Food Wastes

Feedstock for Value-Added

Products

Printed Edition of the Special Issue Published in Fermentation

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Diomi Mamma

Edited by

Food Wastes • Diomi Mamma

Food Wastes

Feedstock for Value-Added Products

Special Issue Editor

Diomi Mamma

MDPI • Basel • Beijing • Wuhan • Barcelona • Belgrade • Manchester • Tokyo • Cluj • Tianjin

Special Issue Editor

Diomi Mamma

National Technical University of Athens

Greece

Editorial Ofﬁce

MDPI

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Contents

About the Special Issue Editor ...................................... vii

Diomi Mamma

Food Wastes: Feedstock for Value-Added Products

Reprinted from: Fermentation 2020, 6, 47, doi:10.3390/fermentation6020047 ............. 1

Daniel Bustamante, Marta Tortajada, Daniel Ram´on and Antonia Rojas

Production of D-Lactic Acid by the Fermentation of Orange Peel Waste Hydrolysate by Lactic

Acid Bacteria

Reprinted from: Fermentation 2020, 6, 1, doi:10.3390/fermentation6010001 ............. 5

Hisao Tomita and Yutaka Tamaru

The Second-Generation Biomethane from Mandarin Orange Peel under Cocultivation with

Methanogens and the Armed Clostridium cellulovorans

Reprinted from: Fermentation 2019, 5, 95, doi:10.3390/fermentation5040095 ............. 17

Nathan D. Schwalm III, Wais Mojadedi, Elliot S. Gerlach, Marcus Benyamin, Matthew A.

Perisin and Katherine L. Akingbade

Developing a Microbial Consortium for Enhanced Metabolite Production from Simulated

Food Waste

Reprinted from: Fermentation 2019, 5, 98, doi:10.3390/fermentation5040098 ............. 29

George Prasoulas, Aggelos Gentikis, Aikaterini Konti, Styliani Kalantzi, Dimitris Kekos and

Diomi Mamma

Bioethanol Production from Food Waste Applying the Multienzyme System Produced On-Site

by Fusarium oxysporum F3 and Mixed Microbial Cultures

Reprinted from: Fermentation 2020, 6, 39, doi:10.3390/fermentation6020039 ............. 49

Vinicio Carri ´on-Paladines, Andreas Fries, Rosa Elena Caballero, Pablo P´erez Dani¨els and

Roberto Garc´ıa-Ruiz

Biodegradation of Residues from the Palo Santo (Bursera graveolens) Essential Oil Extraction and

Their Potential for Enzyme Production Using Native Xylaria Fungi from Southern Ecuador

Reprinted from: Fermentation 2019, 5, 76, doi:10.3390/fermentation5030076 ............. 61

Friedrich Felix Jacob, Lisa Striegel, Michael Rychlik, Mathias Hutzler and Frank-J ¨urgen

Methner

Spent Yeast from Brewing Processes: A Biodiverse Starting Material for Yeast Extract Production

Reprinted from: Fermentation 2019, 5, 51, doi:10.3390/fermentation5020051 ............. 81

Zehra Gulsunoglu, Smitha Aravind, Yuchen Bai, Lipu Wang, H. Randy Kutcher and Takuji

Tanaka

Deoxynivalenol (DON) Accumulation and Nutrient Recovery in Black Soldier Fly Larvae

(Hermetia illucens) Fed Wheat Infected with Fusarium spp.

Reprinted from: Fermentation 2019, 5, 83, doi:10.3390/fermentation5030083 ............. 99

Vassileios Varelas

Food Wastes as a Potential New Source for Edible Insect Mass Production for Food and Feed:

A review

Reprinted from: Fermentation 2019, 5, 81, doi:10.3390/fermentation5030081 .............109

About the Special Issue Editor

Diomi Mamma, Ph.D., is an Assistant Professor of Bioprocess Engineering at the School of

Chemical Engineering, National Technical University of Athens, Greece. She studied Chemical

Engineering at the School of Chemical Engineering, National Technical University of Athens, where

she obtained her Ph.D. (2002). She teaches subjects related to bioprocess engineering. Her research

interests focus on microbial production and characterization of enzymes, bioconversion of biomass to

ethanol applying different fermentation strategies, and environmental biotechnology, with emphasis

on the design of appropriate biological processes for the complete removal of xenobiotics.

vii

fermentation

Editorial

Food Wastes: Feedstock for Value-Added Products

Diomi Mamma

Biotechnology Laboratory, School of Chemical Engineering, National Technical University of Athens Zografou

Campus, 15780 Athens, Greece; dmamma@chemeng.ntua.gr

Received: 22 April 2020; Accepted: 24 April 2020; Published: 27 April 2020

Food is a precious commodity, and its production can be resource-intensive. According to the Food

and Agriculture Organization of the United Nations, nearly 1.3 billion tons of food products per year are

lost along the food supply chain, and in the next 25 years the amount of food waste has been projected

to increase exponentially. Food waste is produced at any stage of the supply chain, which extends from

the agricultural site to the processing plant and ﬁnally the retail market. The management of food waste

should follow certain policies based on the 3R’s concept, i.e., reduce, reuse, and recycle [

]. Generally,

food waste is composed of a heterogeneous mixture formed by carbohydrates (starch, cellulose,

hemicellulose, or lignin), proteins, lipids, organic acids, and smaller inorganic parts. Currently, most

food wastes are recycled, mainly as animal feed and compost. The remaining quantities are incinerated

and disposed in landﬁlls, causing serious emissions of methane (CH

), which is 23 times more potent

than carbon dioxide (CO

) as a greenhouse gas and signiﬁcantly contributes to climate change [

Valorizing food waste components could in fact lead to numerous possibilities for the production of

valuable chemicals, fuels, and products [1].

The present Special Issue compiles a wide spectrum of aspects of research and technology in the

area of “food waste exploitation”, and highlights prominent current research directions in the ﬁeld

for the production of value-added products such as polylactic acid, hydrogen, ethanol, enzymes, and

edible insects.

Polylactic acid (PLA) is a biodegradable polymer with great potential in replacing petrochemical

polymers. The morphological, mechanical, and thermal properties of the polymer are determined by

the presence of diﬀerent amounts of l- and d-lactic acid monomers or oligomers [

]. The microbial

production of optically pure lactic acid has extensively been studied, because chemically synthesized

lactic acid is a racemic mixture. Optimizing culture conditions and selecting the LAB strains capable of

producing d-lactic acid with high yield and optical purity from orange peel waste as raw material can

contribute to the development of biowaste reﬁneries. Bustamante et al. [

] evaluated six strains of

the species Lactobacillus delbrueckii ssp. bulgaricus for the production of d-lactic acid from orange peel

waste hydrolysate. L. delbrueckii ssp. bulgaricus CECT 5037 had the best performance, with a yield of

84% w/w for D-LA production and up to 95% enantiomeric excess (optical purity).

Biomethanation (methane fermentation) is a complex biological process, which can be divided in

four phases of biomass degradation and conversion, namely, hydrolysis, acidogenesis, acetogenesis,

and methanation. The individual phases are carried out by diﬀerent groups of micro-organisms

(bacteria), which partly stand in syntrophic interrelation and place diﬀerent requirements on the

environment. Undissolved compounds like cellulose, proteins, and fats are hydrolyzed into monomers

by enzymes produced by facultative and obligatorily anaerobic bacteria [

]. The use of a microbial

consortium consisting of the microbial ﬂora of methane production and microorganisms that can

degrade cellulosic biomass like Clostridium cellulovorans was proven eﬃcient in degraded mandarin

orange peel without any pretreatments and produced methane that accounted for 66.2% of the total

produced gas [6].

Fermentation 2020, 6, 47; doi:10.3390/fermentation6020047 www.mdpi.com/journal/fermentation

Fermentation 2020, 6,47

Hydrogen is a noncarbonaceous fuel and energy carrier possessing higher net caloriﬁc value

compared to other fuels (120 MJ/kg versus 46.7 MJ/kg for gasoline). Microbes primarily produce

hydrogen via photofermentation by the purple nonsulfur bacteria Rhodobacter and Rhodopseudomonas,

and during dark fermentation by strictly anaerobic Clostridium species [

]. Depending upon the

availability of substrate, the selection of functional microorganisms necessary for hydrogen production

is an important step. Simulation of the exchange metabolic ﬂuxes of monocultures and pairwise

coculturesusing genome-scale metabolic models on artiﬁcial garbage slurry resulted in the identiﬁcation

of one of the top hydrogen producing cocultures comprising Clostridium beijerinckii NCIMB 8052 and

Yokenella regensburgei ATCC 43003. The consortium produced a similar amount of hydrogen gas and

increased butyrate (attributed to cross-feeding of lactate produced by Y. regensburgei), compared to the

C. beijerinckii monoculture, when grown on the artiﬁcial garbage slurry [9].

Household food waste is a complex biomass containing various components that make it a source

of potential fermentative substrates. The general scheme of bioethanol production from such complex

materials involves a pretreatment step that increases the digestibility of the material—enzymatic

hydrolysis—to liberate the monosaccharides and fermentation of these sugars to ethanol. In terms

of cost, the most demanding step, which signiﬁcantly increases the total cost of the production of

bioethanol and is identiﬁed as a barrier in the further deployment of ethanol production, is enzymatic

hydrolysis. If the necessary enzymes could be eﬃciently produced on-site, the cost could be signiﬁcantly

reduced. A recent study has estimated that the cellulase cost can be reduced from 0.78 to 0.58$/gallon

by shifting from the oﬀ-site to the on-site approach of cellulase production [

]. The mesophilic fungus

Fusarium oxysporum F3 grown under solid state cultivation on wheat bran produced a multienzyme

system capable of hydrolyzing the carbohydrates present in household food waste. The use of

mixed-microbial cultures in bioethanol production step consisting of F. oxysporum solid state culture

and the yeast Saccharomyces cerevisiae increased bioethanol volumetric productivity, compared to

mono-culture of the fungus. Bioethanol production increased by approximately 23% when the mixed

microbial culture was supplemented with low dosages of commercial glucoamylase [11].

Carri

n-Paladines et al. [

] evaluated two Xylaria spp. of the dry forest areas of southern Ecuador,

for ligninase and cellulase production under solid state fermentation using residues obtained from

the Palo Santo essential oil extraction. The Palo Santo is considered a vital resource for the local

communities of the dry forest, as diﬀerent parts of the tree are used in traditional medicine, as well

as for the extraction of essential oil. The essential oil extraction process generates abundant organic

waste, which is commonly discarded directly into the natural ecosystems or burned. Laccase, cellulose,

and xylanase activities of Xylaria feejeensis and Xylaria cf. microceras were generally higher than those

of the control fungus Trametes versicolor (L.) Lloyd, furthering the understanding of the potential use of

native fungi as ecologic lignocellulosic decomposers and for industrial proposes.

Beer production generates large quantities of spent yeast during the fermentation and lagering

process. The spent yeast is an eﬃcient starting material to produce yeast extract, which is generally

deﬁned as the soluble content of a yeast cell that remains once the cell wall has been destroyed and

removed. The variety of diﬀerent physiologically valuable substances in yeast cells oﬀer the possibility

of use as a yeast extract in diﬀerent areas of the food industry. Jacob et al. [

] demonstrated that the

composition of various physiologically valuable substance groups of a yeast extract depends on the

biodiversity of the spent yeast from beer production, indicating that brewer’s spent yeast should be

carefully selected to produce a yeast extract with a deﬁned nutritional composition.

In many cases, food wastes are diﬃcult to utilize for the recovery of value-added products due to

their biologicalinstability or potentially pathogenic nature. Fusarium head blight (FHB), a fungaldisease

caused by several Fusarium spp., is one of the most signiﬁcant causes of economic loss in cereal crops.

Fusarium spp. produce various amounts and types of trichothecene mycotoxins, with deoxynivalenol

being the major one, which are highly toxic to humans and livestock. A method to recover the

nutrients from the aﬀected cereals, without the mycotoxins, was reported by Gulsunoglu et al. [

The infected grains were initially fermented under solid state cultivation with Aspergillus oryzae and/or

Fermentation 2020, 6,47

Lactobacillus plantarum. The fermented material was provided to black soldier ﬂy larvae, which

consumed deoxynivalenol-contaminated materials and converted them in insect biomass without

accumulating deoxynivalenol in their bodies. This treatment technology using black soldier ﬂy larvae

may contribute to reducing the burden of animal protein shortages in the animal feed market.

Varelas [

] compiled up-to-date information on the mass rearing of edible insects for food and

feed based on food wastes. Edible insects are insect species that can be used for human consumption

but also for livestock feed as a whole, parts of them, and/or protein, and lipid extract.

Funding: This research received no external funding.

Acknowledgments:

The editor wish to thank our article contributors, Editorial Board members, Reviewers,

and Assistant Editors of this journal, whose contributions made the publication of this Special Issue possible.

Conﬂicts of Interest: The authors declare no conﬂict of interest.

References

Nayak, A.; Bhushan, B. An overview of the recent trends on the waste valorization techniques for food

wastes. J. Environ. Manag. 2019, 233, 352–370. [CrossRef][PubMed]

Lin, C.S.K.; Pfaltzgraﬀ, L.A.; Herrero-Davila, L.; Mubofu, E.B.; Abderrahim, S.; Clark, J.H.; Koutinas, A.A.;

Kopsahelis, N.; Stamatelatou, K.; Dickson, F.; et al. Food waste as a valuable resource for the production

of chemicals, materials and fuels. Current situation and global perspective. Energy Environ. Sci.

2013

, 6,

426–464. [CrossRef]

Singhvi, M.; Zendo, T.; Sonomoto, K. Free lactic acid production under acidic conditions by lactic acid

bacteria strains: Challenges and future prospects. Appl. Microbiol. Biotechnol.

2018

, 102, 1–14. [CrossRef]

[PubMed]

Bustamante, D.; Tortajada, M.; Ram

n, D.; Rojas, A. Production of D-Lactic Acid by the Fermentation of

Orange Peel Waste Hydrolysate by Lactic Acid Bacteria. Fermentation 2020, 6,1.[CrossRef]

Xu, N.; Liu, S.; Xin, F.; Zhou, J.; Jia, H.; Xu, J.; Jiang, M.; Dong, W. Biomethane Production from Lignocellulose:

Biomass Recalcitrance and Its Impacts on Anaerobic Digestion. Front. Bioeng. Biotechnol.

2019

, 7, 191.

[CrossRef][PubMed]

Tomita, H.; Tamaru, Y. The Second-Generation Biomethane from Mandarin Orange Peel under Cocultivation

with Methanogens and the Armed Clostridium Cellulovorans. Fermentation 2019, 5, 95. [CrossRef]

Łukajtis, R.; Hołowacz, I.; Kucharska, K.; Glinka, M.; Rybarczyk, P.; Przyjazny, A.; Kami´nskia, M. Hydrogen

production from biomass using dark fermentation. Renew. Sustain. Energy Rev.

2018

, 91, 665–694. [CrossRef]

Keskin, T.; Abo-Hashesh, M.; Hallenbeck, P.C. Photofermentative hydrogen production from wastes.

Bioresour. Technol. 2011, 102, 8557–8568. [CrossRef][PubMed]

Schwalm, N.D., III; Mojadedi, W.; Gerlach, E.S.; Benyamin, M.; Perisin, M.A.; Akingbade, K.L. Developing a

Microbial Consortium for Enhanced Metabolite Production from Simulated Food Waste. Fermentation

2019

5, 98. [CrossRef]

10.

Johnson, E. Integrated enzyme production lowers the cost of cellulosic ethanol. Biofuels Bioprod. Bioreﬁn.

2016, 10, 164–174. [CrossRef]

11.

Prasoulas, G.; Gentikis, A.; Konti, A.; Kalantzi, S.; Kekos, D.; Mamma, D. Bioethanol Production from Food

Waste Applying the Multienzyme System Produced On-Site by Fusarium oxysporum F3 and Mixed Microbial

Cultures. Fermentation 2020, 6, 39. [CrossRef]

12.

Carri

n-Paladines, V.; Fries, A.; Caballero, R.E.; P

rez Daniëls, P.; Garc

a-Ruiz, R. Biodegradation of Residues

from the Palo Santo (Bursera graveolens) Essential Oil Extraction and Their Potential for Enzyme Production

Using Native Xylaria Fungi from Southern Ecuador. Fermentation 2019, 5, 76. [CrossRef]

13.

Jacob, F.F.; Striegel, L.; Rychlik, M.; Hutzler, M.; Methner, F.-J. Spent Yeast from Brewing Processes:

A Biodiverse Starting Material for Yeast Extract Production. Fermentation 2019, 5, 51. [CrossRef]

Fermentation 2020, 6,47

14.

Gulsunoglu, Z.; Aravind, S.; Bai, Y.; Wang, L.; Kutcher, H.R.; Tanaka, T. Deoxynivalenol (DON) Accumulation

and Nutrient Recovery in Black Soldier Fly Larvae (Hermetia illucens) Fed Wheat Infected with Fusarium spp.

Fermentation 2019, 5, 83. [CrossRef]

15.

Varelas, V. Food Wastes as a Potential New Source for Edible Insect Mass Production for Food and Feed:

A review. Fermentation 2019, 5, 81. [CrossRef]

2020 by the author. Licensee MDPI, Basel, Switzerland. This article is an open access

article distributed under the terms and conditions of the Creative Commons Attribution

(CC BY) license (http://creativecommons.org/licenses/by/4.0/).

fermentation

Article

Production of D-Lactic Acid by the Fermentation

of Orange Peel Waste Hydrolysate by Lactic

Acid Bacteria

Daniel Bustamante

1,2

, Marta Tortajada

, Daniel Ramón

and Antonia Rojas

Current address: National Renewable Energy Centre (CENER), Av. Ciudad de la Innovación, 7,

31621 Sarriguren, Spain; dbustamante@cener.com

ADM-BIOPOLIS, Parc Cient

ﬁc Universitat de Val

ncia, C/Catedr

tico Agust

n Escardino, 9, 46980 Paterna,

Spain; marta.tortajada@adm.com (M.T.); daniel.ramonvidal@adm.com (D.R.)

* Correspondence: antonia.rojas@adm.com

Received: 31 October 2019; Accepted: 16 December 2019; Published: 18 December 2019

Abstract:

Lactic acid is one the most interesting monomer candidates to replace some petroleum-

based monomers. The application of conventional poly-lactic acid (PLA) is limited due to insuﬃcient

thermal properties. This limitation can be overcome by blending poly-D and poly-L-lactic acid.

The main problem is the limited knowledge of D-lactic acid (D-LA) production. Eﬃcient biochemical

processes are being developed in order to synthesize D-LA from orange peel waste (OPW). OPW is

an interesting renewable raw material for bioreﬁnery processes of biocatalytic, catalytic or thermal

nature owing to its low lignin and ash content. Bioprocessing of the pretreated OPW is carried out by

enzymatic hydrolysis and fermentation of the released sugars to produce D-LA. Several strains of the

species Lactobacillus delbrueckii ssp. bulgaricus have been evaluated for the production of D-LA from

OPW hydrolysate using Lactobacillus delbrueckii ssp. delbrueckii CECT 286 as a reference strain since its

performance in this kind of substrate have been widely reported in previous studies. Preliminary

results show that Lactobacillus delbrueckii ssp. bulgaricus CECT 5037 had the best performance with a

yield of 84% w/w for D-LA production and up to 95% (e.e.).

Keywords: added value product; D-lactic acid; LAB strains; food waste; orange peel waste

1. Introduction

Lactic acid is an important chemical and has attracted a great attention due its widespread

applications in the food, pharmaceutical, cosmetic, and textile industries. Polylactic acid (PLA) is a

biodegradable polymer with great potential in replacing petrochemical polymers and therefore, L-and

D-lactic acids are prominent monomers of the bioplastic industry [

]. The morphological, mechanical

and thermal properties of the polymer are determined by the presence of diﬀerent amounts of L- and

D-lactic acid monomers or oligomers [

–

]. Microbial production of optically pure lactic acid has

extensively been studied because chemically synthesized lactic acid is a racemic mixture [

]. In fact,

the optimization of operation conditions is very eﬀective to achieve high selectivity to the isomer of

interest [

]. Although the L-isomer has been studied in detail, information on biosynthesis of D-lactic

acid (D-LA) is still limited [5,9].

PLA market demand accounts for 11.4% of total bioplastic production worldwide, approximately

18 × 10

metrictonsper year andthe PLA demand isestimated to growby 28% peryear until 2025. However,

production costs of PLA are still high, mainly due to expensive fermentation media components.

To overcome this problem, several residues have been employed as raw material

[3,5,7,10–12]

Production of D-LA from liquid pineapple wastes [

], date juice [

], corn stover [

], hardwood pulp

hydrolysate [

] and brown rice [

] has been studied. In this sense, the valorization of food waste to

Fermentation 2020, 6, 1; doi:10.3390/fermentation6010001 www.mdpi.com/journal/fermentation

Fermentation 2020, 6,1

useful products such as D-LA is a good alternative [

]. In particular, orange peel and pulp waste

(OPW) can be used to produce D-LA after adequate pre-treatment processes [20–22].

Orange waste is the most abundant citrus waste with up to 50 million metric tons of oranges

consumed every year [

]. This huge amount of waste accounts for 45%–60% of the total fruit weight,

and therefore, a lot of potential applications have been studied for their valorization to date [

The main application of this residue is as an ingredient for cattle feed or as pelletized dry solid fuel, but

its processing results in highly polluted wastewater [

]. The use ofcitrus waste to produce compounds of

high added value, essential oils, fertilizer, pectin, industrial enzymes, ethanol and absorbents has recently

been described [

–

]. In addition, orange waste present low levels of lignin and a large amount of

sugars [

], which make it an ideal substrate for fermentation processes after the implementation of the

required pre-treatment and enzymatic hydrolysis stages.

Lactic acid is produced in high amounts by lactic acid bacteria (LAB) which can do so in a

homofermentative way employing the Embden-Meyerhof pathway where lactic acid is the only acid

produced, or by the heterofermentative way following the phosphogluconate and phosphoketolase

pathway where lactic acid is one of the products and yields of 0.5 g g

−1

of hexose. LABs produce either

one or the two forms of lactate [

]. The species Lactobacillus delbrueckii ssp. delbrueckii has been

reported as a homofermentative producer of D-LA using several agro-industrial residues [

]. This

bacterium yields 90% D-LA from sugarcane molasses, 95% D-LA from sugarcane juice, 88% D-LA

from sugar beet juice [

] and 88% D-LA from orange peel waste (OPW) [

]. Moreover, the species

Lactobacillus delbrueckii subsp. bulgaricus has been used in the dairy industry to transform milk into

yogurt and some strains are able to produce highly pure D-LA [

]. Therefore, lactose and whey have

been widely studied as raw materials for lactic acid production [

–

], even cloning the D-lactate

dehydrogenase gene in Escherichia coli [

]. Other studies included wheat ﬂour, molasses, sorghum and

lignocellulosic hydrolysates as feedstocks for the production of lactic acid by Lactobacillus delbrueckii

subsp. bulgaricus, especially for L-LA isomer production [

]. This fact means that some strains

of Lactobacillus delbrueckii subsp. bulgaricus could be potential candidates for D-LA production from

sustainable feedstocks.

The aim of this work was to ﬁnd LAB strains capable of producing D-LA with high yield and

optical purity from OPW as raw material to contribute in the development of biowaste-reﬁneries.

For this purpose, several Lactobacillus delbrueckii ssp. bulgaricus strains were evaluated in comparison

to the reference strain Lactobacillus delbrueckii ssp. delbrueckii CECT 286 which has been reported as a

high yield producer of D-LA from biowaste and OPW hydrolysate in particular.

2. Materials and Methods

2.1. Bacterial Strains, Media and Growth Conditions

The bacterial strains employed in this study are listed in Table 1 and Lactobacillus delbrueckii ssp.

delbrueckii CECT 286 was used as reference strain. The selected strains were purchased from the

Spanish Type Culture Collection (CECT). After being received they were recovered in MRS medium

and stored in 20% glycerol at

−

◦

C for long-term preservation. Precultures were prepared in

tubes containing MRS medium with a small headspace and incubated overnight at 37

◦

C and static

micro-aerobic conditions.

Table 1. Lactic acid bateria (LAB) strains selected for D-lactic acid production screening.

Microorganism Strain Code

L. delbrueckii ssp. bulgaricus CECT 4005

L. delbrueckii ssp. bulgaricus CECT 4006

L. delbrueckii ssp. bulgaricus CECT 5035

L. delbrueckii ssp. bulgaricus CECT 5036

L. delbrueckii ssp. bulgaricus CECT 5037

L. delbrueckii ssp. bulgaricus CECT 5038

Fermentation 2020, 6,1

Screening of LAB strains was performed in 15 mL tubes at 37

◦

C and using a medium with sugars

resembling OPW hydrolysate as follows: MRS broth plus glucose 30 g L

−1

, fructose 20 g L

−1

, galactose

5gL

−1

and arabinose 6 g L

−1

. Cultures were inoculated in duplicate with 5% v/v of preculture and

were incubated in orbital shaker at 200 rpm. Aerobic and micro-aerobic conditions were tested at

pH 6.2 for 40 h.

2.2. OPW Hyrolysate Tolerance Assays

Tolerance assays were performed in triplicate using selected strains and preparing a multi-well

plate with 200

L of MRS with OPW hydrolysate diluted at 50%, 85% and 100% v/v as culture

medium for each condition. Precultures were prepared in MRS and inoculated at 10% of total volume.

A microplate incubator spectrophotometer was used with temperature set at 37

◦

C for 45 h. The plate

was shaken every hour for 5 seconds before each OD

600

measurement to obtain the growth curves of

the strains.

2.3. Fermentation Assays

Strains were cultured in 50 mL tubes containing MRS with 85% v/v OPW hydrolysate at pH 6.2,

◦

C and 45

◦

C in micro-aerobic conditions. An additional assay was done by adjusting pH at 5.8 each

24 h with NaOH 5 M. All runs started by inoculating 15% v/v of preculture and then incubated in an

orbital shaker at 200 rpm for 120 h.

The experiments in the bioreactor setup were performed in 1.5 L Applikon

in batch mode with

OPW hydrolysate at 85% v/v with MRS and 5 g L

−1

meat extract as additional nitrogen source. The OPW

hydrolysate was sterilized using sterile glass ﬁber and cellulose acetate membrane ﬁlters with 0.2

of pore size, and then added to the bioreactor. Before the inoculum addition, the anaerobic atmosphere

was obtained by stripping the oxygen oﬀ with a nitrogen stream. The experimental conditions were set

up at 37

◦

C, 200 rpm, and pH of 5.8, adding NaOH 5 M or HCl 2 M for pH control during fermentation.

2.4. OPW Pretreatments

The substrate used in this study was OPW obtained from juice elaboration. These residues were

blade-milled to a ﬁnal particle diameter of around 5 mm and then, samples were subsequently stored

in a freezer at −20

◦

C until use. The characterization of the raw material was performed according to

the NREL procedures for determination of structural carbohydrates and free sugars, in addition to

extractives [

–

], while moisture was assessed by using an infrared drying balance at temperatures

between 70 and 90

◦

C until constant weight. The results obtained by applying the NREL methodology

are compiled in Table 2. For D-LA production assays, OPW was milled down to 1–2 mm particle

size and hydrolysis was carried out at 10% w/w of dry solid, 50

◦

C, 300 rpm and initial pH of 5.2

using enzyme cocktails with cellulases,

-glucosidase, xylanase,

-xylosidase, pectinase, and auxiliary

activities (Celluclast 1.5 l, Novozym 188, Pectinex Ultra SP-L gifted by Novozymes) as described by

de la Torre and colleagues [22].

Table 2. OPW composition analysis according to NREL protocols.

Component % Dry Weight (w/w)

Total solids 19.2 ± 0.5

Ash 3.9 ± 0.2

Fats n.d.

Water

extractives

37.5 ± 0.4

Free sugars 36.4 ± 0.6

Glucan 19.1 ± 0.1

Hemicellulose 14.8 ± 0.2

Lignin 6.2± 0.5

Pectin 17.9 ± 1.5

Fermentation 2020, 6,1

2.5. Analytical Procedures

The content of sugars and organic acids was determined by HPLC liquid chromatography

(2695 HPLC with a refractive Index Detector 2414; Waters, Cerdanyola del Vall

s, Spain) using a Rezex

ROA Organic acid column, with H

at 2.5 mM and 0.5 mL min

−1

ﬂow. The optical purity of

D-LA was determined by HPLC (Agilent Technologies 1100 Series, Waldbronn, Germany) using a

DAD detector, a Chirex 3126 (D)-penicillamine (250

4.6; Phenomenex) column working at room

temperature, and a CuSO

1 mM solution as mobile phase ﬂowing at 1.2 mL min

−1

3. Results and Discussion

3.1. Screening of LAB Strains for D-LA Production

Lactic acid production was tested in 15 mL tubes containing 3 mL of culture resembling OPW

hydrolysate for aerobic conditions and 14 mL of culture for micro-aerobic conditions to compare

the behavior of the diﬀerent LAB strains. Results are shown in Figure 1. Lactobacillus delbrueckii

ssp. bulgaricus CECT 4005 and CECT 5038 did not produce a signiﬁcant amount of lactic acid while

L. delbrueckii ssp. bulgaricus CECT 5036 produced up to 14 g L

−1

of lactic acid racemic mixture in aerobic

and micro-aerobic conditions. Furthermore, three strains, L. delbrueckii ssp. bulgaricus CECT 4006,

CECT 5035 and CECT 5037 transformed sugars into lactic acid in micro-aerobic condition with D-LA

enantiomeric excess in the same way as L. delbrueckii ssp. delbrueckii CECT 286. Those strains produced

around 15 g L

−1

of lactic acid with around 75% (e.e.) of D-LA while L. delbrueckii ssp. delbrueckii CECT

286 reached 92% (e.e.) of D-LA. Therefore, those three strains were selected to study D-LA production

from OPW hydrolysate in micro-aerobic conditions.

100

CECT

286

CECT

4005

CECT

4006

CECT

5035

CECT

5036

CECT

5037

CECT

5038

Sugars consumed (%)

Yield (%)

D-LA (%)

100

CECT

286

CECT

4005

CECT

4006

CECT

5035

CECT

5036

CECT

5037

CECT

5038

Sugars consumed (%)

Yield (%)

D-LA (%)

Figure 1.

D-LA production in 15 mL tubes with MRS medium containing sugars resembling OPW

hydrolysateusing LAB strains selected for screening. A. Aerobicconditions. B. Micro-aerobic conditions.

Previous reports showed that lactose rather than glucose markedly increases the growth rate of

L. delbrueckii ssp. bulgaricus strains [

]. Therefore, transport systems of sugars other than lactose are

likely to vary among these strains and hence, some strains, such as L. delbrueckii ssp. bulgaricus CECT

4005 and CECT 5038, appear to have diﬃculties to assimilate the sugars tested in this work. Moreover,

strains such as L. delbrueckii ssp. bulgaricus CECT 5035 and CECT 5037 show low yield in assays at

aerobic conditions in the same way as L. delbrueckii ssp. delbrueckii CECT 286. It is known that during

growth, toxic oxygen derivatives are produced for LAB strains in aerobic conditions, but the enzymes

required to eliminate them seem not to be expressed in some L. delbrueckii ssp. bulgaricus strains [

Reducing agents may provide protection against toxic products, particularly if growth conditions are

not strictly anaerobic. However, with exception of L. delbrueckii ssp. bulgaricus CECT 5036, the other

strains showed higher selectivity to D-LA than L. delbrueckii ssp. delbrueckii CECT 286 in aerobic

conditions and as mentioned above, L. delbrueckii ssp. bulgaricus CECT 5036 have similar results at

Fermentation 2020, 6,1

aerobic and micro-anaerobic conditions but produced racemic mixture in both cases. L. delbrueckii ssp.

bulgaricus CECT 4005 appears to prefer aerobic conditions but yields are still low.

3.2. Use of OPW Hydrolysate for D-LA Production by Selected Strains

The OPW hydrolysates were prepared following the methodology described in Section 2.4.

and developed by de la Torre and colleagues [

] obtaining a glucose yield around 60% w/w which

corresponds to around 30 g L

−1

, and obtaining a total sugar concentration above 50 g L

−1

. Therefore,

OPW is a good source of several monosaccharides but also have essential oils rich in limonene and

containing terpenes and phenolics with some antimicrobial activity [

]. The tolerance of the strains

to the substrate was tested with diﬀerent concentrations of OPW hydrolysate ranging from 50% to

100% v/v diluted with MRS broth. Growth monitoring was performed in a micro-plate incubator

for 48 h (Figure 2). Microorganisms grew up well at 50% v/v hydrolysate content, but the strain

L. delbrueckii ssp. delbrueckii CECT 286 tolerated the hydrolysate and was able to grow even when

hydrolysate content was 100% v/v. Lactobacillus delbrueckii ssp. bulgaricus CECT 4006 appears to be

more sensitive to OPW hydrolysate while L. delbrueckii ssp. bulgaricus CECT 5037 was able to grow

up at any OPW concentration; however, the higher the hydrolysate concentration, the higher the lag

phase and the lower the growth. Diﬀerences lied on the performance of the strains, which is slightly

lower when using OPW hydrolysates, probably due to the presence of essential oil components, either

terpenes or phenolics. However, Lactobacilli are able to withstand relatively high concentrations of

citrus extracts [43].

0,2

0,4

0,6

0,8

1,2

1,4

1,6

1,8

0 1020304050

OD 600nm

Time (h)

50% OPW hydrolysate

85% OPW hydrolysate

100% OPW hydrolysate

0,2

0,4

0,6

0,8

1,2

1,4

1,6

1,8

0 1020304050

OD 600nm

Time (h)

50% OPW hydrolysate

85% OPW hydrolysate

100% OPW hydrolysate

0,2

0,4

0,6

0,8

1,2

1,4

1,6

1,8

0 1020304050

OD 600nm

Time (h)

50% OPW hydrolysate

85% OPW hydrolysate

100% OPW hydrolysate

0,2

0,4

0,6

0,8

1,2

1,4

1,6

1,8

0 1020304050

OD 600nm

Time (h)

50% OPW hydrolysate

85% OPW hydrolysate

100% OPW hydrolysate

Figure 2.

Growth curves for tolerance assays to OPW hydrolysate in microplates and microarebic

conditions. (

) Lactobacillus delbrueckii ssp. delbreckii CECT 286. (

) Lactobacillus delbrueckii ssp.

bulgaricus CECT 4006. (

) Lactobacillus delbrueckii ssp. bulgaricus CECT 5035. (

) Lactobacillus delbrueckii

ssp. bulgaricus CECT 5037. The results were obtained as the average of three replicates and standard

deviation was lower than 0.5%.

Concerning the nutritional requirements, previous studies showed that niacin, calcium pantothenate,

riboflavin, and vitamin B12 were essential for the growth of L. delbrueckii ssp. bulgaricus, and that folic acid,

pyridoxal, and CaCl

were important for eﬃcient growth [

]. There could be discrepancies due to

Fermentation 2020, 6,1

diﬀerences in medium composition or to strain-speciﬁc requirements as in the case of L. delbrueckii ssp.

bulgaricus CECT 5037, which not only seems to tolerate hydrolysate, but also seems to grow with less

strict nutritional requirements. Although L. delbrueckii ssp. delbueckii CECT 286 and L. delbrueckii ssp.

bulgaricus CECT 5037 have shown highest robustness cultured in OPW hydrolysate, the next assays

were performed using the four selected strains and inoculating the cells recovered from 15% v/v of

preculture with respect to the volume of culture at 85% v/v OPW hydrolysate diluted with MRS medium

and micro-aerobic conditions. The inoculum amount was increased to compare the performance of

the selected strains with the maximum concentration of OPW hydrolysate during the preliminary

fermentation trials.

The optimal growth temperature for Lactobacilli ranges from 30 to 40

◦

C, although some

thermophilic strains grow well and have highly activated metabolism at temperatures around 45

◦

The four Lactobacillus strains selected were cultured at 37

◦

C and 45

◦

C during 120 h to test their

activity at conditions as close as possible to those of hydrolysis stage and therefore, to evaluate if

the hydrolysis and fermentation stages could be done simultaneously (SSF) as a preliminary result

for the future optimization and scale-up of the process. In general, the SSF process oﬀers better

yields because it avoids product inhibition and results in higher productivity [

]. Aghababaie and

colleagues [

] reported that optimum temperature and pH for growth and lactate production from

whey for L. delbrueckii ssp. bulgaricus were 44

◦

C and 5.7, respectively. However, the results in Figure 3

show that the strains selected in this study produced D-LA up to 90% (e.e.) in all cases, but the

performance of the strains was still better at 37

◦

C using OPW hydrolysates.

100

CECT 286

(37ᵒC)

CECT 286

(45ᵒC)

CECT 4006

(37ᵒC)

CECT 4006

(45ᵒC)

CECT 5035

(37ᵒC)

CECT 5035

(45ᵒC)

CECT 5037

(37ᵒC)

CECT 5037

(45ᵒC)

Sugar consumed (%)

Yield (%)

D-LA (%)

Figure 3.

D-LA production results of three L. delbrueckii ssp. bulgaricus selected in front of L. delbrueckii

ssp. delbrueckii CECT 286 using OPW hydrolysate at 85% v/v and incubated at 37

◦

C and 45

◦

Cto

compare strains performance at diﬀerent temperatures.

Similarly to temperature, the eﬀect of pH change on growth characteristics varied between

diﬀerent species of LAB and in most cases, a decrease of lactate production with a decrease of pH were

observed [

]. Therefore, the strains were cultured in 85% v/v OPW hydrolysate and pH was adjusted

to 5.8 each 24 h during fermentation to test their capacity of production with pH regulation. Cultures

were incubated at 37

◦

C and micro-aerobiosis for 120 h. The results show that sugar consumption

and yields were higher when pH was adjusted, and D-LA up to 95% (e.e.) was produced (Figure 4).

L. delbrueckii ssp. bulgaricus CECT 5037 showed the best results in comparison to the other L. delbrueckii

ssp. bulgaricus strains and its performance was comparable to L. delbrueckii ssp. delbrueckii CECT 286

strain using OPW hydrolysate, whose productivities were between 0.23 and 0.29 g L

−1

, respectively.

Due to the homofermentation of L. delbrueckii ssp. delbrueckii and L. delbrueckii ssp. bulgaricus [

only lactic acid could be produced. Nevertheless, a small increase in ethanol concentration onwards of

48 h of fermentation was observed during pH regulation trials. The explanation for this fact, according

to the literature [

], is that some homofermenters, when grown in limited sugar environment or in

the presence of diﬀerent sugars, can lead to other end products. The main diﬀerence is in pyruvate

metabolism, but the homofermentation pathway is still used. Additionally, the accumulation of ethanol