting
91 in complex substances such as humic acid, fulvic acid, humin and its polymers (Kaur et al.,
92 2019). Provided the high amount of FW produced yearly and globally, besides the need to
93 preserve environment, a reliable tool to predict FW valorization through recycling is needed.
94 The target of the present study is to provide a FW characterization protocol, which includes
95 FW chemical composition, typological classification and statistical variability analysis,

3
 96 towards its eventual use as biorefinery feedstock. In this way, different residue valorization
97 paths may be proposed to achieve an efficient biorefinery design. As may be seen from
98 previously mentioned literature, the main innovation of this work is the proposal of a
99 systematic study of food residue composition, considering its variability. This study includes
100 both residue classification and statistical analysis, which will definitely give an added value
101 to, eventually, filter and select the best food waste valorization option.
102 2. Materials and methods
103 2.1 Sampling
104 A description of the sampling and characterization processes applied to FW samples is
105 presented below.
106 2.1.1 Sample collection and preparation
107 FW samples were collected from the hospitality sector in the city of Cordoba (Spain). Four
108 different establishments kindly supplied organic wastes from their kitchens. They were
109 collected according to specific sampling criteria for characterization and test variability.
110 Hospitality sector restaurants involved in the study were a grill restaurant (GR), mostly
111 serving meat, a fine dining restaurant (FDR), offering season meals, an Italian restaurant (IR),
112 mostly serving pasta and pizza and a University campus cafeteria (UCC), that serves seasonal
113 daily menus and fast food. To achieve extensive sample diversity and enough representation,
114 samples were collected in two different periods. First period encompassed a daily sampling of
115 each restaurant during a week, accounting for 132 samples (33 samples/restaurant). The
116 second period lasted 3 months in which samples were collected every Wednesday, accounting
117 for 84 more samples (21 samples/restaurant); both periods resulting in a total of 216 samples.
118 Although globalization makes possible to have almost the same food along seasons, sample
119 collection covered 5 months and different seasons, so any seasonal variability was analyzed.
120 Garbage bags were picked up from restaurants according to the above described sampling
121 plan. Inorganic residues were discarded and remaining OF was described based on the
122 typological classification proposed by Esteves and Davlin (2010).
123 2.1.2 Food waste classification: discarding causes
124 As mentioned above, FW samples were qualitatively described according to its composition
125 (foodstuff profile). FW was therefore classified in four groups, including (1) fruit and
126 vegetables, (2) starchy foodstuff, (3) meat, fish and byproducts and (4) others (e.g. dairy
127 products, sweets or non-edible products, i.e. bones, shell, skin, etc.). Even though the above-
128 mentioned authors (Esteves and Davlin, 2010) proposed a quantitative description based on
129 the percentage (on a wet basis) of each foodstuff, for this study a qualitative description was

4
130 adopted, followed by a further classification based on discarding causes. According to Pirani
131 and Arafat (2016), four types of foodstuff discarding causes might be considered:
132  Type #1: FW mainly contains out of date and non-processed food, as well as not
133 consumed full meals. Causes of discard might be deficient logistic control, implying
134 wrong forecasting and planning predictions about number of expected customers.
135 Special attention to highly perishable and seasonal products (e.g. fish or special meals
136 included in the menu) shall be paid.
137  Type #2 includes surplus production of processed food, normally bounded to buffet
138 restaurants or to a wrong forecast in hospital-, school- or prison-catering services.
139  Type #3 is linked to inappropriate food handling according to hygienic, quality and
140 storage standards, as well as legal concerns on use and handling of remaining FW.
141 This category may contain lots of edible foodstuff, such as low-quality meats, wrong
142 processed and discarded meals, or degraded and stinking foodstuff, among others.
143  Type #4 is related to excessive meal portion sizes.
144 2.1.3 Homogenization
145 Finally, OF was ground, for a proper homogenization, using an AM80ZBA2YH mill from
146 AEG (Germany). Samples were subsequently lyophilized for three days in a lyophilizer
147 LyoQuest model Telstar 55 (Spain) and stored at -20 ºC to preserve samples from degradation
148 by either microorganisms or chemical reactions.
149 2.2. Sample analysis and characterization
150 Moisture and ash content were carried out following the European Directive for Official
151 Control of Feeding stuff (EU, 1971). Samples were dried at 105ºC and weighted several times
152 along the process, until constant weight; moisture percentage was calculated by weight
153 difference. For ash content calculations, samples were calcined at 550ºC and a gravimetric
154 analysis was performed.
155 Total starch content was analyzed using the “Total starch” test kit from Megazyme®, usually
156 used for the measurement and analysis of total starch in cereal flours and food products,
157 following AOAC method no. 76.13 (Englyst et al., 1992), from the Association of Official
158 Analytical Chemists (known as AOAC International). This methodology uses thermostable -
159 amylase (Megazyme cat. no. E-BSTAA) and amyloglucosidase (10 mL, 3,300 U/mL on
160 soluble starch). The kit includes potassium phosphate buffer (pH 7.4), p-hydroxybenzoic acid,
161 sodium azide (0.09% w/v), glucose oxidase plus peroxidase and 4-aminoantipyrine. Sample

5
162 absorbance was measured using a Biochrom WPA lightwave II/ Visible Spectrophotometer
163 (series number 115407, UK).
164 Sugars were calculated according to the “Official analysis method for juices, fruits and other
165 vegetables and its derivates” reported in the Spanish standard RD 2081/87. The method is
166 carried out by direct titration with sodium thiosulphate 0.1 M (0.1 N), in aqueous solution
167 (AVS titrinorm volumetric solution, VWR BDH chemical) in the presence of phenolphthalein
168 (99% purity, ACS reagent from Sigma-Aldrich, Germany) as indicator.
169 Fiber content, calculated as neutral detergent fiber (NDF), acid detent fiber (ADF) and lignin,
170 was analyzed through the method proposed by Van Soest (1963), and carried out according to
171 AOAC official methodology no. 973.18. Fibre content may be also represented as the sum of
172 cellulose, hemicellulose and lignin; correspondence with above mentioned calculations might
173 be summarized as:
174 NDF = neutral detergent fiber
175 Hemicellulose = NDF- ADF
176 Cellulose = ADF- Lignin
177 Soxhlet method was used for oil quantification using a 500 mL Pyrex® Soxhlet apparatus.
178 This analysis was carried out using hexane (95% purity, J.T. Baker, from Merk®, USA) as
179 organic solvent, followed by a hexane removal step in a rotary evaporator under vacuum
180 conditions at 65ºC. After complete removal of hexane, oil dry content was quantified by
181 weighing difference. Liquid and solid lipidic phases were separated by a final winterization
182 process, carried out in a temperature-controlled centrifuge (MPW-351, Poland) at 0ºC and
183 2000 rpm for 10 min.
184 Subsequently, solid and liquid oily phases were firstly quantified by weight, for each
185 establishment and finally blended with the same phase samples from the rest of
186 establishments. For further analysis, each phase (solid and liquid) was derived to free fatty
187 acid methyl ester by a two-step transesterification reaction (basic and acid conditions). For the
188 first step, sodium methylate 0.5 M (Panreac®, Spain) was used as basic catalyst, followed by
189 HCl (37% purity, Scharlab®, Spain) for the acid step, both blended in methanol (99% purity,
190 J.T. Baker, from Merk®, USA) with a molar ratio of 1:9.5. Both reactions took place under
191 the following conditions: 20 min, at 70ºC and 400 rpm in a water bath and reflux column with
192 phenolphthalein (99% purity, ACS reagent from Sigma-Aldrich, Germany) as indicator. Fatty
193 acid profile of the transformed samples was carried out by gas chromatography using a
194 Perking Elmer gas chromatograph Clarus 500 (Waltham, MA, USA), equipped with a

6
195 PerkinElmer capillary column of 30 m x 0.25 mm Elite 5ms (0.25 µm particle diameter) and a
196 flame ionization detector (FID).
197 Total Kjeldahl Nitrogen (TKN) content in solid samples was determined by AOAC method
198 no. 955.04, using a Kjeldahl pro-Nitro M distiller from JP Selecta S.A. (Spain). Protein may
199 be estimated as 6.25 times the obtained TKN value. In the present study, total nitrogen (TN)
200 was reported as elemental N (by means of CHNS elemental analyzer); protein content was
201 calculated from TKN; and the remaining nitrogen compounds were calculated as the
202 difference of both parameters and sorted as inorganic nitrogen.
203 Metal content was measured by inductively coupled plasma mass spectrometry (ICP-MS)
204 NexION 350X, from PerkinElmer (Waltham, MA, USA), while elemental analysis of C and
205 N was performed after the Dynamic Flash Combustion Principle, using a CHNS Elemental
206 Analyzer (Germany).
207 2.3 Statistical methodology
208 The main target of statistical analysis was the comparison between restaurant samples, to find
209 out either similarities or differences between them, which may help defining a biorefinery
210 based on FW valorization processes. For this purpose, IBM SPSS Statistics 22 software
211 (USA) was used. Pearson coefficient of variation (CV) was calculated to discern the
212 representation of the mean (dispersion). According to Fisgativa et al. (2016), samples may be
213 classified into three groups as a function of its CV: (1) samples with CV values below 16%
214 may be included in the low variation group; (2) if CV varies from 16% to 33%, the mean
215 would be included in the second group, considering that obtained mean shows many errors;
216 (3) finally, if CV is above 33%, mean should not be considered as representative.
217 On the other hand, lack of symmetry (skew ness) and pointiness (Kurtosis) were tested to
218 evaluate the distribution as first criterion of normality. Tests were conducted following the
219 protocol proposed by Ghasemi and Zahediasl (2012). Furthermore, a Kolmogorov-Smirnov
220 test (K-S) was carried out as a second criterion to evaluate data normality (with a confidence
221 level of 95%), taking into account that p-values higher than 0.05 may be considered within a
222 normal distribution.
223 In case there is lack of normality between samples, to improve statistical description of
224 collected samples, a non-parametric analysis may be performed. Application of U-test from
225 Mann-Whitney Wilcoxon allows to work with non-homogeneity of variance. Also, it may be
226 used to compare means from properties that were not representative in previous tests. U-test
227 was, therefore, performed to identify differences between establishment samples, providing
228 insight about shared common features and differences between samples; the null hypothesis

7
229 expresses no differences between establishment samples, while alternative hypothesis
230 indicates differences between them.
231 Finally, as a complement to previous analysis, thus allowing a visual sample classification, a
232 principal component analysis (PCA) was carried out. This multivariate methodology is used
233 to decompose the complex data matrix created with samples from each establishment, into a
234 bilinear mathematical model, as well as to show patterns, clusters, trends and outliers of a
235 sample set. To analyze variables from each establishment by PCA, samples were divided into
236 three groups: the first one including macronutrients (e.g. starch or protein), the second one
237 including metals and the third one considering temporary variations between samples.
238 2.4. Reagents and methods for FW valorization
239 To valorize starch and protein fractions for microbial lipid production, oleaginous yeast strain
240 Rodosporidium kratochvilova Y13 3/8 was used for microbial oil production, while
241 Aspergillus awamori, kindly provided by Professor Colin Webb (The University of
242 Manchester, UK) was used to produce crude enzymes through solid state fermentation (SSF).
243 Yeasts were stored at 4ºC on both agar slopes and Petri dishes, containing glucose (10 g/L),
244 peptone (10 g/L), yeast extract (10 g/L) and agar (20 g/L). Inoculum was prepared in liquid
245 medium containing glucose (10 g/L), peptone (10 g/L) and yeast extract (10 g/L) and
246 cultivated for 24 h at 28ºC in a rotatory shaker (ZHHWY-211C Series Floor Model Incubator,
247 PR, China) at 180±5 rpm. SSF of A. awamori was carried out in 250 mL flasks containing 5 g
248 of dried potato peel (PP) after 48 h incubation at 30°C. PP composition was 38% (w/w, db)
249 starch, 66% (w/w, db) NDF and 3% (w/w, db) protein content. Fermented solids from SSF
250 were macerated and suspended in sterilized tap water. Hydrolyzates were produced by mixing
251 varying quantities of FW streams with the enzyme-rich suspension in 1 L-Duran bottle.
252 Fermentations were carried out in 250 mL Erlenmeyer flasks, with a working volume of 50
253 mL of FW hydrolyzate inoculated with 1 mL of a 24 h exponential pre-culture of R.
254 Kratochvilova. To valorize cellulose, hemicellulose and lignin rich fractions into bio-oil, four
255 grams of solid hydrolizate were mixed with the appropriate ratio of polyethylene
256 glycol/glycerol, with a solvent /biomass ratio of 5; mixture was heated and stirred for 3 min
257 and then 3.5% w/w sulfuric acid were added. The mixture was continuously ultrasonicated in
258 batch mode (100% duty cycle and 40% amplitude, during 20 min). At the end of the process,
259 100 mL of acetone were added, and the solid fraction was discarded. Subsequently, the liquid
260 fraction was heated at 50ºC to remove acetone. 100 mL of distilled water was added and the

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261 product was stirred four h at 150 rpm and 20ºC. The final product was centrifuged at 7000
262 rpm for 20 min and the solid precipitate (bio-oil) was dried at 40ºC for 12 h.
263 3. Results and discussion
264 The combination of green chemistry and circular economy has allowed the development of
265 several pathways for FW recovery and valorization. The first step in every valorization
266 process should include raw material description including chemical composition,
267 biodegradability and statistical variability. However, nowadays, there is no specific
268 international standard to apply to FW, but adaptation of methodologies to both raw material
269 and process specific needs. This is of main importance for decision-making.
270 3.1. Samples description and discarding causes: typology and categorization
271 FW management problem might be solved through different strategies, from which
272 prevention and post-valorization are two of the most important solutions known to date.
273 Sample valorization, within the biorefinery concept, requires a prior detailed description of
274 available raw material composition. The proportion of foodstuffs within each sample will
275 determine its chemical composition and, therefore, potential valorization routes. Also,
276 discarding causes for every analyzed food sample will determine necessary prevention actions
277 to reduce the amount of generated FW (Berman et al., 2019; Esteves and Davlin, 2010;
278 Knezevic et al., 2019).
279 For each establishment included in the present study, a qualitative description of organic FW
280 based on the work by Esteves and Davlin (2010) and Pirani and Arafat (2016), that results in a
281 typological description of FW was carried out. In general, a high presence of starchy FW,
282 namely bread, pasta and rice, followed by vegetable and meat, was found.
283 In this sense, IR might be categorized as type 4 (FW related to excessive portion sizes) as
284 main FW consisted of discarded portions of pizza, pasta and desserts. Other important FW
285 from this kitchen were concentrated sauces (such as tomato or béchamel sauces) rich in oil,
286 egg and flour. This type of restaurant represents a good candidate for special programs of
287 preventive treatment in FW generation (Esteves and Davlin, 2010; Pirani and Arafat, 2016).
288 FDR includes buffet restaurant and special catering service for exceptional events (e.g.
289 weddings) which fulfills requirements of type 2. It has also been included in type 1, due to
290 strong seasonal impact on FW. Besides, crude fish and fruits were found in major proportion
291 than other restaurants.
292 UCC FW might be included in the first and categories, as daily foresight is difficult to follow
293 in this kind of restaurants, where customers -mainly students- have rotating schedules during

9
294 the whole academic year (type 1). Meal portion size is also shown as a potential cause of food
295 disposal (type 4), with special attention to bread portions, among the majority of waste found.
296 GR has meat dishes as main offer, which are considerably affected by type 3 of the proposed
297 categorization: meat wastes were found both cooked and raw, the latter possibly discarded
298 due to hygienic and quality parameters. FW found in these samples might be, therefore, the
299 result of a combination between logistic control (types 1 and 2) and storage legal aspects (type
300 3). A relatively high amount of non-edible vegetables, such as peels, was also found among
301 GR FW samples (Kaur et al., 2019; Priefer et al., 2013).
302 3.2 Chemical composition of FW samples
303 According to Carmona-Cabello et al. (2018), FW sample chemical composition is the base of
304 any valorization process included in the biorefinery concept and will be discussed in detail in
305 this section. Chemical profiles analyzed in this research depict a heterogeneous composition
306 of FW, with a moisture content from 52.1 to 73.9% (w/w, wb). Remaining 27 to 47% (w/w,
307 wb) is the range of all FW samples and is mainly composed of OF and minimal trace element
308 fraction.
309 There is a direct correlation between high moisture/water content, water activity and high
310 biodegradability. High moisture/water content observed in FW samples considered in this
311 study may have two effects. One effect may be the loss of nutrients in the draining process. In
312 fact, due to high nutrient solubility, more than 74% of nutrients might be lost (Kim et al.,
313 2015). The second effect may be positively considered; initial moisture/water content of FW
314 may promote contamination and hydrolytic processes. Moreover, in terms of energy recovery
315 efficiency, high moisture content reduces raw material lower calorific value. You et al. (2017)
316 reported values for lower heating values of SUR between 3,599.21 kJ/kg and 4,956.77 kJ/kg,
317 while the heat minimum limit considered suitable for waste incineration is 3,344 kJ/kg.
318 Different criteria might be used to categorize the previously mentioned range of OF and trace
319 elements present in FW samples; attending to its biodegradability, an easily biodegradable
320 organic matter fraction (including proteins, lipids, simple sugars, starch) and a poorly
321 biodegradable fraction (including lignocellulosic content, i.e. lignin, cellulose and
322 hemicellulose) could be defined. Moreover, to evaluate biochemical conversion potential,
323 another classification may be adopted in accordance with the chemical composition of the dry
324 matter fraction, resulting in four different groups: (1) carbohydrates, accounting for 14-20%
325 (w/w, wb), this group being composed of simple sugar, starch and fibre; (2) lipids,
326 representing 7.2 to 11.8 % (w/w, wb); (3) 4.6-11.4% (w/w, wb) proteins and (4) inorganic
327 material, including ash content, accounting for the remaining 1.3-2.6% (w/w, wb).

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328 3.2.1. Carbohydrates
329 Table 1 shows carbohydrate distribution for every establishment considered in the present
330 study. High deviation values are explained in section 3.3 (statistical analysis of FW samples).
331 Carbohydrates, accounting for 47.4% (w/w, db) of the total biomass, were sorted out into
332 different categories: simple sugars, reducing sugars, starch and fibre (including cellulose,
333 hemicellulose and lignin). Starch represents the major component of the carbohydrate family,
334 being 24.1% (w/w, db). Therefore, FW might be considered as “starchy waste” with a high
335 valorization potential, i.e. bioethanol, microbial oil, bioplastic, etc.
336 Sugars represent an average of 10.7% (w/w, db) of total dry biomass of FW. The highest
337 concentration of sugar was found in FDR samples (14%, w/w, db), followed by IR and UCC
338 samples, with 9.7% (w/w, db) and 9% (w/w, db), respectively, and finally GR samples, with
339 8% (w/w, db). According to FW categorization already mentioned (Esteves and Davlin, 2010;
340 Pirani and Arafat, 2016), it might be assumed that FW from restaurants offering fruits and
341 sweet desserts in their menus will include higher concentrations of sugars.
342 Reducing sugars (RS) is a group of carbohydrates (including all monosaccharides and some
343 di-, oligo- and poly-saccharides) which is able to act as a reducing agent. This group of sugars
344 has the potential to reduce the biostability of organic FW, due to the presence of a free
345 aldehyde or ketone group and may originate parallel reactions in recovery processes. An
346 example of this type of reactions is the formation of N-glycoside bonds between reducing
347 sugars and either proteins, peptides or amino acids. This reaction depends on the temperature,
348 water activity and reaction time (Esteves and Davlin, 2010). Reducing sugars represent an
349 average of 2.9 % (w/w, db) of total dry biomass for all considered samples (Table 1). FDR
350 samples showed a maximum value of 3.8% (w/w), followed by UCC with 3.1 % (w/w, db)
351 and IR and GR, with values of 2.5 % (w/w, db) and 2.4 % (w/w, db), respectively.
352 Starch is a polysaccharide, composed of amylose and amylopectin, used as energy reserve in
353 different plants, such as corn, potato, legumes, etc. It is stored in the form of granules within
354 the cell organelles, known as amyloplasts. The average starch content of all dry samples
355 (Table 1) is 24.1 % (w/w, db). Samples with higher starch content belong to UCC, 29.4%
356 (w/w, db), while the minimum value was found for FDR samples, 16.2% (w/w, db), around
357 two times below that of UCC.
358 As reported in Table 1, carbohydrates (sugars and starch) showed a joint average
359 concentration of 34.8% (w/w, db). The presence of sugars and starch in FW may be valorized
360 in different sectors, i.e. biotechnological industries or in environmental remediation (Dahiya
361 et al., 2018; Patel et al., 2019). This kind of carbohydrates constitute a source of natural

11
362 carbon and energy source for microorganisms and are involved in many metabolic pathways.
363 Easily assimilated carbohydrates are employed in anaerobic digestion, bioethanol production,
364 lipid production, etc. (Carmona-Cabello et al., 2018; V. Kumar et al., 1998). As an example,
365 Kwan et al. (2016) reported the use of Aspergillus awamori fungi to hydrolyze the starch
366 content from FW into glucose.
367 Fibres analyzed in this study were cellulose, hemicellulose and lignin, resulting in 10.3% (db)
368 of the total dry biomass. This group of polysaccharides is included within dietary fibre, an
369 element of plant cell walls with structural functions, which is present in vegetables, fruits and
370 cereals. It may be transformed into simple sugars (e.g. glucose), yet through more complex
371 treatments than those required for other polymers, such as starch (Galanakis, 2015). To break
372 fibre internal structure, current tendencies involve the use of an acid or basic chemical
373 pretreatment, followed by an enzymatic treatment, as well as a thermophilic pretreatment.
374 Other emergent pretreatments include different power sources, namely ultrasound or
375 microwave energy, instead of the above mentioned chemical pretreatments, for the production
376 of new biofuels, i.e. bio-oil (Beneroso et al., 2017).
377 Cellulose is a polysaccharide composed of β-glucopyranosyl residues connected by 1-4
378 linkages. It shows a semi-crystalline structure with amorphous gel regions, whose proportion
379 depends on the moisture content. Samples of FDR and GR showed maximum values for
380 cellulose content, 4.3 and 4.1% (w/w, db) respectively, followed by UCC, 3.6% (w/w, db) and
381 IR, with 3.5% (w/w, db).
382 Hemicellulose is a polysaccharide with an internal structure modification; interspaces are
383 occupied by carbohydrates, as xyloglucans (in dicotyledoneae), arabinoxylans (in
384 monodicotyledonaeae) and β-glucans (in barley and oats). Average hemicellulose content for
385 all samples is 6.4% (w/w, db); GR and FDR showed maximum values of 8.5 and 7.4% (w/w,
386 db), respectively, while IR and UCC presented lower values than the average value.
387 Lignin is a heterogeneous and amorphous polymer, composed of monomeric units of phenyl
388 propanoic acid. Maximum lignin value was found for GR, 2.4% (w/w, db), higher than the
389 average value of 1.5 % (w/w, db) for all considered samples.
390 Finally, Table 1 also reports cellulose-to-lignin ratio, that describes fibre distribution. This
391 parameter is used to evaluate biodegradability of FW samples (Lesteur et al., 2010). It may
392 also be used as a degradation indicator for FW during storage. Zheng et al. (2015) reported a
393 cellulose-to-lignin ratio close to 0.6 of samples from SUR, while data shown in Table 1
394 present values above 1.5 and a maximum value of 3.2 for UCC. This ratio depends on initial
395 fibre concentration and it may be used to determine adequate pretreatment.

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396 3.2.2. Lipids
397 Lipidic fraction, the second largest component of studied FW samples, reaches 28.1% (w/w,
398 db) of the total dry biomass, and it presents a distinctive profile, as shown in Table 2.
399 Maximum lipidic value was provided by FDR with 33.2% (w/w, db), followed by GR, UCC
400 and IR with 27.3, 26.2 and 25.7 % (w/w, db), respectively.
401 Liquid and solid lipidic phases, representing an average of 20 and 81% (w/w, db) of all dry
402 biomass, respectively, are closely linked to the typological composition of samples and,
403 therefore, to the presence of meats, used frying oils, or sauces. Highest concentrations of solid
404 lipidic fractions were found in GR and IR samples, being 29.9 and 23.4 % (w/w, db),
405 respectively. According to Esteves and Davlin (2010) and Pirani and Arafat (2016), these are
406 restaurants offering dishes rich in meat, fish and sauces. On the contrary, UCC and FDR
407 showed higher values of liquid lipidic fraction,90.3 and 86.4 % (w/w, db), respectively. Most
408 of their FW showed high presence of processed food, with the ability of accumulating oils
409 during cooking processes. This typological difference represented 2.5 times the ratio between
410 solid and liquid fractions in GR, and vice versa for FDR.
411 Fatty acid profile may help selecting valorization pathways in a biorefinery. Oleic acid
412 (C18:1) is majority, with a solid/liquid phase average proportion of 45.8% (w/w, db). Both
413 lipidic phases show a heterogeneous fatty acid profile, yet with important differences in
414 linoleic (C18:2) and palmitic (16:0) acids content: linoleic acid is 1.63 times higher in the
415 liquid fraction, while palmitic acid was 1.56 times higher in the solid fraction. These profiles
416 may influence total unsaturation degree (TUD) and explain the presence of the above
417 mentioned two phases: fraction with a TUD of 0.9 was liquid at room temperature, while solid
418 fraction presented a TUD value of 0.7. Each fraction may be used in different routines in
419 biorefineries. As an example, liquid fraction may be used in biodiesel production, while solid
420 fraction, due to high viscosity, may be used in biolubricants production (Carmona-Cabello et
421 al., 2018).
422 Lipids in FW samples may also be divided in three different groups, according to their fatty
423 acid chain length: (1) volatile fatty acids (VFA) with a number of carbons between 4 and 11,
424 easily assimilable by microorganisms (Lesteur et al., 2010); (2) medium chain fatty acids
425 (MCFA) with 12-14 carbon atoms; and (3) long chain fatty acids (LCFA), with 14 to 24
426 carbon atoms in their molecules, showing an amphipathic character. The apolar chain of fatty
427 acids, belonging to groups 2 and 3, is able to damage the cellular membrane of
428 microorganisms. Moreover, LCFA (group 3) have the ability to generate vesicular structures,
429 which may trap essential metabolites that are implicated in cellular metabolism and produce a

13
430 negative effect in by-product yield (Neves et al., 2008). According to data from Table 2, both
431 lipidic phases present fatty acids belonging to the three above mentioned groups, yet with
432 higher presence of MCFA and LCFA. Values of LCFA are similar in both phases (around 17)
433 and only the solid phase presents more variability of VFA (e.g. C8, C10, C12).
434 Fatty acids profile is directly related to FW typology, and therefore, to the presence of meat,
435 fish or vegetables in FW samples. Meat and fish present more saturated fatty acids profiles,
436 with higher tendency to be solid greases at room temperature; while vegetables and vegetable
437 oils show higher unsaturation degrees, with higher tendency to be liquid. Lipidic profile
438 reported in this study may be classified as LCFA-dominant and, therefore, highly suitable as
439 raw material for biofuels, according to recent studies (Pirani and Arafat, 2016; Yang et al.,
440 2014).
441 3.2.3. Protein and total nitrogen content
442 Proteins derived from FW management depict a high potential in biotechnology applications.
443 Nutraceutical, cosmetic and pharmaceutical industries use protein compounds, such as
444 collagen, chitosan, elastin or keratin. Moreover, some antioxidants are produced by hydrolysis
445 of proteins, producing a peptide sequence with antioxidant properties. On the other hand,
446 nitrogen plays a central role in microbial fermentation due to its implication in cell growth. To
447 hydrolyze protein to nitrogen sources assimilable by microorganisms, several applications
448 have been developed. One example could be the use of filamentous fungi to produce
449 proteolytic enzymes, which degrades the protein into free amino nitrogen (FAN) (Du et al.,
450 2008).
451 Processed foodstuff present in FW samples is subjected to many cooking processes such as
452 frying, boiling or steaming. Moreover, part of the N present in protein molecules may be
453 degraded by some microorganisms to other chemical species, such as NH4+ or NH3 (Fisgativa
454 et al., 2017). Other forms of N that can be found in FW are nitrates and nitrites, both used as
455 additives, helping to preserve the red color of meat and acting as antimicrobial agents (Belitz
456 et al., 2009).
457 Protein origin, according to the typological classification of tested FW samples, was mainly
458 related to the presence of meat and fish dishes. Thus, even though many vegetables (such as
459 soybean, lentils or chickpeas) are richer in proteins, they were not present in the evaluated
460 samples. Average protein concentration for all FW samples is 20.6 % (w/w, db); GR was the
461 establishment with largest amount of proteins, followed by FDR with a concentration slightly
462 lower (23.5 and 21.5%, w/w, db respectively); IR and UCC samples presented a concentration
463 below 20% (w/w, db).

14
464 Elemental or total nitrogen average for all FW samples is 3.5 % (w/w, db) with a maximum
465 value of 3.9 % (w/w, db) for GR. Values for TN and TKN, reported in Table 3, allow to
466 calculate that an average of 83.5%(w/w, db) of total nitrogen present in FW samples came
467 from proteins. The largest difference between TN and TKN was found in samples from GR,
468 with only a 78% (w/w, db) of nitrogen belonging to protein molecules. Nitrogen origin might
469 be an important issue in biorefinery valorization processes. Thus, as an example, certain
470 amounts of ammonium may have a negative effect on reaction yield in anaerobic digestion.
471 3.2.4. Elemental carbon content and C/N ratio
472 Elemental carbon is present in lipids, carbohydrates and proteins, with an average content of
473 52.5% (w/w, db) for all considered FW samples (Table 3). UCC and GR were the
474 establishments with highest and lowest carbon content (55 and 50.2%, w/w db, respectively)
475 as shown in Table 3.
476 C/N ratio is a parameter that allow getting information about OF from FW and may be used as
477 an indicator in valorization processes. It is used as degradation indicator for biological
478 material and also as reference value in bioprocesses (e.g. anaerobic digestion). It may help to
479 determine the biodegradability of any biomaterial from two perspectives; on one hand, the
480 extent of decomposition and, on the other hand, the capacity of the biomass to self-degrade
481 within FW recovery chain (Lü et al., 2018). Average C/N ratio for all FW samples is 14.9,
482 with a maximum value of 16 for IR and a minimum one of 12.8, corresponding to GR.
483 In this study, the lowest C/N ratio from GR correlates with the highest value for elemental N
484 and minimum elemental C value, due to high presence of meat and low quantity of food rich
485 in carbohydrates (e.g. starch). However, C/N ratio provides limited information about
486 nutrients proportion. As an example, UCC, FDR and IR samples presented similar C/N ratios
487 (15.8, 15.2 and 16.0, respectively), however their nutrient profiles are quite different. UCC
488 had a higher concentration of starch 29.4% (w/w, db), while FDR had higher concentration of
489 carbohydrates, namely simple sugars (14%, w/w), lipids (33.2%, w/w, db) and fibres (11.7%,
490 w/w, db). IR presented a homogeneous nutrient profile (see Tables 2-4). These are significant
491 differences that may have important implications in FW recovery (Carmona-Cabello et al.,
492 2018). For example, fibre needs more difficult pre-treatments than starch. On the other hand,
493 fibre may produce richer by-products, such as antioxidant.
494 3.2.5. Ash and trace element content
495 Ash content represents mineral and metal fraction present in organic samples and help
496 understanding the mineralization process of FW. Gravimetric analysis revealed that ashes
497 represent an average of 4.5 (w/w, db) for all FW samples (Table 3). Maximum value was

15
498 5.3% (w/w, db) from FDR, followed by IR with 4.6% (w/w, db), while GR and UCC
499 presented values below the average.
500 A relationship between typological classification of FW samples and ash content is not easy
501 to elucidate. Thus, every foodstuff shows a different mineral profile. To study minerals
502 present in FW samples, they were sorted in three categories, according to their concentration
503 level, namely main elements, trace elements and ultra-trace elements (Belitz et al., 2009).
504 Three well-defined measure scales, according to these ranges of concentration, may be
505 appreciated in Figure 1. The first group of minerals, including Na, K, Ca and Mg, represent in
506 average 0.32% (w/w, db), 0.26% (w/w, db), 0.11% (w/w, db) and 0.03 % (w/w, db) of the
507 total biomass on a dry basis, respectively, and may be considered as main components of the
508 metal group (high range of ppm). The second group is composed of Zn, Mn and Fe; their
509 values (8.01, 3.76 and 26.15 mg/kg, respectively) are in the low range of ppm and can be
510 considered as trace elements. The third group is considered in the range of ultra-trace
511 elements and is composed of heavy metals, namely Cu, Ni and As (0.9, 0.21 and 0.1 mg/kg,
512 respectively). Their concentration values are below 1 ppm, as well as Pb and Cd, that have not
513 been represented in Table 3 as their concentration were lower than the detection limit. Heavy
514 metal concentration in FW samples is less than the maximum value allowed by the European
515 directive 199/31/EC.
516 Trace elements are implicated in synergistic and inhibitor routes in microorganism
517 fermentations through metabolic pathways, enzymatic stabilizations and cofactors (Schmidt et
518 al., 2014). It has been estimated that to produce 1 kg of biodiesel from microalgae
519 Auxenochlorella protothecoides, 0.03 kg of magnesium and 0.06 kg of potassium are
520 required. The importance of Mg, Fe, Zn, Cu and Mn in lipogenesis has been reported for
521 concentrations of 1.5g/l (Mg) and 0.001 g/l (Fe, Zn, Cu or Mn) (Bohutskyi et al., 2014).
522 In anaerobic digestions, metals like Fe and Ca, may help stabilizing the bioprocess; also, Fe
523 has a significant role in cell redox homeostasis due to its ability to transport electrons and act
524 as a buffer (Kaur et al., 2019). On the other hand, Ca is a cofactor that helps to capture LCFA,
525 thus reducing the above mentioned negative impact of these fatty acids (Muhid et al., 2008).
526 Nevertheless, in a valorization process, bioaccessibility of mineral nutrients must be
527 considered, taking into account that metal solubility might be reduced as the result of
528 interactions with substances such as phytates, oxalates, tannins and fibres. On the other hand,
529 cooking processes may improve mineral bioaccessibility, although each one exhibits different
530 behavior, so this assessment may not be a general rule. All samples analyzed in this study
531 showed similar concentration ranges to those reported in literature.

16
532 3.3. Statistical analysis of FW samples
533 Statistical tools, i.e. dispersion and distribution analysis or principal component analysis
534 (PCA), were used to highlight similarities and differences between samples from different
535 establishments. Statistical methodology allows the integration of typology profile, physical
536 and chemical properties and biodegradability information. This methodology influences the
537 decision-making process in the valorization of the waste. Fisgativa et al. (2017) demonstrated
538 this impact in predictions of FW behavior in anaerobic digestion processes. The present
539 statistical study may help predicting development of future valorization processes.
540 3.3.1. Evaluation of sample distribution and normality
541 To provide insight about shared common features and differences between samples, four
542 different tests were carried out. Considered data show a high dispersion; also, skewness and
543 kuortosis values indicate a non-normal distribution. Furthermore, the Kolmogorov-Smirnov
544 test confirmed asymmetric distribution of each parameter with the exception of fibre. As a
545 result, it was necessary to use the U Mann-Whitney test (a non-parametric analysis), used to
546 evaluate the differences between restaurant samples. Table 4 summarizes dispersion of all
547 considered samples, showing their inter- and intra-variabilities.
548 Sample dispersion degree was studied through the coefficient of variation (CV). Overall data
549 showed high values of CV, which means that reported values were very dispersed due to
550 internal factors (i.e. chemical matrix or typological distribution), as well as external factors,
551 i.e. analytical methodologies. According to Table 4, within this group of nutrients, total fibre
552 and protein presented the greatest dispersion (CV equal to 40%) followed by starch (CV equal
553 to 33%), simple sugar (CV of 30%) and ash. However, high dispersion is not reflected in
554 elemental carbon dispersion, with a CV of 10%. This value, together with CV calculated for
555 elemental N (15%) indicate that C/N ratio is not influenced by variations of composition in
556 studied FW samples. Nevertheless, C/N ratio may not show the real potential for efficient
557 valorization, because it does not differentiate between raw material components. Variability is
558 an essential parameter to consider, when seeking for an efficient valorization pathway. An
559 example that contributes to the variability study was found in the protein content related to
560 proportion of meat in FW samples, that was not reflected in variation of N values. As a
561 conclusion, this variability in protein content may not be translated into elemental nitrogen
562 variability, thus there are other compounds containing nitrogen (e.g. nitrites, ammonium) that
563 also participate of C/N ratio. Finally, both ash content and trace element CV reported a value
564 equal to or slightly higher than 30%, with the exception of iron and calcium (Table 4).
565 Variability of these elements is correlated to important differences in typological composition

17
566 and how a certain food was made. For example, food derived from milk will have a higher
567 amount of calcium. While manufactured food depicts higher Na and K concentration that non-
568 processed food.
569 3.3.2. Sample distribution and classification considering composition
570 Finally, PCA showed clusters of samples and their distribution according to composition.
571 Figures 2-4 summarize distribution of all considered samples, showing their inter- and intra-
572 variabilities. With the objective of understanding sample variation, a principal component
573 analysis (PCA) was carried out from three different points of view (Knezevic et al., 2019).
574 Figure 2 shows the three principal macronutrient components that explain 70% of sample
575 variability.
576 PC1 (x-axis) explains the distribution of restaurant samples as a function of protein and ash
577 content. In this case, important variation between sample restaurants was observed; thus, only
578 GR showed more dispersed samples. PC2, corresponding to z-axis, explains the distribution
579 of polymers, i.e. starch, fibres or lipids. UCC showed less dispersion than other restaurants,
580 while GR and FDR depicted the highest dispersion. PC3 (y-axis) reflects simple sugars
581 distribution and shows maximal dispersion for FDR samples.
582 PCA allows to analyze samples in a collective way, besides visualizing typology differences.
583 As an example, protein and ash patrons were homogeneous in all restaurants with the
584 exception of GR, that serves meat specialties. Besides, simple sugars show higher dispersion
585 in FDR (menu includes a high amount of sweet foodstuff and fruits) than in the rest of
586 establishments. As a general rule, it might be assumed that the more specialized the
587 restaurant, the higher the dispersion of one component, in relation to the other restaurants.
588 The U Mann-Whitney test allows to find out similarities and differences between sample
589 restaurants, in terms of chemical composition. Data shown in Table 4 confirm the first PCA
590 result about low variability for ash and protein, while the U Mann-Whitney test was more
591 specific and indicated both that IR showed higher ash content variability and that GR
592 exhibited the highest protein dispersion degree. Simple sugars also presented low variability
593 between establishments and confirmed the specific pattern of FDR, elucidated from PCA,
594 while lipids did not present significant differences between restaurants. Finally, starch
595 dispersion showed in PCA was confirmed by the U Mann-Whitney test, being all restaurants
596 statistically different.
597 As mentioned above, metals (shown in Figure 3 and Table 4) are presented in three different
598 scales of concentration. Trace element PCA explained more than 70% of variation of this
599 group of nutrients. In this PCA, PC1 (x-axes) includes main metallic elements, namely Na,

18
600 Mg and Ca, without important variations, with the exception of outliers. PC2 (z-axes) shows
601 trace element (K, Zn, Mg and Fe) variation, with concentrations close to ppm and ppb. GR
602 samples presented higher variability in PC2 with respect to other establishments, showing a
603 cluster that has a correlation between meat presence and iron concentration from myoglobin,
604 that was corroborated by the U test. Finally, PC3 (y-axes) described variations of ultra-trace
605 metallic elements, with concentrations close to ppb. In this case, no significant variations
606 were found, and the U Mann-Whitney test only presented partial variations between
607 restaurants, as reported in Table 4.
608 A final PCA to consider time variations was carried out and results are shown in Figure 4. As
609 mentioned in section 2, food waste samples were collected during five months, covering two
610 seasons (winter and spring) aiming to analyze time variations. The most significant variation
611 was found in PC2 (z-axis) that shows protein variations and PC1 (y-axis) that exhibits
612 variations in simple sugars over time. Finally, PC3 (x-axis) explains starch variations,
613 showing significant monthly deviations. Data show not only typology composition issues, but
614 also reports information about user eating habits.
615 In this study, statistical analysis shows high correlation between typology and chemical
616 composition. PCA shows high degree of sensitivity, while detecting small changes in patterns,
617 such as protein variability in GR and special FDR pattern. PCA also shows metal variability
618 and confirms concentration scales and seasonal differences between components. These
619 results are in agreement with all statistical tests.
620 An interesting result reported in this study, in agreement to that reported by Fisgativa et al.
621 (2016), is the high CV, that PCA analysis cannot explain beyond 70%. High CV values
622 indicate that mean includes dispersed values that might be considered as errors. As a result, to
623 achieve statistically reliable conclusions, mean cannot be used. However, considering food
624 waste case, variations in typology may partly explain sample deviation, while other factors,
625 i.e. seasonal variations, complex chemical matrices or non-specific analysis, may explain the
626 remaining deviation.
627 In each country, FW is regulated by a specific directive. The current standard “RSB EU RES
628 standard for certification of biofuels based on waste and residues” describes how to
629 characterize this biomass. Several tests, including elemental analysis and calorific value tests,
630 are described by this standard. Nevertheless, this standard does not include a chemical
631 component test. And this is unfortunate, as part of the unexplained variability may originate in
632 different interactions within the chemical matrix. Carmona et al. (2018) reported several

19
633 interactions in FW matrix, i.e. amino acid reactions carried out by the Maillard reaction, redox
634 reactivity from reducing sugars and fibre degradation from pre-treatment.
635 3.4. Preliminary experimental results to show the valorization of food waste based on its
636 composition
637 To show the feasibility of the proposed biorefinery, three different valorization processes
638 based on restaurant FW composition were carried. During the first process, oil was extracted
639 from FW and used as raw material to produce biodiesel. Methodology and results may be
640 found in Carmona et al. (2019) and are summarized in Table 5, showing a fatty acid methyl
641 ester (FAME) conversion above 90%, although bellow standard EN 14214 threshold. Other
642 biodiesel properties, i.e. low calorific power (LCV), carbon residue (CR), cetane number
643 (CN) and oxidation stability (OXS) were also analyzed. FW complex chemical matrix may be
644 the detrimental cause of low OXS and yield, considering European standard thresholds.
645 The second process is focused on the production of fermentation media rich in carbon and
646 nitrogen sources that could be used for microbial production of bio-based chemicals and
647 polymers. So, the presence of starch and protein in the FW is essential. Hydrolysis was
648 carried out by crude enzyme consortia produced by fungal solid-state fermentation carried out
649 on wheat milling by-products. A starch to glucose conversion yield of 98.23% has been
650 achieved leading to 99 g/L glucose production. Protein hydrolysis by protease led to 574
651 mg/L of free amino nitrogen (FAN) production. Inorganic phosphorus production was 700
652 mg/L. The FW derived hydrolysate has been used in shake flask fermentations for the
653 production of microbial lipids using the oleaginous yeast strain Rhodosporidium
654 kratochvilova. Table 5 presents total dry weight (TDW) production (20.9 g/L) and lipid
655 accumulation (3 g/L) at the end of shake flask cultures.
656 The third process includes solid fraction valorization. The solid fraction contains mainly
657 lignin, cellulose and hemicellulose, not valorized in the previous stages. Initial high calorific
658 value (HCV) of this fraction was 21.4 MJ/kg (Table 5). Liquefaction processes allow to
659 degrade this fraction into aromatic hydrocarbons, aliphatic compounds, ester, ketones, alcohol
660 and carboxylic acid. In the present study, a mixture (5:1) of polyethylene glycol and crude
661 glycerol (considered waste from previous transesterification) was used as solvent and
662 ultrasound (US, amplitude 40%) was used as auxiliary energy. During liquefaction, a bio-oil
663 conversion yield of 20.15% was achieved, while HCV increased to 38.17%. Results are
664 summarized in Table 5.
665 4. Conclusions

20
666 Qualitative analysis of hospitality food waste (FW) typology profile showed a high presence
667 of rich-in-starch foodstuff. FW chemical profile showed high moisture content and high
668 degradation potential of organic fraction. Statistical analysis reported differences in
669 restaurant-to-restaurant profile and showed a complex matrix. In this study, non-normality
670 distribution shows the importance of developing a specific standard model for FW analysis.
671 The main conclusion of this work shows that a systematic study about FW composition and
672 variability, besides statistical information, allows finding out the best valorization pathway.
673 Proposed methodology provides a good starting point to set up the basis of FW biorefinery.
674 Acknowledgments
675 Authors are thankful to the Spanish Ministry of Economy and Competitiveness for grant no.
676 ENE2013-47769R and EU INTERREG V POCTEP Program for grant no.
677 0022_BIOMASSTEP_5_E.
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24
801 Figure captions
802 Figure 1. Trace element concentration in food waste samples
803 Figure 2. Principal component analysis for macronutrients for different restaurant food waste.
804 UCC: University campus cafeteria. FDR: fine dining restaurant IR: Italian restaurant GR: grill
805 restaurant
806 Figure 3. Principal component analysis for metal content in different restaurant food waste.
807 UCC: University campus cafeteria. FDR: fine dining restaurant IR: Italian restaurant GR: grill
808 restaurant
809 Figure 4. Principal component analysis along the year in which sampling was carried
810 out.UCC: University campus cafeteria. FDR: fine dining restaurant IR: Italian restaurant GR:
811 grill restaurant
812
813
814 Table captions
815 Table 1. Carbohydrate distribution in collected samples sorted by restaurant
816 Table 2. Lipid content and fatty acid profile of food waste samples, based on matter state of
817 aggregation
818 Table 3. Nitrogen, carbon and ash content in food waste samples
819 Table 4. Statistical test for macronutrients and trace elements considering variability provided
820 by each establishment
821 Table 5. Preliminary experimental results to show the valorization of food waste based on its
822 composition
823
824

25
825

826

827
828 Figure 1.
829

26
830

831
832 Figure 2.
833

27
834

835
836 Figure 3.
837

28
838

839
840

841

842 Figure 4.
843
844
845
846
847
848
849
850
851
852
853
854
855
856
857
858
859

860

29
 861 Table 1.

Restaurant UCC1 FDR2 IR3 GR4 AVERAGE

% (w/w, db)

Total carbohydrates 49.9 45.7 43.9 50.9 47.4±3.3

Sugars 9.0±1.0 14.0±1.0 9.7±0.9 8.0±1.0 10.7±2.2

Reducing
3.1±0.2 3.8±0.3 2.5±0.2 2.4±0.4 2.9±0.6
sugars

Starch 29.4±4.1 16.2±5.1 23.1±3.1 28.1±3.1 24.1±6.1

Total fibre 8.4±0.8 11.7±0.6 8.2±0.3 12.4±0.8 10.3±2.1

Cellulose 3.6±0.4 4.3±0.3 3.5±0.4 4.1±0.5 3.8±0.4

Fibre
Hemicellulose 4.7±0.2 7.4±0.3 5.1±0.2 8.5±0.2 6.4±2.2
types
Lignin 1.1±0.1 1.4±0.2 1.5±0.1 2.4±0.4 1.5±0.1

Cellulose/lignin ratio 3.2 2.9 2.4 1.64 2.4 ±0.7

(1) UCC: University campus cafeteria; (2) FDR: fine dining restaurant; (3) IR: Italian restaurant; (4) GR: grill restaurant

862
863

30
864 Table 2.
865

Lipid fraction (%,w/w, db)

RESTAURANT TOTAL Liquid phase Solid phase

UCC1 26.2±1.2 86.5±1.8 13.5±0.1

FDR2 33.2 ±2.2 90.2±1.2 11.3±0.3

IR3 25.7±2.2 77.4±0.8 23.4±0.1

GR4 27.3±2.8 70.1±1.8 29.9±0.8

AVERAGE 28.1±1.8 81.1±6.9 20±6.03

Fatty acid profile
Fatty acid (%, w/w, db) Liquid phase Solid phase
C8:0 (Caprylic acid) 0.2±0.1
C10:0 (Capric acid) 0.3±0.1
C12:0 (Lauric acid) 1.7±0.1 0.2±0.02
C14:0 (Myristic acid) 0.2±0.1 2.5±0.2
C16:0 (Palmitic acid) 16.6±0.5 25.9±0.1
C16:1 (Palmitoleic acid) 1.7±0.1 1.6± 0.1
C18:0 (Stearic acid) 9.2±0.3 7.9±0.1
C18:1 (Oleic acid) 45.0±1.9 46.5 ±0.1
C18:2 (Linoleic acid) 23.0±1.1 14.1±0.1
C18:3 (Linolenic acid) 1.1±0.1 --
Carbon chain properties
LC 5 17.3 17.1
TUD 6 0.9 0.7
1) UCC: University campus cafeteria; (2) FDR: fine dining restaurant; (3) IR: Italian restaurant; (4) GR:
grill restaurant
5: Length of chain, LC = Σ(n Cn)/100, where n is the number of carbon atoms of each fatty acid and Cn is
the weight percentage of each fatty acid methyl ester.
6: Total unsaturation degree, TUD = (1 %MU + 2 %DU + 3 %TU)/100, where %MU are monounsaturated
methyl esters (%, w/w), %DU are diunsaturated methyl esters (%, w/w) and %TU are triunsaturated
methyl esters (%, w/w).
866

867

868

31
869 Table 3.

RESTAURANTS UCC1 FDR2 IR3 GR4 AVERAGE

% (w/w, db)

TN5 3.4±0.2 3.4±0.1 3.3±0.2 3.9±0.1 3.5±0.2

TKN 3.0±0.6 2.8±0.3 3.1±0.6 3.1±0.2 2.9±0.1

IN6 0.5±0.1 0.6±0.1 0.2 ±0.1 0.9±0.1 0.6±0.1

Elemental carbon 55.0±2 52.1± 0.3 53.0±0.2 50.2±0.1 52.5±0.2

Protein 18.3±0.2 21.5±1.5 19.5±0.2 23.5±2.1 20.6±2.2

C/N ratio 15.8 15.2 16.0 12.8 14.9 ±1.4

Ash 4.3±0.9 5.3±0.1 4.6±0.1 4.25±0.2 4.5±0.3

(1) UCC: University campus cafeteria; (2) FDR: fine dining restaurant; (3) IR: Italian restaurant; (4) GR: grill
restaurant (5) TN: Total nitrogen (elemental); (6) IN: Inorganic nitrogen; (7) TKN: Total Kjeldahl Nitrogen.

870
871

32
872 Table 4.

ELEMENTS AND Normality Non-parametric

Dispersion Distribution
MACRONUTRIENTS tests analysis

Average value CV K-V1 p ≤ U Mann-Whitney

Skewness Kurtois
(%, w/w) (%) 0.05 p≤ 0.05

MACRONUTRIENTS

Low variability
Ash (%, w/w) 4.5 30 2.2 12.3 ≥ 0.001
IR ≠ GR, UCC

Low variability
Protein (%, w/w) 20.6 40 0.2 0.1 ≥ 0.001
GR ≠ FDR, IR, UCC

Low variability
Simple sugar (%, w/w) 10.7 30 1.3 3 ≥ 0.001
FDR

High variability
Starch (%, w/w) 24.2 33 -0.4 -0.7 ≥ 0.001
UCC ≠ FDR ≠ IR ≠ GR

Low variability
FND (%, w/w) 10.1 40 0.8 0.1 0.20
FDR ≠ IR, GR, UCC

Lipids (%, w/w) 28 20 0.4 1.2 0.07 No differences

ELEMENTS

Low variability
C (%, w/w) 53 10 1.3 6.7 0.019
UCC

N (%, w/w) 3.5 15 1.2 2.5 ≥ 0.001 No differences

Na (ppm) 3282.9 30 0.2 -0.9 ≥ 0.001 High variability

K (ppm) 2620.6 40 1.2 0.7 ≥ 0.001 High variability

Mg (ppm) 228.2 30 4.8 30.2 ≥ 0.001 No differences

Ca (ppm) 1167 2 5.1 29.5 ≥ 0.001 High variability

High variability
Cu (ppm) 1 35 3.2 16.2 ≥ 0.001
UCC = GR

High variability
Zn (ppm) 8.1 35 1.7 3.7 ≥ 0.001
UCC = FDR = GR

33
 Variability
Mn (ppm) 3.7 33 7.5 58.5 ≥ 0.001
UCC = FDR = GR

High variability
Fe (ppm) 26.15 5 3.2 13.8 ≥ 0.001
UCC = IR

Variability
Ni (ppm) 0.21 30 6.1 42.3 ≥ 0.001
UCC = FDR = IR

Variability
As (ppm) 0.1 30 6.7 47.6 ≥ 0.001
UCC = GR = IR

UCC: University campus cafeteria. FDR: fine dining restaurant IR: Italian restaurant GR: grill restaurant

873

874

34
875 Table 5

Lipid fraction conversion into biodiesel

Properties

Product Yield  at 40ºC LCV OXS CN CR

(%) (mm2/s) (kJ/kg) (h) (%)

Biodiesel 93 4.23 39,572 3.25 62.42 0.03

Starch and protein fraction utilization for microbial lipid production

Step 1: Hydrolysis
Temperature: 55ºC Starch to glucose conversion Glucose TKN to FAN FAN IP
yield (%) (g/L) conversion yield (%) (mg/L)
FW: 200 g/L (mg/L)

Hydrolysate GA: 0.61 (U/mL) 99 PA: 3.02 (U/mL) 574 700

production 98.23 6.54

Step 2: fermentation for microbial oil production using Rodosporidium Kratochvilova Y13 3/8
C/N ratio Time (h) TDW (g/L) Lipid production Lipid content (%) Productivity (g/L/h)
(g/L)
100 120 20.9 3.00 15.00 0.03

Cellulose, hemicellulose and lignin rich fraction conversion into bio-oil

Initial value Step 3: liquefaction process Results

Product HCV MJ/kg US Amplitude (%) Solvent/biomass Time (min) Yield (%) HCV (MJ/kg)
ratio
Bio-oil 21.4 40 5 20 20.15 29.57

876 Note: : kinematic viscosity; LCV: low calorific value; HCV: high calorific value; OXS: oxidation stability; CN: cetane number; TDW: total dry weight; CR:
877 carbon residue; TKN: Total Kjeldahl nitrogen; FAN: free amino nitrogen; IP: inorganic phosphorous; GA: glucoamylase activity; PA: protease activity
878 Food waste from restaurants exhibits a rich composition in starch, protein and lipid.

35
879 Combination of typological, chemical and statistical tests support its valorization.
880 A new tool to assess biorefinery development from food waste has been provided.
881

36