ACTA MICROBIOLOGICA BULGARICA · в продължение на 3 месеца, а степента...

ACTAMICROBIOLOGICABULGARICA

Volume 35, Issue 3, 2019

Bulgarian Society for MicrobiologyUnion of Scientists in Bulgaria

ISSN 0204-8809

ACTAMICROBIOLOGICABULGARICA

Acta MicrobiologicaBulgarica

The journal publishes editorials, original research works, research reports, reviews, short communications, letters to the editor, historical notes, etc. from all areas of microbiology

An Official Publicationof the Bulgarian Society for Microbiology

(Union of Scientists in Bulgaria)

Volume 35 / 3 (2019)

Editor-in-ChiefAngel S. Galabov

Press Product LineSofia

Editor-in-ChiefAngel S. Galabov, The Stephan Angeloff Institute of Microbiology, Sofia, Bulgaria

Editors

Maria Angelova, The Stephan Angeloff Institute of Microbiology, Sofia, BulgariaHristo Najdenski, The Stephan Angeloff Institute of Microbiology, Sofia, Bulgaria

Sohret Aydemir, Ege University, İzmir, TurkeyLyudmila Boyanova, Medical University, Sofia, BulgariaElisabeth Carniel, Institut Pasteur, Paris, FranceMilton da Costa, University of Coimbra, PorugalErik De Clercq, Rega Institute for Medical Research, Leuven, BelgiumFrancis Delpeyroux, Institut Pasteur, Paris, FranceStefan Denev, Trakia University, Stara Zagora, BulgariaDietmar Fuchs, Innsbruck Medical University, AustriaStoyan Groudev, University of Mining and Geology, Sofia, BulgariaIlia Iliev, Plovdiv University “Paisii Hilendarski”, BulgariaGabriel Ionescu, Cantacuzino National Institute of Research, Bucarest, RomaniaLiliya Ivanova, Medical University of Varna, BulgariaZeynep Çiğdem Kayacan, Istanbul University, Istanbul, Turkey Ivan Mitov, Medical University, Sofia, BulgariaIgor Mokrousov, St. Petersburg Pasteur Institute, RussiaMariana Murdjeva, Medical University, Plovdiv, BulgariaAlexander Netrusov, Moscow Lomonosov State University, RussiaBauke Oudega, Vrije, University of Amsterdam, NetherlandsRayko Peshev, National Diagnostic & Research Veterinary Medical Institute„Prof. Dr. G. Pavlov“,Sofia, BulgariaMilena Petrovska, “Ss. Cyril and Methodius” University, Skopje, Republic of North MacedoniaŠpiro Radulović, University of Belgrade, SerbiaLazar Ranin, University of Belgrade, SerbiaPeter Raspor, Biotechnical Faculty, University of Ljubljana, SloveniaBeatrice Riteau, Aix-Marseille Université, FranceJean Rommelaere, German Cancer Research Center, Heidelberg, GermanyArzu Sayiner, Dokuz Eylul University, School of Medicine, Izmir, TurkeyGalina Satchanska, New Bulgarian Univers ity, Sofia, BulgariaEncho Savov, Military Medical Academy, Sofia, BulgariaMaria Stoyanova, Institute of Soil Science, Agrotechnologies andPlant Protection “N. Pushkarov”, Sofia, BulgariaAntoniy Stoev, Institute of Soil Science, Agrotechnologies andPlant Protection „N. Pushkarov“, Sofia, BulgariaStoyanka Stoitsova, The Stephan Angeloff Institute of Microbiology, Sofia, BulgariaTatiana Tcherveniakova, Medical University, Sofia, BulgariaEnzo Tramontano, University of Cagliari, ItalyAthanassios Tsakris, Medical School, University of Athens, GreeceFabian Wild, Expert in the Centre of WHO, Lion, France

ACTA MICROBIOLOGICA BULGARICA

CONTENTS

EventsFifth National Congress of Virology with International ParticipationDays of Virology in Bulgaria with international participation 2-4 October 2019, Sofia, Bulgaria 79

Program of Fifth National Congress of Virology with International Participation 83

Abstracts from Fifth National Congress of Virology with International Participation 92

Review ArticlesFungi Useful for the Purification of the Lignin Fraction from Residues of Bioethanol Production 125Chiara Daccò, Marta Elisabetta Eleonora Temporiti, Lidia Nicola, Solveig Tosi

Solid-State Fermentation using a Strain of Trichoderma asperellum Improves the Saccharification of Rice Straw 133Sonia Accossato, Mirco Granata, Matteo Faè, Rino Cella, Solveig Tosi, Anna Maria Picco

Antimicrobial Susceptibility/Resistance of Escherichia coli among the Outpatients with Urinary Tract Infections in Mostar 141Mufida Aljicevic, Amina Omerika, Velma Rebic, Sabina Mahmutovic, Amila Abduzaimovic

Microbiological Safety of Commercial Pasteurized Apple and Orange Juices 147Galina Satchanska, Margarita Tsenova, Aleksandra Sasheva

Tolerance of Yeast to Formic Acid during Ethanol Fermentation 155Folake T. Afolabi, Godwin E. Oduokpaha, Abiodun A. Onilude

Volume 35, Issue 3 2019

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ACTA MICROBIOLOGICA BULGARICA Volume 35 / 3 (2019)

Fungi Useful for the Purification of the Lignin Fraction from Residues of Bioethanol Production

Chiara Daccò, Marta Elisabetta Eleonora Temporiti, Lidia Nicola, Solveig Tosi*

Laboratory of Mycology, Department of Earth and Environmental Sciences, University of Pavia, Italy

AbstractLignocellulosic residues, deriving from the production of biofuels, represent a precious source and

being able to purify and exploit them is crucial for a more sustainable supply of lignin. The aim of this work was to increase the efficiency of the biological treatment of lignocellulosic residues to obtain purified lignin which can be used in biotechnological applications. The study evaluated the activity of two Ascomycota fungal strains on a bioethanol production waste material rich in lignocellulosic residues (LRR). The tested fungi were Trichoderma asperellum 1020, isolated from a hydroponic plant culture and Paecilomyces variotii 1030, isolated from a sample of lignin purified by chemical methods. The degradation ability of the selected fungi was verified by measuring their consumption of reducing sugars over 3 months, while the purification was assessed by measuring the degradation of reducing sugars and the activity of the secretome (endo-β-1,4-glucanase, β-glucosidase, β-1,3-glucanase, endo-1,4-β-xylanase, pectinase). Both strains appear to use almost all the available reducing sugars in the first 4 days after inoculation and a decrease of the reducing sugars up to 70% was obtained in the samples treated with T. asperellum 1020. The quantification of enzymes secreted revealed that P. variotii 1030 has greater potential in the degradation of cellulose and pectin, while T. asperellum 1020 degrades preferably pectin and carbohydrate residues. These strains act on the components of LRR and are particularly promising to processes lignocellulosic residues.Keywords: Fungi, Ascomycetes, lignin-rich residues, enzymes, biodegradation, bioethanol

РезюмеЛигноцелулозните остатъци, получени от производството на биогорива, представляват скъпоценен

източник и възможността за тяхното пречистване и експлоатация е от решаващо значение за по-устойчи-вото производство на лигнин. Целта на тази работа е да се повиши ефективността на биологичното трети-ране на лигноцелулозни остатъци за получаване на пречистен лигнин, който може да се използва в биотех-нологични производства. Проучена е активността на два щама гъби от отдел Ascomycota върху отпадъчен материал, богат на лигноцелулозни остатъци (LRR) за производство на биоетанол. Изследваните щамове са Trichoderma asperellum 1020, изолиран от хидропонна растителна култура и Paecilomyces variotii 1030, изолиран от проба лигнин, пречистен по химически метод. Способността на избраните щамове да раз-граждат лигно-целулозни материали се установява чрез определяне консумация на редуциращи захари в продължение на 3 месеца, а степента на пречистване се оценява чрез измерване разграждането на ре-дуциращите захари и активността на секретома (ендо-β-1,4-глюканаза, β -глюкозидаза, β-1,3-глюканаза, ендо-1,4-β-ксиланаза, пектиназа). Двата щама използват почти всички налични редуциращи захари през първите 4 дни след инокулацията, а в пробите, третирани с T. asperellum 1020 се установява понижава-не количеството на редуциращите захари до 70%. Количественото определяне на секретираните ензими показа, че P. variotii 1030 има по-голям потенциал при разграждането на целулозата и пектина, докато T. asperellum 1020 разгражда предимно пектиновите и въглехидратните остатъци. Тези щамове действат върху компонентите на LRR и са особено обещаващи за процесите на лигноцелулозни остатъци.

* Corresponding author: [email protected]

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IntroductionBioethanol is a biofuel that has received

most interest because of the possibility to use it as a replacement of gasoline in the transport fuel market (Lewis, 1996). It can be derived from lignocellulosic materials which are renewable, low cost and abundantly available ( Lang et al., 2002; Bjerre et al., 2006). These materials include crop residues, grasses, sawdust, wood chips, but also rice straw, wheat straw, corn straw and sugarcane bagasse which are the major agricultural wastes in terms of quantity of biomass available (Kim and Dale, 2004). However, the production of bioethanol from lignocellulosic wastes is not able to consume the whole raw material, leaving behind a large amount of residual lignocellulosic waste, whose further exploitation could be improved from the energetic and chemical-industrial point of view, especially for the lignin supply. Lignin is an extremely versatile, non-toxic and renewable material and it can be used, for example, as rubber intensifier, rubber packing, in composite materials, as a food additive, as a sequestering agent, blend, dispersants, binder and in other applications (Agrawal et al., 2014). Lignocellulosic residues from bioethanol production are mainly composed by cellulose, hemicellulose, pectin and lignin (Taiz and Zeiger, 2008). Cellulose and hemicellulose are macromolecules formed from different sugars, while lignin is an aromatic polymer synthesized from phenylpropanoid precursors. Cellulose is a linear polymer that is composed of D-glucose subunits linked by β-1,4 glycosidic bonds forming the dimer cellobiose; linear cellulose molecules are organised in parallel joined by hydrogen bonds (Taiz and Zeiger, 2008). Hemicellulose contributes to forming the cell wall by acting as an essential element to the concatenation of cellulose fibrils. It is formed from D-xylose, D-mannose, D-galactose, D-glucose, L-arabinose, 4-O-methyl-glucuronic, D-galacturonic and D-glucuronic acids. Sugars are linked together by β-1,4- and sometimes by β-1,3-glycosidic bonds (Kuhad et al. 1997, Ling-Ping et al., 2013). Pectins are heteropolymers present in the plant cell wall with not yet fully known structures and functions and they are formed by the main skeleton of α-1,4-D-galacturonic acid residues (Ochoa-Villarreal et al., 2012).

Many microorganisms are capable of degrading and utilizing cellulose, hemicellulose and pectins as carbon and energy sources, but fungi play an important role in the biotreatment of lignocellulosic wastes because of their typical

mycelial growth that allows the fungus to transport scarce nutrients such as nitrogen and iron to a distant nutrient-poor lignocellulosic substrate that constitutes its carbon source (Hammel, 1997). The fungal strains mostly known for lignocellulose degradation belong to the basidiomycetes (Bennett et al., 2002; Rabinovich et al., 2004). Little is known about the degradation mechanisms of lignocellulose by ascomycetes; most of the data are related to Trichoderma reesei and its mutants used for the commercial production of hemicellulases and cellulases (Esterbauer et al., 1991; Nieves et al., 1998; Jørgensen et al., 2003). Fungi can grow successfully on a wide variety of lignocellulosic residues such as cereal straws, soybean, cotton stalk, and almost any lignocellulosic substrate that has a substantial cellulose component (Quintero et al., 2006; Rani et al., 2008). In addition, fungi are able to degrade also different persistent environmental pollutants, such as chlorinated aromatic compounds, aliphatic and aromatic hydrocarbons and synthetic high polymers (Bennett et al., 2002). In fact, fungi possess a variety of extracellular enzymes with different specificities, useful to attack cellulose and lignin, but also to degrade recalcitrant compounds.

Cellulases, the enzymes responsible for the hydrolysis of cellulose, are composed of a complex mixture of enzymes with different specificities to hydrolyze β-1,4-glycosidic linkages bonds. Cellulases can be divided into three major enzyme activity classes: endoglucanases or endo-1-4-β-glucanase (EC 3.2.1.4), cellobiohydrolase (EC 3.2.1.91) and β-glucosidase (EC 3.2.1.21) (Goyal et al., 1991; Rabinovich et al., 2002a, b). Endoglucanases, often called also carboxymethylcelluloses, are thought to initiate attack randomly at multiple internal sites in the amorphous regions of the cellulose fibre which opens sites for a subsequent attack by the cellobiohydrolases (Lynd et al., 1991; Deobald and Crawford, 1997). Cellobiohydrolases remove monomers and dimers from the end of the glucan chain. β- glucosidase hydrolyses glucose dimers and, in some cases, cellulose oligosaccharides to glucose. Similar enzymes are involved in cellulose and hemicellulose biodegradation. Xylan is the main carbohydrate found in hemicellulose. Its complete degradation requires the cooperative action of a variety of hydrolytic enzymes, of which the most important are the endo-1,4-b-xylanases (EC 3.2.1.8) and 1,4-β-xylosidase (EC 3.2.1.37). These enzymes degrade xylan to a short chain of xylo-oligosaccharides and then, for the complete

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hydrolysis to xylose monomers, xylanases cooperate with b-xylosidases (Jeffries, 1994; Krengel and Dijkstra, 1996). The degradation of pectins requires a series of hydrolytic enzymes to completely degrade the polymer. Pectinases are an extensive family of enzymes able to attack the various and complicated polygalacturonic structures. Pectinases are classified according to the type of binding they hydrolyze in the pectin structure: polygalacturonase (EC 3.2.1.15) that hydrolyzed the α-1,4 bonds between the units of galacturonic acid, pectinesterase (EC 3.1.1.11) that remove the methyl group of pectins by hydrolyzing the bond carboxyl ester of galacturone and pectinase (EC 4.2.2.10) that hydrolyze the α-1,4 bonds between the galacturonic acid producing units unsaturated galatturonates or methyl galatturonates (Rodrigo de Souza, 2013). Pectinases are most commonly extracted from Aspergillus niger, an ascomycete (Singh Jayani et al, 2005). One of the most used methods to analyse the degradative potential of fungal strains on lignocellulosic substrates is the study of the concentration of reducing sugars performing 3,5-dinitrosalicylic acid (DNS) test. The concentration of reducing sugars is an index of the fungal degradation of the cellulosic components of lignocellulosic residues. DNS test measures the reduction of the reducing sugars concentration in a supernatant. DNS is an aromatic compound that reacts with reducing sugars and other molecules to create 3-amino-5-nitrosalicylic acid, a reduced form of DNS. This acid effectively absorbs light at 540nm (Miller, 1959).

The aim of this work was to evaluate the activity of two fungal strains (Trichoderma asperellum 1020 and Paecilomyces variotii 1030) on a bioethanol production waste material rich in lignocellulosic residues (LRR) in order to obtain purified lignin that could be used in biotechnological applications. The degradation and purification abilities of the selected fungi were verified by the degradation of reducing sugars and the activity of the different enzymes involved.Materials and MethodsLignin rich residue substrate

The substrate used in this study was a lignin-rich residue (LRR), which is a waste material derived from the industrial production of bioethanol. In Table 1 is reported its composition made by the University Politecnico di Milano (Italy). It can be noted that, despite the material deriving from the bioethanol extraction processes, polysaccharides such as glucans and xylans are also present in large

quantities. Before being used, LRR was manually fragmented to be more easily accessible to the fungal species and then sterilized for 2 h in the autoclave at 121°C.

Table 1. Lignin rich residue composition

Water content 49,26 % Initial sample composition (dry wight)

Glucose 0,05%Xylose 0,06%Glucose oligomers 0,10%Xylose oligomers 0,14%Insoluble glucans 20,74%Insoluble xylans 3,11%Klason lignin 45,51%Acetic acid 0,22%Other insoluble 21,87%

Strains and inoculum preparation The tested strains used in this study are T.

asperellum 1020 and P. variotii 1030 and were obtained from the Collection of the Mycology Laboratory of the University of Pavia (Italy) (Table 2) and used in this work for their known abilities to grow on recalcitrant substrates like petroleum and oils.

Table 1. Isolation substrates of the tasted strains

Species OriginT. asperellum 1020 Plant hydroponic cultureP. variotii 1030 Pure lignin purified by chemical

methods

The fungi were preserved in water supplemented with 15% glycerol at -80°C and revitalized from frozen stocks by cultivation on Malt Extract Agar for microbiology (MEA) (Sigma-Aldrich, St. Louis, USA) for 7 days at 25°C. After this time, all the mycelium grown on the plate was collected in a tube containing water-agar 0,15% to prepare the fungal suspension. To evaluate the degradative capacity of the fungal strains, 100 mL flasks were prepared containing 50 mL sterile distilled water and 5 g of LRR. One mL of fungal suspension was added in the flasks except for the ones used as a control. To ensure statistical significance, the test was conducted in a triple copy. For all the test period (2 months) the flasks were maintained at 25°C. For the DNS test, the flasks were monitored every month and subsequently, the DNS test was repeated to evaluate the trend of

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reducing sugars in the first 9 days of fungal activity. The enzymatic tests were performed extracting the supernatant, after one week from the inoculum, from the same flasks.DNS test for measuring the concentration of reducing sugars

DNS was prepared in laboratory following the protocol reported by Miller (1959): distilled water – 1.416 L; 3,5-Dinitrosalicylic acid – 10.6 g; NaOH – 19.8. The components are dissolved and then added: Rochelle salt - 306 g; phenol – 7.6 g; sodium metabisulfite – 8.3 g (Ghose, 1987). To perform the test, 40 μl of supernatant was taken from each flask and insert in a 0.5 mL tube with 40 μl of DNS. The mix was heated at 95°C for 5 minutes in a thermocycler. The concentration of reducing sugars was evaluated with the use of a spectrophotometer, measuring the absorbance at two different wavelengths: 540 nm, which measures the absorbance of the reduced form of the DNS, and 700 nm, which measures the absorbance of the turbidity of the sample. To obtain a more precise measurement, the value from the reading at 540 nm was subtracted to the one from 700 nm reading.

Study of lignocellulolytic enzyme activity The study of enzymatic activities was carried

out in collaboration with the Laboratory of Plant Molecular Biology of the University of Pavia. After 1 week from the inoculum, 5 mL of the supernatant was transferred in 10 mL tubes and centrifuged at 3000 rpm for 5 min to divide the liquid part, in which the enzymes should be, from possible residues of LRR or spores and mycelium. The liquid part was transferred into new tubes and the supernatant was diluted before being tested.

Endo-β-1,4-glucanase (EC 3.2.1.4) assay Endo-β-1,4-glucanase was performed using

a modified protocol of Islam and Roy (2018). 40 μl of the diluted sample (1:2) were placed in 0.5 mL microtubes with 40 μl of 1% CMC (Sigma-Aldrich, St. Louis, USA) in 1N citrate buffer (pH 5.0) as substrate. The mix was incubated at 45°C for 30 minutes in a thermocycler. 80 μl of DNS was added and the mix is reinserted into thermocycler for 5 min at 95°C. The CMCase activity was assessed measuring the absorbance at 540 nm and 700 nm with a spectrophotometer. Two replicates of the same sample were analyzed. One unit of CMCase activity in 1 mL of culture broth was defined as the amount of enzyme that catalyzed the release of one micromole of CMC per minute.

β-glucosidase (EC 3.2.1.21) assay. β-Glucosidase was performed using a modified

protocol of Zhang et al. (2018). 40 μl of the diluted sample (1:2) were placed in 0.5 mL microtubes with 75 μl of 1% para-nitrophenyl-β-D-glucopyranoside (p-NP-β-gluc) in water as substrate. The mix was incubated at 25°C for 30 minutes in a thermocycler. 100 μl of NaOH 0.1 M were added. The β-glucosidase activity was assessed measuring the absorbance at 405 nm with a spectrophotometer. Two replicates of the same sample were analyzed. One unit of β-glucosidase activity in 1 mL of culture broth was defined as the amount of enzyme that catalyzed the release of one micromole of p-NP-β-gluc per minute.

β-1,3-glucanase (EC 3.2.1.58) assay.β-1,3-Glucanase was performed using the

DNS method by El-Katatny et al. (2001). Forty μl of the diluted sample (1:2) were placed in 0.5 mL microtubes with 40 μl of 0.75% laminarin (Sigma-Aldrich, St. Louis, USA) in sodium acetate (NaAc) (pH 5.0), as substrate. The mix was incubated at 45°C for 30 minutes in a thermocycler. 80 μl of DNS were added and the mix was reinserted into thermocycler for 5 min at 95°C. The β-1,3-glucanase activity was assessed measuring the absorbance at 540 nm and 700 nm with a spectrophotometer. Two replicates of the same sample were analyzed. One unit of β-1,3-glucanase activity in 1 mL of culture broth was defined as the amount of enzyme that catalyzed the release of one micromole of laminarin per minute.

Endo-1,4-β-xylanase (EC 3.2.1.8.) assay.Endo-1,4-β-xylanase was performed using

the DNS method by Royer and Naka (1989). Forty μl of the diluted sample (1:2) were placed in 0.5 mL microtubes with 40 mL of 1% xylan in 0,05M citrate-HCl buffer (pH 4.8) as substrate. The mix was incubated at 50°C for 30 minutes in a thermocycler. Eighty μl of DNS were added and the mix was reinserted into thermocycler for 5 minutes at 95°C. The xylanase activity was defined as the measure of the absorbance at 540 nm and 700 nm with a spectrophotometer. Two replicates of the same sample were analyzed. One unit of xylanase activity in 1 mL of culture broth was defined as the amount of enzyme that catalyzed the release of one micromole of xylose per minute.

Pectinase (EC 3.2.1.8) assayPectinase was performed using DNS method

by Oumer and Abate (2018). Forty μl of the diluted sample (1:2) were placed in 0.5 mL microtubes with

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40 μl of 1% pectin (Sigma-Aldrich, St. Louis, USA) in 0.1 M phosphate buffer (pH 7.5) as the substrate. The mix was incubated at 50°C for 10 minutes in a thermocycler. Eighty μl of DNS were added and the mix was reinserted into thermocycler for 10 min at 95°C. The pectinase activity was assessed measuring the absorbance at 540 nm and 700 nm with a spectrophotometer. Two replicates of the same sample were analyzed. One unit of pectinase activity in 1 mL of culture broth was defined as the amount of enzyme that catalyzed the release of one micromole of pectin per minute.Statistical analysis

Statistical analyses were performed using Microsoft Excel software (Microsoft Office 2013) and SigmaStat 4.0 (Systat Software, Inc.). The means and the standard deviation of the mean were calculated., and then the Analysis of Variance (ANOVA) an, in some cases, the Student t-test were applied to the data. A P-value of <0.05 was considered statistically significant.

Results and DiscussionBiological treatment of LRR: DNS test

The DNS test allowed to observe the degradative activity of T. asperellum 1020 and P. variotii 1030. The concentration of reducing sugars decreased considerably between the time of the inoculation and the following 2 months, in all the samples (Fig. 1 and 2).

This is an indication that the sugars were used by fungi to carry out their metabolic activities. T. asperellum 1020 was the most efficient strain, with a reduction of reducing sugars of about 70% in the first month, while P. variotii 1030 caused a 55% reduction. The increased concentration of reducing sugars in the control (just water and LRR with no fungal strain) after 1 and 2 months is a normal

consequence of their release in the supernatant by the lignocellulosic biomass. The tested strains reduced the concentration of sugars by 53% compared to the control, in the first 4 days from the inoculum, until reaching a value close to 0 (Fig. 3).

On day 2 the curve of T. asperellum 1020 showed a significantly different trend compared to the curves of P. variotii 1030 (P<0.05). This behaviour could indicate a greater efficiency of this strain related to the growth condition on a liquid substrate, coherently with the isolation conditions (hydroponic condition). The maximum activity of these fungi in the reduction of reducing sugars took place in the first 4 days. Looking at the figures (Fig. 1; Fig. 2; Fig. 3) in all the samples treated the concentration of reducing sugars approaches a value close to 0 without ever reaching it. This result could be due to the difficulty of the tested strains to completely degrade the cellulosic compounds contained in the LRR biomass or for the release in the supernatant of compounds containing an aldehyde or ketone group detectable by the DNS test.

Study of the lignocellulosic enzymatic activity of the strains

In all the samples, there was a significant (P<0,05) increase, up to the 600%, in enzyme activity in the presence of LRR compared to the control (Table 3). This result indicates that fungi were stimulated in producing lignocellulosic enzymes in presence of LRR. β-Glucosidase and xylanase are the most produced enzymes by the two strains. in P. variotii 1030, the β-glucosidase activity increased of the 421% and the xylanase of 608%; in T. asperellum 1020 the β-glucosidase activity increased of the 267% and the xylanase of 609%.

Pectinase also showed significant results (P<0.05), although its activity was lower than the

Fig. 1. Concentration of the reducing sugar in two months: T. asperellum 1020 in water and LRR

Fig. 2. Concentration of the reducing sugar in two months: P. variotii 1030 in water and LRR

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two above mentioned enzymes. In fact, pectinase activity increased of the 375% in and of the 175% T. asperellum 1020.

Our results are in accordance with the data reported in literature. Several studies showed that Trichoderma and Paecilomyces genera are excellent producers of cellulolytic enzymes (Sarkar et al., 2012; Tosi et al., 2014). Trichoderma species can degrade cellulose by a synergistic combination of different cellulase activities (Reese, 1956; Mandels er at., 1971). T. viride MMS 3, T. reesei, T. virens (Ang et al., 2015), T. asperellum MR1, T. virens UKM1, and T. tubingensis NKBP-55 (Prajapati et al., 2018) have been studied for their production of cellulases and xylanases. T. viride VKF3 achieved a high level of xylanase by utilizing coconut oil cake as a substrate. T. reesei Rut C-30 is the most well-known Trichoderma strain producing several xylanases and cellulases (Peterson and Nevalainen, 2012) but Marx et al. (2013), in a comparison study between the lignocellulolytic enzyme profiles of T. asperellum S4F8 and T. reesei Rut C30, showed

that S4F8 had significantly higher hemicellulase and β-glucosidase enzyme activities. Paecilomyces spp. are indicated as good producers of cellulase

enzymes (Paganini Marques et al., 2018; Ingle, 2019). In their work Hussain et al. (2012) showed that P. variotii has the ability to produce high activities of all three main components of cellulase in low cost substrate and how physical and chemical factors, influenced their production.Conclusions

In conclusion, measuring the concentration of reducing sugars in the LRR samples, T. asperellum 1020 showed the best performance, with reducing sugars rate decreasing by about 70%. The two strains, however, seem to use almost all the available material in the first 4 days from the inoculum. The quantification of enzymes secreted confirmed that the two strains act on the specific components of LRR. The obtained results indicate that LRR biotransformation by Trichoderma and Paecilomyces has interesting potential and that lignin purification by this method, with the

Table 3. Concentrations of the tested enzymes determined with spectrophotometric protocols

β-1,3-glucanase endo-β-1,4-glucanase β-glucosidase Pectinase (U/ml) Xilanase (U/ml)

Water Water + LRR Water Water +

LRR Water Water + LRR Water Water

+ LRR Water Water + LRR

T. asperellum

1020Traces† Traces 0,015 ±

0,0020,026 ±0,001

1,00 ±0,02

*2,67 ±0,22

0,04 ±0,005

*0,07 ±0,003

0,044 ± 0,011

*0,268 ± 0,055

P. variotii 1030 Traces Traces Traces 0,029 ±

0,0011,46 ±0,11

*6,16 ±0,02

0,024 ±0,01

*0,09 ±0,004

0,035 ± 0,002

*0,213 ± 0,078

†Activities lower than 0.02 U/ml, The strains grew in water only and in water + LRR.

Fig. 3. Concentration of the reducing sugar in 9 days: T. asperellum 1020 and P. variotii 1030 in water and LRR

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appropriate improvements, could reach the same purification percentage obtained with traditional chemical methods.

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Solid-State Fermentation using a Strain of Trichoderma asperellum Improves the Saccharification of Rice Straw

Sonia Accossato1§*, Mirco Granata2, Matteo Faè1, Rino Cella1, Solveig Tosi2, Anna Maria Picco2

1 Department of Biology and Biotechnology, University of Pavia, via Ferrata 9, Pavia, Italy2 Laboratory of Mycology, Department of Earth and Environmental Sciences, University of Pavia, via Epifanio 14, Pavia, Italy§ present address: Laboratory of Plant Physiology, University of Neuchâtel, Rue Emile-Argand 11, 2000 Neuchâtel, Switzerland

AbstractRice straw is a cheap and widely available agro-waste biomass that can be used to generate renewable

biofuel. However, due to its composition, it is particularly recalcitrant to enzymatic degradation. Here, prior to enzymatic hydrolysis, biological pre-treatment of rice straw for saccharification by solid-state fermentation (SSF) was performed using a new isolate of Trichoderma asperellum called UNIPV1. More specifically, the study aimed to investigate the effect of adding the fungus to rice straw on the temporal activity of secreted enzymes, and reducing sugar formation. As excepted, under long-term solid-state fermentation (SSF), depolymerizing enzymes such as xylanases and exo/endo-cellulases were secreted by Trichoderma asperellum UNIPV1 during its growth. T. asperellum was an excellent producer of cellulolytic enzymes; a significant peak in reducing sugars occurred between 10-20 days. Trichoderma initially showed a high preference for the secretion of xylanases to degrade the linear polysaccharide beta-1,4-xylan into xylose. Afterwards, endoglucanase and exoglucanase enzymes were secreted to complete the hydrolytic activity on the substrate. This result is consistent with the trend of total protein accumulated in the secretome and the fungal metabolic activity measured by CO2 production. Overall, our findings suggest that a short fungal pre-treatment of rice straw might be useful to begin the degradation of cell-wall polymers, and can therefore effectively improve saccharification. That said, further work is required to improve the fungal pre-treatment, since alone does not entirely complete the degradation of lignocellulosic insoluble material.Keywords: rice straw, solid-state fermentation (SSF), Trichoderma, saccharification, cell-wall degrading enzymes.

РезюмеОризовата слама е евтина и широко достъпна отпадъчна биомаса от селското стопанство, която

може да се използва за генериране на възобновяеми биогорива. Поради състава си, обаче, тя е трудно податлива на ензимно разграждане. В настоящото изследване, преди ензимната хидролиза на оризовата слама се извършва предварителна биологична обработка с цел озахаряване в условията на твърдо фазова ферментация (ТФФ), за която се използва новоизолиран щам от Trichoderma asperellum, наречен UNIPV1. По-конкретно, изследването има за цел да проучи ефекта от добавянето на гъбата към оризовата слама върху активност на секретираните ензими и образуването на редуциращи захари. Както се очаква, при дълговременна ТФФ, деполимеризиращите ензими като ксиланази и екзо/ендоцелулази се секретират от T. asperellum UNIPV1 по време на растеж. T. asperellum е високоефективен продуцент на целулолитични ензими, а пик на количеството редуциращи захари настъпва между 10 и 20 ден. В началото на процеса, новият щам Trichoderma показва ускорена секрецията на ксиланази, за да разгради линейния полизахарид -1,4-ксилан в ксилоза. След това се секретират ендо- и екзо-глюканазните ензими, които завършват хи-дролитичното действие върху субстрата. Този резултат е в съответствие с установената тенденция на нат-


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рупване на общия белтък в секретомата, както и с метаболитна активност на щама, измерена чрез ко-личеството на CO2. В заключение, установените от нас резултати предполагат, че кратката предвари-телна обработка на оризовата слама с гъбния щам може да бъде полезна за започване разграждането на полимерите в клетъчната стена и следователно може ефективно да подобри озахаряването. Въпре-ки това, необходимо е по-нататъшно оптимизиране на предварително третиране с гъби, тъй като този процес не може да осъществи пълно разграждане на неразтворимите лигноцелулозни материали.

IntroductionLignocellulosic biomasses such as wheat

bran, sugarcane bagasse, corncob, and rice straw constitute an extremely abundant and inexpensive global resource. Via the degradation of their cellulosic material by microbial conversion, a wide variety of value-added products can be derived (Elisashvili et al., 2009), including renewable fuels, chemical feedstocks, and raw material for chemical reactions in an environmentally friendly fashion. Rice straw is a particularly cheap and underutilized agro-waste biomass. For instance, Italy is the largest European rice producer with 1.5 million tons annually, but only approximately 30% of the resultant rice straw is used in industrial processes (Ministry of Agriculture, Food and Forestry of Italy, 2014).

Such underutilization of agro-wastes is largely due to certain persistent practical problems. In particular, the lignocellulosic biomass in its natural form is a tough feedstock for hydrolysis due to the crystallinity of cellulose, presence of hemicellulose, and lignin in the plant material (Hu et al., 2013). Biomass pre-treatment processes are therefore necessary to reduce the paracrystalline cellulose structure and to make cellulose more available to the enzymes by removing hemicellulose and lignin (Cai et al., 2008). A number of different pre-treatment methods have been extensively investigated, including steam explosion (Nakamura et al., 2001), organosolv extraction (Pan et al., 2005), subcritical and supercritical water treatment (Schmieder et al., 1999; Yoshida et al., 2004), biological pre-treatment with white-rot fungi (Hatakka et al., 1983; Itoh et al., 2003), as well as various physical and thermomechanical processes (Chahal et al., 1999). However, biological pre-treatments excepted, most approaches have expensive energy requirements, albeit with some variability depending on the complexity (Wingren et al., 2004). Presently, the

efficient depolymerization of lignocellulosic using biological pre-treatment is considered to be a safe and environmentally friendly method of breaking down the lignin and overcoming the resistance of cellulose to hydrolytic cleavage. To enhance susceptibility to enzymatic hydrolysis and thus digestibility, various pre-treatments have been exploited white-rot fungi over the years, mainly considering fungi belonging to Basidiomycota.

Amongst the many promising micro-organisms used for biological pre-treatment, Trichoderma, a genus of filamentous fungi belonging to the phylum Ascomycota (Druzhinina et al., 2016) has been extensively studied due to its diverse physiological characteristics and biotechnological applications. Within the species, Trichoderma asperellum stands out as a particularly versatile organism with remarkable degrading abilities (Zafra and Cortes-Espinosa, 2015). This fungus is commonly found in soils worldwide, and it is characterized by rapid growth and high rate of sporulation (Eveleigh, 1999). Interestingly, the secretome of T. asperellum comprises a wide range of potential useful enzymes, such as cellulases, pectinases, chitinases, and laminarinases (Eveleigh, 1999). In particular, cellulases (exo/endo-glucanases and xylanases) are a family of enzymes that act at several stages on the cellulose: firstly, through endocellulases cutting randomly inside the macromolecule, in order to generate a new end of the polysaccharide chain; secondly, these ends are subjected to the action of exocellulases that make disaccharides available. Ultimately, beta-glucosidases act on these products releasing glucose. Most fungi prefer to use xylanases to degrade hemicellulose and consequently make cellulose more accessible to the enzymatic action of cellulases (Longoni et al., 2015).

Another characteristic of the Trichoderma species is that, thanks to its hyperparasitic nature, it is well-known for its antagonistic action against pathogenic fungi, as well as for its potential use in the production of antibiotic substances and enzymes (Thrane et al., 1997; Dengkolb et al., 2003; Osbourn, 2010). For this reason, considerable attention has been devoted to the use of Trichoderma as biological control agents of plant diseases, with many commercial formulations available on the market. Therefore, its environmental release is not expected to pose any hazard.

Processing rice straw waste presents a particular challenge. Of its total dry weight, 40% is given by cellulose, 18% by hemicellulose, and

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5.5% lignin (Takagi, 1987). This constitution, along with high silica content, makes it particularly recalcitrant to degradation. In light of the high enzymatic activity of the T. asperellum secretome, the aim of this paper is to evaluate how solid-state fermentation (SSF) using this fungus could contribute to the pre-treatment of rice straw with a view to its subsequent usage for biogas production by anaerobic biodigestion. Three classes of enzymes were considered: i) xylanases (EC 3.2.1.8 Endo-1,4-β-xylanase), which catalyzes the hydrolysis of β-1,4 bonds of xylan backbone in xylose thus breaking down hemicellulose, one of the major components of plant cell walls (Rasala, 2012; Gupta, 2000); ii) endoglucanases (EC 3.2.1.4), which break the intra-chain beta bonds of cellulose and belongs to the family of Hydrolases (Adlakha, 2011); and iii) exoglucanases (EC 3.2.1.91), which hydrolyze 1,4-beta-D-glucosidic bonds releasing cellobiose from both ends of the glucan chain.Materials and MethodsFungal strain

A strain of T. asperellum UNIPV1 was isolated as a hyperparasite of a Fusarium strain growing on a freshly harvested sample of rice seeds that were collected in northern Italy in March 2013. This strain was selected on account of its heavy growth on cellulosic materials, seeds, and agro-residues (demonstrated by previous experiments in our laboratories). The fungus was maintained in pure culture on Malt Extract Agar (MEA, Oxoid, UK) in slant tubes and stored under mineral oil at room temperature (25°C) in the mycological collection of Pavia University. It was assigned the code T. asperellum UNIPV1. Its optimum temperature for growth was detected at 25°C. The taxonomical identification was based on morpho-dimensional analysis following dichotomous key reported by Samuels et al. (1999) and Bisset (1991a, 1991b). The molecular characterization was performed by amplifying and analysing the sequences of Internal Transcribed spacer gene 1 and 2 (ITS) and translation elongation factor 1-alpha encoding gene (tef1). Identifications were made using the BLAST interface in TrichOKEY and TrichoBLAST sequence of the gene encoding for Elongation Factor-1α (eEF1α) (Druzhinina et al., 2005; Kopchinskiy et al., 2005). The nucleotide alignment of the obtained eEF1a sequence in NCBI (National Center for Biotechnology Information), Mycobank and TrichoMARK (Trichoderma genus-specific database) confirmed that the strain UNIPV1 belongs to T. asperellum.

Rice straw biomass

Rice straw collected in the Lombardy region (northern Italy) was air-dried and stored at room temperature for 3-6 months before starting the experimental phase. Subsequently, it was milled and sub-samples of 30 g were put in polypropylene gas-permeable bags with filters (SacO2, Microsacs, Belgium) to allow gas exchange. They were then wetted with 30 mL of sterile distilled water. Before proceeding with the experiment, the sub-samples of rice straw were pre-treated with a strong sterilization in autoclave at 120°C for 50 minutes, which was repeated after 24 hours. The materials were then stored at room temperature. Inoculum preparation and solid-state fermentation (SSF)

UNIPV1 was grown on Potato-dextrose-agar (PDA; Oxoid) medium in sterile Petri dishes for 10 days in a dark growth chamber at 25°C. A suspension of actively growing mycelium was prepared to initiate fermentation. A mother suspension was obtained by gently excising 10 disks of 10 mm diameter of a 10-day-old colony and putting them in a sterile flask with 100 mL of agar gel prepared with sterilized water and 0.02% of agar. The suspension was shaken on a magnetic stirrer for 10 min. A volume of 30 mL of the suspension was then added to each bag containing sterile rice straw. Subsequently, for the SSF process, the plastic bags were sealed and kept in the dark growth chamber at 25°C for 0, 10, 15, 20 and 45 days. Three replicates were performed for each time condition, and three untreated samples, constituted by rice straw with 30 mL of sterile distilled water without the fungus, represented the control. All the experimental procedures were carried out under the flow cabinet to maintain aseptic conditions.Biomass pre-treatment and enzymatic activity assay

Cellulolytic enzymes, i.e. xylanases and exo/endo-cellulases, derived from the secretome of T. asperellum UNIPV1 were obtained during its growth on rice straw under SSF. A sub-sample of 2.5 g from each treated sample (i.e. rice straw colonized by UNIPV1), was put in a 100 mL flask and subjected to 15 mL of extraction buffer (100 mM sodium acetate buffer, pH 5 – ratio 1:1), kept for 24 hours at 4ºC in an orbital shaker at 180 rpm. The entire volume of liquid derived by the rice straw pre-treatment (13-14 mL) was collected in 50 mL falcons and subjected to a double centrifugation for 20 min at 4ºC at 4,000 rpm. The supernatant of each falcon was collected, filtered (pore size 0.2 µm), and stored at -20ºC. The enzymatic activity assay was

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performed on both untreated and treated rice straw. It was carried out in PCR tubes in a final volume of 180 µL, using 10 µL of total enzymatic extract derived from the filtered broths of the UNIPV1 secretome. Three different substrates were utilized to check the different enzymatic activities: 1% xylan from beechwood (w/w) (Sigma-Aldrich, Germany) for xylanase activity, 1% carboxymethyl-cellulose (CMC) (Panreac, Spain), for endoglucanase activity, and 4% microcrystalline cellulose (MCC) (Acros Organics, USA) for exoglucanase activity. The analyses were performed in triplicate. To determine enzymatic activity, aliquots of 10 µL of total enzymatic extract were added to 10 µL of synthetic substrate and diluted in a final volume of 70 µL sodium acetate buffer (100 mM, pH 5). Afterwards, reactions were incubated for 60 min at 55ºC in a thermal cycler (Eppendorf). At regular intervals (10, 15, 20, 25, 30, 35, 40, 45, 50, 55 and 60 mins), reactions were performed by analyzing samples to evaluate the optimum reaction time between enzyme and substrate. Sterilized water was used as a control. After incubation, samples were boiled for 5 min to inactivate the enzymes. The enzymatic activity was measured by adding an equal volume (90 µL) of 3,5-dinitrosalicylic acid (DNS) reagent to the samples and then boiling the mixture at 95ºC for 5 min in a thermal cycler, following the method described by Ghose (1987). The reactions were then transferred into a 1 mL cuvette for spectrophotometric analyses, adding 180 µl of water. Absorbance of the samples was read at 540 nm (corresponding to absorbance peak of reacted DNS) and 700 nm (the absorbance peak of the sample turbidity). The net absorbance of each sample was calculated by subtracting the absorbance value at 700 nm from that at 540 nm. Using a calibration curve developed with known dilutions of glucose, absorbance values were converted to mg/mL glucose equivalents. Enzymatic activity was expressed in U/mL, where one Unit is the amount of enzyme that releases 1 µmol of reducing sugar equivalent per minute from the substrate under the assay conditions. Saccharification and protein quantification

Saccharification experiments were required to determine the hydrolytic ability of enzymes produced by UNIPV1 growing on lignocellulosic agro-wastes. The method proposed by Jeya et al. (2009) was followed. Tests to evaluate the degradation of rice straw were carried out at different times of incubation with the fungus, respectively at 0, 10, 15, 20 and 45 days, both on treated (with

UNIPV1) and untreated rice straw. Time courses were followed by two different assays: the DNS assay (Ghose, 1987) to evaluate the reducing sugar ends, whereas samples from three biological replicates were analyzed in triplicate, and the oxygraphic method specific for glucose. In parallel, proteins were quantified using Bicinchoninic acid (BCA) from protein assay kit (Sigma-Aldrich), followed by spectrophotometrical analysis at λ = 562 nm. Fungal metabolic activity measured by CO2 production

In order to monitor the metabolic activity of UNIPV1 growing on rice straw, CO2 production was evaluated during the course of the experiments. The CO2 flux (µmol m-2 s-1) was measured with portable CO2 analyser (ADCPro-sd with sample chamber). This device consists primarily of a dual-channel, non-dispersive infrared (NDIR) sensor (TelaireT6615) (General Electric Company, Billerica, MA, USA), an aspirator pump (FM1001; Chengdu, China), and a display screen. The aspirator pump pulls air from the sample through silicone pipes (2×4 mm) to the sensor; the maximum gas flow through the CO2 sensor was 50 mL min–1. After 3 min, the CO2 concentration was recorded. Throughout, the data were directly collected from the plastic bags containing rice straw and the fungus growing on it and compared to the control.Fiber content analysis

In order to evaluate the effect of SSF on the mobilization of recalcitrant polymers such as cellulose and lignin, a gravimetric analysis at different times following fungal addition was performed. After 10, 15, 20, and 45 days, the resulting pre-treated rice straw substrate was placed in a 70°C dryer overnight to prevent the growth of spores. Subsequently, the dried substrate was transferred into pre-weighed crucibles for fiber content analysis, and solvent extraction was used to separate the soluble from insoluble fiber according to the procedures proposed by Official Methods of analysis of AOAC International, 16th Edition, vol. II 1995.

Results and DiscussionIsolation and identification of fungal isolate T. asperellum UNIPV1

Morpho-dimensional and biomolecular analysis confirmed that the fungal strain UNIPV1 belongs to T. asperellum (Samuel et al., 1999), a cosmopolitan soil borne species with teleomorph in Hypocrea (Fungi, Dicarya, Ascomycota, Pezi-

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zomycotina, Sordariomycetes, Hypocreomyceti-dae, Hypocreales, Hypocreaceae). T. asperellum is known to be an outstanding environmental oppor-tunist with behaviour ranging from saprotrophy to biotrophy. It is also able to antagonize other fun-gi by a necrotrophic hyperparasitism. Therefore, this species is utilized as a biological control agent against a wide spectrum of plant-pathogenic fun-gi and oomycetes (Tondje et al., 2007; Mbarga et al., 2012; El_Komy et al., 2015). T. asperellum has also been particularly investigated for its ability to stimulate the plants defence reactions against plant pathogens (Segarra et al. 2007; Viterbo et al., 2010; Yoshioka et al., 2011; Herrera-Télle et al., 2019). Beside their biotrophic nature, species of Tricho-derma are particularly fast decomposers of cellu-lose rich substrates. This feature can be exploited for many industrial perspectives such as the pro-duction of biofuels. Preliminary experiments in our laboratories (Picco A. M., unpublished data) suggested that the strain UNIPV1 may strongly de-grade rice straw. Based on this, we decided to spe-cifically investigate its possible utility to improve saccharification of rice straw in biofuel industry. Secretome analysis

The time course of xylanase and cellulase secretion was analyzed during SSF over a period of 45 days. The results of this analysis, which are plotted in Fig. 1, show that xylanases were initially

rapidly produced, reaching a peak at day 15 (25 000 U/g IDS, Initial Dry Substrate), decreasing thereaf-ter to a very low level by day 45.

Endoglucanase and exoglucanase activities were assessed, respectively, using CMC and MCC substrates. Endoglucanase showed a specific activ-ity of 2 600 U/g IDS after 10 days due to fungal addition which remained constant until day 20, declining thereafter. On the contrary, exoglucanas-

es slowly increased until day 20 (2 000 U/g IDS), reaching a specific activity of 12 000 U/g IDS at day 45, possibly due to further process oligosac-charides. A similar trend was observed in the ac-cumulation of Trichoderma exoglucanases in batch fermentation by Elshafei et al. (2014). The pattern of enzymatic activity clearly reveals that the fungus uses xylanases first, probably to facilitate the ac-cess of cellulases to cellulose. The trend observed in our experiments is consistent with that reported previously in relation to Chaetomium globosum, whereby xylanases were the first secreted enzymes to act upon poplar wood, followed by the action of endo- and exo-cellulases (Longoni et al. 2015).

The kinetic of the total soluble protein release in the secretome, meanwhile, is reported in Fig. 2. The maximum protein concentration was observed at day 20 and decreased thereafter. The pattern of total protein secretion is in accordance with cell-wall degrading enzyme activities. The release of proteins observed in the untreated sample is proba-bly due to maceration.Respiratory activity during SSF

The pattern of enzyme secretion was also consistent with the fungal respiratory activity as assessed by CO2 evolution during the SSF of rice straw. As shown in Fig. 3, the peak of CO2 occurred between 10 and 20 days, before levelling off at a slightly lower plateau value.

Reducing sugar production during SSFFigure 4 illustrates the time course of reduc-

ing sugar production, assayed by DNS, following the SSF of rice straw by T. asperellum. Total re-ducing sugars produced by the saccharification of rice straw polymers reached a peak between 10 and 15 days from the beginning of SSF. Between day 20 and the end of the experiment, these val-ues decreased from 0.3 mg/mL to 0.1 mg/mL. Con-

Fig. 1. Time course of enzymatic secretion of T. asperellum UNIPV1 grown on milled rice straw

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versely, CO2 production remained high from day 20 onwards, suggesting that from this point onwards all reducing sugars released were uptaken by the fungus for growth.Gravimetric analyses of biomass deconstruction

Results of gravimetric analysis reported in Fig. 5 show that in the sample treated with T. as-

perellum, the amount of soluble fibers started to significantly decrease (P < 0.05) at day 15 of SSF, and then continued to decline until day 45 when a value equal to one third of the initial soluble fib-er weight was detected. In contrast, the decrease in the weight of insoluble fibers was much more de-layed, with a significant decrease (P < 0.05) with

Fig. 4. Time course of reducing sugar accumulation during SSF of milled rice straw inoculated with T. asperellum UNIPV1

Fig. 3. Rate of CO2 evolution during SSF of T. asperellum UNIPV1 on milled rice straw

Fig. 2. Time course of total protein accumulation in the fungal secretome during SSF of milled rice straw

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respect to the previous time point occurring only at day 45. This finding is most likely due to an in-itial preference of T. asperellum for soluble fibers, readily available for the fungal metabolism, while the more recalcitrant insoluble fibers were degrad-ed only subsequently, when simple sugars were no longer available.

ConclusionIn this work, enzymatic tests were performed

via the inoculation of T. asperellum UNIPV1 on sterile rice straw. The results revealed that in treated straw, i) a significant production of CO2 occurs, ii) peak production of enzymes occurred between 10 and 20 days after inoculation, and iii) an increase in the total free reducing sugars of rice straw occurred alongside a decrease in the soluble component. These findings reveal how T. asperellum UNIPV1 can degrade cellulose and hemicellulose and are consistent with those of Bech et al. (2015). Overall, we conclude that adding the fungus to rice straw may lead to the formulation of "bioactive agro-ma-trices” that are more easily degradable. According-ly, T. asperellum UNIPV1 seems to be an excellent candidate for future practical applications. Howev-er, use of the fungal pre-treatment alone does not appear to be sufficient to fully complete the deg-radation of lignocellulosic insoluble material, sug-gesting a need for further research in this area.

Acknowledgements This work was supported by a grant from

Lombardy Region and Ministry of Education, Uni-versity and Research (Italy), D.d.u.o. 29 luglio 2011 - n. 7128. We thank Mr. James Thornton for proofreading.

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Antimicrobial Susceptibility/Resistance of Escherichia coli among the Outpatients with Urinary Tract Infections in Mostar

Mufida Aljicevic*, Amina Omerika, Velma Rebic, Sabina Mahmutovic, Amila AbduzaimoviciInstitute of Microbiology, Faculty of Medicine, University of Sarajevo, Sarajevo, Bosnia and Herzegovina

AbstractUrinary tract infections (UTIs) are considered to be the most common bacterial infection. Escherichia

coli is the most common uropathogen in uncomplicated upper and lower urinary tract infections (70–95% of cases). The aim of this study was to examine the presence of antimicrobial susceptibility/resistance among E. coli's strains in outpatients. This retrospective study was carried out from January1st to December 31th 2017, at the Department of Microbiology, Faculty of Medicine, University of Sarajevo, in cooperation with the Microbiological laboratory of the Cantonal Hospital „Dr. Safet Mujić“, Mostar. During the research, a total of 3 148 urine samples of outpatients were examined. Our study showed that E. coli had the highest resistance to ampicillin (58%), followed by trimethoprim-sulfamethoxazole (38.4%) and amoxicillin-clavulanic acid (38.4 %), and the lowest resistance to nitrofurantoin (2.7%) and cefuroxime (4.1%). The isolated strains of E. coli showed the highest resistance to ampicillin, and the highest susceptibility to nitrofurantoin. Gender distribution of positive E. coli isolates showed statistically significant differences in favor of females.Keywords: E. coli, urinary tract infections, resistance, susceptibility, infection, uropathogen.

РезюмеИнфекциите на пикочните пътища (ИПП) са сред най-разпространените бактериални инфекции,

а Escherichia coli е най-често срещаният уропатоген при неусложнени инфекции на горните и долните пикочни пътища (70–95% от случаите). Целта на настоящото проучване е да се изследва наличието на ан-тимикробната чувствителност/резистентност сред щамовете на E. coli, изолирани от амбулаторно болни. Това е ретроспективно проучване, проведено от 1 януари до 31 декември 2017 г. в Катедрата по микроби-ология при Медицинския факултет на Университета в Сараево в сътрудничество с Ммикробиологичната лаборатория на кантоналната болница „Д-р Сафет Муджич“ в Мостар. Изследвани са общо 3148 проби урина от амбулаторни пациенти. Резултатите показват, че Е. coli има най-висока резистентност към ампи-цилин (58%), следвана от тази към триметоприм-сулфаметоксазол (38.4%) и амоксицилин-клавулонова киселина (38.4%), а най-ниската резистентност - към нитрофурантоин (2.7%) и цефуроксим (4.1%). В същото време те показват най-висока устойчивост на ампицилин и най-висока чувствителност към нитро-фурантоин. Разпределението по пол на положителните изолати на E. coli показа статистически значими разлики в полза на жените.

IntroductionUrinary tract infections (UTIs) are the most common infections worldwide accounting for nearly 25%

of all infections. There are different etiological agents, but Escherichia coli is the most common, with a frequency of 70 to 95% (Flores-Mireles et al., 2015). Relapses and recurrent infections are very common. Strains of E. coli that cause UTI act as opportunistic intracellular pathogens that exploit the sensitivity of the host using the spectrum of various virulence factors for the colonization of the urinary tract (Foxman, 2003). By entering into the urinary tract of the host, uropathogenic strains of E. coli (UPEC) generally


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colonize the bladder mucous membrane causing cystitis. Ascending spread through the ureter reaches the kidney and can lead to the development of pyelonephritis. UPEC ability to bind to host tissue is one of the most important factors that facilitate the colonization of the urinary tract, allowing the bacteria to withstand the higher flow of urine and support cell invasion (Eto et al., 2007).

Most urinary infections are caused by the ascending spread of microorganisms through the urethra, although some microorganisms can reach the urinary tract through the blood and lymph (Krkić-Dautović, 2011). The largest number of urinary infections is caused by a single pathogen. In 70-95% of cases, the causative agent is E. coli, and in 5-10% of cases the cause is Staphylococcus saprophyticus, and slightly less Proteus mirabilis and Klebsiella spp. In complicated infections, strains of Pseudomonas, Staphylococcus, Serratia and Providencia may also be isolated (Dąbrowski et al., 2016). Urinary tract infections can range from asymptomatic bacteriuria, which is characterized by the presence of bacteria in the urine in the absence of symptoms, cystitis, in which the infection is restricted to the bladder, and pyelonephritis, in which the kidneys are involved (Bauman, 2015). It is common that cystitis subsides without consequences, while pyelonephritis can cause permanent damage and even lead to death (Marrs et al., 2005; Barišić 2011). Around the world, antimicrobial resistance among UPEC infection is a major health problem because of the increasing development of resistance to different classes of antibiotics. Bacteria have developed excellent mechanisms of genetic adaptation and the consequences of the use of antibiotics are the development of bacterial resistance to them. In the treatment of infection, antibiotics do not distinguish the pathogenic bacteria that cause the infection from the non-pathogenic bacteria of the normal flora, so resistance also develops into bacteria of the normal flora, thus creating reservoirs of the resistance gene in nature (Brumbaugh and Mobley 2012; Sanchez et al., 2012; Al-Badr and Al-Shaikh 2013; Mohseni et al., 2013; Picozzi et al., 2014). Antibiotics remain, despite antibiotic resistance, crucial drugs in medicine, where their use reduces child mortality and extends life expectancy. New bacterial resistance mechanisms are constantly being described, and new ways of transferring resistant genes are discovered daily. Due to the insufficient development of new antibiotics and the increasing levels of resistance, many countries have become aware of the problem and therefore, at the request of the Council of the

European Union, the fight against bacterial resistance to antibiotics is one of the priorities of the World Health Organization (Paphitou, 2013). Material and Methods

The study included 3148 urine samples of outpatients collected at Hospital "Dr. Safet Mujić", Mostar, of which 295 samples were found positive for E. coli. The research was conducted in the period from January1st to December 31th 2017.

Bacteriological analysis of urine samples included cultivation, standard biochemical testing and antimicrobial susceptibility testing. Midstream, clean-catch urine samples were processed in the laboratory for standard urine analysis and culture. Urine samples were inoculated on blood agar and Endo agar, with incubation at 37°C for 24 hours. Significant numbers of bacteria in urine (>105/ml) were tested with the basic biochemical reactions characteristic of E. coli, such as double sugar, peptone water, mannitol, urea, citrate and 10% lactose.

Susceptibility testing of isolates to antibiotics and interpretation of the results was carried out according to EUCAST (The European Committee on Antimicrobial Susceptibility Testing) standards. Antimicrobial susceptibility/resistance of E. coli isolates was tested by the disc-diffusion method. The pre-prepared suspension of the tested strain was inoculated onto the surface of Mueller-Hinton (MH) agar, after which the following antibiotic discs were applied: ampicillin (AMP) 10 mg, amoxicillin-clavulanic acid (AMC) 20/10 mg, gentamicin (GARA) 10 mg, nitrofurantoin (FURA) 100 mg, trimetoprim-sulfometoksazol sulfametoksazol (TSH) 1.25-23.75 mg, cefazolin (CZ) 30 mg, cefuroxim (CXM) 30 mg, ciprofloxacin (CIP) 5 mg, norfloxacin (NOR) 10 mg, ceftriaxone (CRO) 30 mg, moxifloxacin (MOX) 5 mg, cefotaxime (CTX) 5 mg, ceftazidime (CAZ) 10 mg, levofloxacin (LEV) 5 mg, meropenem (MRP) 10 mg, imipenem (IMP) 10 mg, tobramycin (TOB) 10 mg, amikacin (AN) 30 mg, piperacillin (PIP) 30 mg, piperacillin/tazobactam (PIP/IT) 30/6 mg, cefepime (FEP) 30 mg and fosfomycin (FOSF) 30 mg.

For the statistical analysis we used SPSS software program (Statistical Package for Social Science version 23.0).

The results were shown by the number of cases, percentage, arithmetic means with standard deviation and range values. Differential testing was done using the chi-square and Student's t=test. The results of these tests were considered statistically significant at a confidence level of 95% or with p <0.05.

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Results The research showed that out of the 3148

urine cultures examined, 295 (9.40% ) were positive and 2 853 (90.60%) tested negative. The incidence of E. coli in the observed period was 9.37%. Table 1 gives data based on gender distribution, positive test for E. coli was recorded in 274 (92.9%) female patients and 21 (7.1%) male patients. There was a statistically significant difference in favor of females (p <0.05). Higher incidence of positive female patients is evident.Table 1. Gender structureGender N %Male 21 7.1Female 274 92.9Total 295 100

χ2 =16,980; p=0,0001As seen in Table 2 and Fig. 1, the average age

of the observed sample was 55.9 ± 23.8 years with the youngest patient aged 1 and the oldest aged 95 years old.Table 2. The age of patients

Age

NCorrect 263Missing 32

Average 55,9316Std. deviation 1,46495Median 63,0000Std. deviation 23,75742Minimum 1,00Maximum 95,00

T=38,180; p=0,0001

Statistical analysis by Student's t-test shows that there is a significant (p <0.05) deviation from the expected distribution in terms of greater representation of older patients - 60 years and older.

The etiological agents of urinary tract infections are different. Table 3 clearly shows that E. coli was isolated in 275 cases (93.2%), but in 20 cases (6.8%) patients with E. coli had another cause of infection (co-infection). The second most common isolate was Proteus mirabilis with 11 (3.7%), followed by Pseudomonas aeruginosa with 4 (1.4%), Klebsiella pneumoniae with 3 (1%), and Enterobacter clocae and Citrobacter diversus in 1 case (0.3 %).

Table 3. Presence of co-infectionCo-infection

N %E. coli 275 93,2E. coli + C. diversus 1 0,3E. coli + E. cloacae 1 0,3E. coli + K. pneumoniae 3 1,0E. coli + P. aeruginosa 4 1,4E. coli + P. mirabilis 11 3,7Total 295 100,0

Figure 2 shows the results of the antibiotic resistance of E. coli in non-hospitalized patients' samples tested throughout the research period.

The highest resistance of E. coli strains was observed to ampicillin (58%). Out of a total of 5 an-tibiograms, resistance to moxifloxacin (MOX) was observed in 80%, however due to the small number of performed antibiograms the resistance is not statisti-cally significant. A slightly lower resistance was man-ifested to trimethoprim-sulfamethoxazole (38.4%),

Fig. 1. The age of patients

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followed by amoxicillin-clavulanic acid (37.3%). The lowest resistance of E. coli was observed to nitro-furantoin (2.7%) and cefuroxime (4.1%).

DiscussionsUrinary tract infections are the most frequent

acute bacterial infections, mostly in women. Females are more susceptible to UTI, because women have a shorter urethra, which enables the ascending spread of bacteria (O’Brien et al., 2015).

Bacteria may trigger inflammation and pain in any or all of the urinary tract, including the urethra, urinary bladder, or kidneys – conditions called ure-thritis, cystitis, and pyelonephritis (Bauman, 2015). A major problem is the inadequate use of antibiot-ics, which results in increased incidence of antibiot-ic resistance. This is a serious problem that leads to the development of severe urinary infections. UTI is the most common reason for prescribing antibiotics in primary care (Masajtis-Zagajewska and Nowicki, 2017). During our study, 3148 urine samples of out-patients were tested, of which 296 were positive for E. coli, whereas 2852 samples were negative. In terms of gender, there were more positive samples in wom-en 274 (92.9%) than men (21.1%). There was a sta-tistically significant difference in favor of females (p ˂0.05). Wadekar and Sathish (2017) in their research conducted in India also proved the prevalence of the female gender (75%). A similar result was obtained in a cross-sectional study conducted in Pakistan, where of the 458 positive urine samples, 69.2% came from female patients (Ali et al., 2017).

In our study, co-infection was recorded in 20 (6.8%) cases. The most common other isolate was P. mirabilis (3.7%), followed by P. aeruginosain (1.4%), K. pneumoniae (1.0%), and C. diversus (0.3%). Abdu-zaimovic et al. (2016) and Mahmutovic et al. (2017)

in their separate studies in Bosnia had similar results regarding co-infection: Proteus spp. 13.27%/9.83%; Pseudomonas spp. 2.88% / 2.45%; Klebsiella pneu-moniae 5.31%/1.63%.

Our research shows that the highest resistance of E. coli was manifested to ampicillin (58%). A slightly lower resistance was exhibited to trimethop-rim-sulfamethoxazole (38.4%), followed by amoxicil-lin-clavulanic acid (37.3%). The lowest resistance of E. coli was observed to nitrofurantoin (2.7%). Hegazy et al. (2018) in their study in Egypt proved the highest resistance of isolated strains of E. coli to ampicillin (100% of the total 98 antibiograms), cefazolin (100%) and trimethoprim-sulfomethoxazole (87.9%). The re-sults of our research on the resistance to these antibi-otics are consistent with this study. In the same study, the highest susceptibility of isolated strains showed amycotine (77.55%), imipenem (76.53%), nitrofuran-toin (77.5%) and gentamicin (71.43%). In a retrospec-tive analysis of the antimicrobial susceptibility of E. coli, followed over 11 years in Ireland, ampicillin and trimethoprim were observed to be the least effective in therapy. The resistance rates for ampicillin and tri-methoprim were 60.8 or 31.5%, respectively, andfor trimethoprim, amoxicillin-clavulanic acid, cefurox-ime, and gentamicin significant trends of increasing resistance were identified over the 11-year period (Cul-len et al., 2012). The results of the research conducted by Abduzaimovic and colleagues (Abduzaimovic et al., 2016) in Bosnia showed E. coli susceptibility to trimethoprim sulfamethoxazole was 38.61%, amox-icillin-clavulanic acid 19.62%, ciprofloxacin 9.49%, gentamicin 8.86%, cephalexin 8.23%, nitrofurantoin 8.23%, cefuroxime 7.52%, ceftazidime 6.33%, amik-acin 4.43%. Yilmaz et al. (2016) in Turkey conducted a study of E. coli susceptibility to antibiotics and ob-

Fig. 2. The resistance of E. coli strains to antibiotics

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tained the following results: ampicillin 66.9%, cefazo-lin 30.9%, cefuroxime 30.9%, ceftazidime 14.9%, ce-fotaxime 28%, cefepime 12%, amoxicillin-clavulanic acid 36.9%, trimethoprim-sulfamethoxazole 20%, ciprofloxacin 49.9%, amikacin 0.3%, gentamicin 24%, nitrofurantoin 0.9% and phosphomycin 4.3%. In Croatia, in the period from 2003 to 2007, tested E. coli strains showed the highest resistance to amoxicillin ranging from 37% to 41%. High resistance was also noted for trimethoprim-sulfamethoxazole, ranging from 19% to 30% (Farkaš et al., 2008). In a retrospec-tive analysis done in India (Shanthi, 2018), isolated E. coli strains showed the highest resistance to cotrimox-azole (75.8%) and cefotaxime (78.27%). The highest sensitivity was observed for nitrofurantoin (85.89%) and amikacin (68.89%). From the above studies it can be concluded that the strains of E. coli showed the greatest resistance to the compositions of penicillin (β-lactam antibiotics), and trimethoprim-sulfametax-azolu cephalosporins 1 and 2 generation. Resistance to penicillin preparations in all studies, as well as in our study, is the most pronounced. In our study, there was no marked resistance to cephalosporins first and second generation (cefazolin and cefuroxime), which was the case with the above studies. The highest sen-sitivity of E. coli in those studies was observed in aminoglycoside antibiotics, nitrofurantoin and car-bapenems. The highest sensitivity in our research is to nitrofurantoin and cefuroxime.Conclusions

Considering the increasing problem of bacte-rial resistance to antibiotics all over the world, it is extremely important to know how susceptible certain causal agents are in order to approach the therapy as rationally as possible. The results of our study suggest the necessity of rational use of antibiotics and the need for multi-disciplinary control of the development of resistance. Monitoring the shift in antimicrobial sus-ceptibility of E. coli is important because of the ration-al prescription of antibiotics in the treatment of urinary tract infection, with the aim of controlling the current level of resistance and making etiological diagnosis.

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Microbiological Safety of Commercial Pasteurized Apple and Orange Juices

Galina Satchanska*, Margarita Tsenova, Aleksandra SashevaDepartment of Natural Sciences, BioLab, New Bulgаrian University, Montevideo 21, 1618 Sofia, Bulgaria

AbstractIn this study we describe the assessment of the microbiological quality of 12 commercial pasteurized

apple and orange juices. Orange juice is the most widely consumed fruit juice in the world and together with apple juice are an essential part of human diet. Although pasteurization is the preferable method for bacterial elimination, some heat-resistant microorganisms and their spores could survive the heat treatment and cause spoilage hence triggering serious outbreaks. Our study revealed that the microbiological quality of all 12 investigated pasteurized juices (6 apple juices and 6 orange juices) meet the national, European and international standards. The results obtained indicated that the juices were free of aerobic mesophilic bacteria and facultative anaerobes, yeasts, molds or of diarrheagenic E. coli and coliform bacteria described in the standards. No heat-resistant spore-forming bacteria had grown during the enrichment experiment. Based on our results, we can conclude that all tested juices are safe for consumption.Keywords: safety of fruit juices, apple, orange, microbiology, E. coli.

РезюмеВ това изследване е проведен анализ на микробиологичното качество на 12 плодови сока - пастьори-

зирани ябълков и портокалов сок, налични в магазинната мрежа. Портокаловият сок е най-кoнсумираният в световен мащаб плодов сок, който заедно с ябълковия сок заемат водещи позиции в диетата на човека. Въпреки, че пастьоризацията е най-предпочитаният и използван метод за предотвратяване развитието на микроорганизми в соковете, някои резистентни към висока температура микроорганизми и техните спо-ри биха могли да я преживеят и да доведат до развала на соковете, а оттам и до нежелани за здравето на човека последствия. Нашето изследване показа, че всички 12 изследвани ябълкови и портокалови сокове са микробиологично чисти и отговарят на националните, европейските и интернационалните стандарти. В изследваните сокове не бяха открити аеробни мезофилни бактерии и факултативни анаероби, дрожди и плесени, както и диарогенни Escherichia coli и колиформи, които да надхвърлят стойностите, наложени в стандартите. Също така, не бяха установени и температурно-устойчиви микроорганизми и техни спори по време на експериментиe с набогатяване. Въз основа на получените резултати, може да се заключи, че всички изследвани сокове са безопасни за консумация от потребителите.

IntroductionFruits and fruit juices represent an important part of human diet supplying the body with carbohydrates,

minerals, vitamins, antioxidants, phenolic compounds, fibers and organic acids. The vast number of organic acids is responsible for the acidic pH values of juices. Due to their rich chemical content, juices play a significant role in the prevention of cardiovascular diseases, diabetes and cancer, and have a pronounced immunomodulatory effect (Slavin and Lloyd, 2012; Liu, 2013). Fruits and fruit juices represent one of the mandatory food groups that any individual should take daily along with vegetables, grains, dairy products, meat, fish, oils, nuts and spices. The high sugar and water contents of fruit juices are favorable for the proliferation of a cohort of bacteria (Caballero et al., 2003; Tribst et al., 2009; Juneja and Sofos, 2010; Montville et al., 2012; Leff and Feirer, 2013). The prevention of microbial growth in fruit juices


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is achieved through pasteurization - the method most commonly applied in industry. Usually, pasteurization for commercial juices is carried out at 82oC for 15 sec (Kemal et al., 2014; Peruttzi, 2017). It is considered that during this process the vegetative microbial cells and spores of molds and spore-forming bacteria are killed but at the same time the method possesses the disadvantage of destroying certain enzymes, particularly phenol oxidases. Pasteurization may also inactivate important substances as phenolic antioxidants (Sun, 2009). Besides common microorganisms, another target of pasteurization are the heat-resistant fungi and acid-tolerant spore-forming bacteria like Clostridium pasteurianum, Bacillus coagulans and Alicyclobacillus acidoterrestris (Walker and Phillips, 2008; Smit et al., 2011; Osopale et al., 2016). It is also known that many of the pathogenic species survive under refrigeration conditions and can be harmful to humans.

Generally, fruit juices are spoiled by three main groups of microorganisms – bacteria, molds and yeasts that are present in the raw material. Tolerating low pH and high sugar content, yeasts spoil juices by producing CO2 and alcohol, enhancing the juice turbidity, causing flocculation and phase separation – all due to the action of microbial enzymes on pectin (Caballero et al., 2003).

Fruit juices of apples, peaches, strawberries and grapes harbor a vast bacterial population dominated by taxa belonging to the Actinobacteria, Bacteroidetes, Firmicutes, and Proteobacteria phyla as reported by Leff and Feirer (2013). Other important human pathogens associated with juices belong to the genera Erwinia, Pseudomonas, Salmonella and the species L. monocytogenes and E. coli These bacteria can cause serious outbreaks. In addition to the bacteria mentioned above, lactic acid bacteria are also considered as common juice spoilers. In the past decade, growing attention has been given to the species belonging to genus Alicyclobacillus, described to be prevalent in fruit-based products due to their ability to survive the acidic juice environment (Walker and Phillips, 2008). Alicyclobacillus is a genus of thermo-acidophilic, endospore-forming bacteria. Furthermore, Alicyclobacillus endospores are resistant to the low pH of fruit juices and the pasteurization treatment (Smit et al., 2011; Osopale et al., 2016). The predominant metabolite synthesized by Alicyclobacillus responsible for juice spoilage is 2-methoxyphenol (guaiacol) (Wall, 2000). Witthuhn et al. (2016) reported that

Alicyclobacillus was detected in 26 out of 225 juice samples investigated, i.e. more than 11% of juices studied were contaminated with this bacterium.

Being mostly aerobic, molds spoil juices via changing the odor and forming mycelial mat on the juice surface (Caballero et al., 2003). The heat-liable asexual spores (conidia) of the most common genera such as Cladosporium, Penicuillium, Botritis, Aspergillus, Mucor, Rhizopus, and Fusarium could be easily killed by heating at 60oC for 5-10 min (Peruttzi, 2017). In contrast, the heat-resistant species of genus Byssochlamys, Neosartorya and Talaromyces survive heat treatment owing to their thick wall. They have been reported in canned blueberries and tomato juice (Kotzekidou, 2009) and have become a serious industrial problem. Extremely harmful for the human health are a few mycotoxins such as byssochlamic acid (isolated from Byssochlamus fulva) (Weinedborner, 2001) (Fig. 1.), Bissotoxin A (isolated from B. fulva) (Kramer et al., 1976) and Patulin (Fig. 2.) (isolated from B. nivea) (Rice et al., 1977). All these toxins have been detected in strains colonizing fruit juices.

One of most infectious bacterium colonizing fruits and invading fruit juices is the diarrheagenic Е. coli O157: H7. First detected in apple cider

in Massachusetts, USA, in 1991, it is reported to cause hemorrhagic diarrhea and hemolytic uremic syndrome (HUS). The complicated etiology of these illnesses combined with the very low infectious

Fig. 1. Structural formula of Byssochlamic acid (PubChem)

Fig. 2. Structural formula of Patulin (PubChem)

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dose (100 cells) identify this bacterium as one of most dangerous food-borne bacterial pathogens (Montville et al., 2012).

The first object of our investigation, apple juice, is produced from the most frequently consumed apple fruit. Apple contains a broad variety of vitamins - A, B1, B2, B3, B6, B9, C, E, H, P, K (highest values are Vit. C and Vit. K); minerals – Fe, K, Mg, Na, S, P, I, Cl, Al, B, V, Co, Mn, Cu, Mo, Ni, Pb, F, Cr, Zn (the highest concentration is K); water, proteins, saturated, monounsaturated and polyunsaturated fatty acids, organic acids (malic-, quinic- and citromalic acid), amino acids including 10 essential amino acids, mono-, oligo- and polysaccharides (primarily glucose and fructose, cellulose, starch; the total CH content is 10.3 g/100g apple), tannins (polyphenols), amides and other nitrogenous compounds, soluble pectin and a diverse range of esters e.g. ethyl and methyl-iso-valerate esters. Esters have been reported to be essential for the typical apple aroma. Potassium is the most abundant mineral in apple juice at 1229 mg/l, and among the amino acids asparagine is the prevailing one (Caballero et al., 2003; Liu, 2013).

Orange juice, the second target of our study, is the most widely consumed fruit juice in the world, particularly appreciated by consumers for its organoleptic properties and its high content of potentially beneficial bioactive components (Liu, 2013). Among the citrus fruits, orange juice is the predominant fruit juice in the human diet. It contains abundance of vitamins - A, B1, B2, B3, B6, B9, B12 C, D, E, K; minerals - K, Ca, I, Mg, P, Na, Zn; phenolic compounds, terpenes, tannins, saponins, flavonoids and alkaloids (Galaverna and Dall’Asta, 2014). Orange juice contains K concentrations that are even higher compared to apple juice – 1971 mg/l. Potassium is essential for human metabolism being responsible for the cardiac activity and together with Na regulates the water balance in the body. Potassium is crucial for the cognitive function and helps the evacuation of toxins from the body. Orange juice has been reported to prevent the formation of kidney stones because of the presence of citrates. The most abundant amino acid in orange juice is proline. The role of proline is important as it is known that this amino acid along with glycine and the proline derivate - hydroxyproline contributes to 57% of the total amino acids in the collagen (Liu, 2013; Li and Wu, 2018).

In this paper we describe the investigation of the microbiological quality of pasteurized commercial apple and orange juices, analyzing the

total number of mesophilic microorganisms and facultative anaerobes, total number of molds and yeasts, and the number of diarrheagenic E. coli and coliform bacteria. The probability for heat-resistant spores to survive pasteurization was assessed as well.Materials and MethodsSampling

A total of 12 juice samples were investigated – three apple juice samples (1 liter Tetra pack), three orange juice samples (1 liter Tetra pack), three apple juice samples (200-250 ml Tetra pack) and three orange juice samples (200-250 ml Tetra pack). Juices were manufactured as follows: BBB juices by Gorna Bania Bottling Complany LtD, Happy day juices by RAUCH Fruchtsäfte GmbH & Co, and Florina juices by Best Fruits 17 Ltd. Samples were purchased from big retail stores in the spring of 2019.

Analysis of total number of microorganisms in one ml of juice

Aerobic mesophilic microorganisms and facultative anaerobes were evaluated using Nutrient agar (Hi-Media, India) and via the Koch’s method, according to the methodology described in ISO 6887-1:2017. Nutrient agar Petri dishes were inoculated with 100 μl of each juice sample and cultivated for 72 h at 30°C. Analysis was performed in duplicate.Analysis of total number of molds and yeasts in one ml of juice

Analysis of molds and yeasts was conducted on Sabouraud agar (Hi-Media, India) using Koch’s

Fig. 3. Apple and orange fruit juices. A) One liter Tetra pack apple andorange juices manufactured by BBB, Happy day and Florina Ltds; B) 200-250 ml Tetra pack apple and orange juices, commonly consumed by children (manufactured by BBB, Happy day and Florina Ltds).

A

B

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method. 100 μl of each juice sample was spread on the agar and grown for 48 h (for fast growing molds and yeasts) to one week (for slowly growing molds) at 30°C. Analysis was performed via the horizontal method for enumeration of molds and yeasts, described in ISO 21527-1:2008. Analysis was done in duplicate.Analysis of E. coli and coliform bacteria

Enumeration of E. coli and coliform bacteria was executed using Deoxycholate citratelactose agar (DCLA) (Scharlau, Spain). One L dH2O contains: meat extract - 10 g, lactose - 10 g, ferric citrate - 1g, sodium citrate - 20 g, sodium deoxycholate - 5 g, metal red - 0.02 g, agar-agar - 15 g. Petri dishes were inoculated with 100 μl of juice sample and cultivated for 24-48 h at 37°C. Enumeration was performed according to ISO 21528-2:2017. Analysis was carried out in duplicate.

DCLA is a selective and differential medium for gut pathogenic bacteria as E. coli, Salmonella and Shigella. It is rich in citrate and deoxycholate medium. Citrate and deoxycholate salts inhibit the growth of Gram (+) bacteria and other normal intestinal microorganisms allowing only the growth of the species of the family Enterobacteriaceae. Colonies of E. coli grow pink in color on DCLA, Salmonella spp. are colorless with or without black center, and Shigella spp. are visualized on the agar as colorless colonies. Presence of spores and spore-forming bacteria

In order to verify if the juices contain heat-resistant spore-forming or gut-colonizing bacteria, an indirect method was applied where juices were enriched in Peptone water. One liter of peptone water contained 10 g peptone from casein (HIMEDIA, India) and 5 g NaCl. The solution was adjusted to pH 7.2 ± 0.2. Erlenmayer flasks with 225 ml peptone water were inoculated with 25 ml juice samples (1:10) and further cultivated overnight for 24 h at 30°C. If present, the cultured bacteria should subsequently be isolated and studied for spore formation according to standard methods.

ResultsAnalysis of total number of microorganisms in one ml of juice

Our analysis showed no bacterial growth of aerobic mesophilic microorganisms in all six samples of one liter Tetra pack apple and orange juices (Table 1).Analysis of total number of molds and yeasts in one ml of juice

The analysis for presence of molds and yeasts in one liter of apple and orange Tetra pack juices, did not establish growth of cells in either apple or orange juices produced by the two manufacturers - Florina and Happy Day. One colony per Petri dish was observed in both apple and orange juice manufactured by BBB Ltd. This result represents 5 CFU/ml juice. As the upper standard limit is 5-10 CFU/ml, this amount does not exceed the standard limits and cannot cause possible outbreaks (Fig. 4).Analysis of E. coli and coliform bacteria

Figure 5 shows the analysis of E. coli and coliform bacteria presence in three orange and three apple juices. As seen from the figure, no single

colonies of E. coli colony or of Salmonella spp. or Shigella spp. were observed in either the apple or orange juices, 1 liter Tetra pack.

The lack of growth of E. coli, Salmonella spp. and Shigella spp. colonies on DCLA confirmed that the tested juices were free of pathogenic gut

Table 1. Presence of co-infection

Microrganisms/Juice type HD apple juice

HD orange juice

BBB apple juice

BBBorange juice

Florinaapple juice

Florina orange juice

Total number of aerobic mesophilic bacteria and FA

0 0 0 0 0 0

Total number of yeasts and molds 0 0 5 5 0 0E. coli and coli-like bacteria 0 0 0 0 0 0

Fig. 4. Analysis of molds and yeasts in three apple and three orange juices (HD, BBB and Florina (1 liter Tetra pack)

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bacteria, especially of the lethal Е. coli O157:H7, and entirely consistent with the ISO 21528-2:2017 standard. E. coli-tests are mandatory for the safety analyses of any kind of food. The amount of permissible colonies per g or per ml of product varies among different foods.

The data presented in Table 1 show optimal microbiological quality of the investigated juice samples. No aerobic mesophilic bacteria or facultative anaerobes were observed in the six investigated juices. According to the standard limits (ISO 6887-2017), the total number of microorganisms in juices should not exceed 50 CFU/ml. Five colonies of yeasts and molds in BBB orange and apple juice per ml does not exceed the Bulgarian, EU and USA standards as the standard limit (ISO 21527-1-2008) for yeast and mold spores is 5-10 CFU/ml. Samples were also free of E. coli and coliform bacteria. According to the standard (ISO 21528-2-2017), the upper limit for E. coli and coliform bacteria is 0.3-1 CFU/ml. The results displayed in Table 1 are convincing evidence that the studied six juices of 1 liter Tetra pack are microbiologically safe for consumption.

Similar assessment was conducted for the 200-250 ml orange and apple Tetra pack juices produced by the same manufacturers (Fig. 6). Due to the predominant consumption of juices in smaller boxes by children, the assessment of juice quality and safety is of significant importance.

The results presented in Fig. 6 show no contamination with microorganisms (mesophylls and facultative anaerobes) of the juices consumed mainly by infants and children. Insignificant growth of a single colony per Petri dish was observed in two of the juice samples (HD orange and Florina apple). Petri dishes that showed no growth whatsoever demonstrated microbiological purity of children juices also regarding E. coli and coliforms (Table 2).

As seen from Table 2, no microbial growth was detected in all 200-250 ml Tetra pack orange and apple juices, except in the BBB apple juice sample (5 CFU/ml) and in Florina apple juice sample (5 CFU/ml). As the standard limit is 50 CFU/ml and the number of bacteria obtained during the experiment is below this value, all 6 apple and orange juices (200-250 ml Tetra pack) can be considered microbiologically safe for consumption.Presence of spores and spore-forming bacteria

Our experiment showed that the peptone waterenriched apple juice samples (produced by BBB Ltd) – 1 liter and 200 ml Tetra packs were negative for bacterial growth, indicating that no heat-resistant spore-forming bacteria survived pasteurization. That is why, no further tests of bacteria and their spores were executed (Fig. 7).

The lack of bacterial growth in the enriched cultures demonstrated that no heat-resistant bacteria, for example Alicyclobacillus acidoterrestris, were

Fig. 5. Analysis of E. coli and coli-bacteria in three apple and three orange juices (HD, BBB and Florina (1 liter Tetra pack)

Fig. 6. Analysis of total number of microorganisms in three apple and three orange juices (HD, BBB and Florina (200-250 ml Tetra pack)

Table 2. Summarized results of microbiota in three apple and three orange juices (CFU/ml), 200-250 ml Tetra packMicrorganisms/Juice type

HD apple juice

HD orange juice

BBB apple juice

BBBorange juice

Florinaapple juice

Florina orange juice

Total number of aerobic mesophilic bacteria and FA

0 5 0 0 5 0

Total number yeasts and molds 0 0 0 0 0 0E. coli and coli-bacteria 0 0 0 0 0 0

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invading the apple fruits or further contaminating the apple juice. Enriched cultures were also free of heat-resistant spores.

DiscussionJuices are of significant importance for the

human health. Detailed information about the beneficial role of fruits and vegetable in the diet was given by Liu (2013). Galaverna et al. (2014) reported the positive effect of antioxidants in orange juice on human health. Both apple and orange juice are known to prevent cardiovascular diseases, diabetes, cancer, and influence the immune system exerting a pronounced immunomodulatory effect. The high potassium concentration in both apple and orange juice helps the maintenance of normal blood pressure. Abundance of vitamins and minerals ensures optimal metabolism. Among vitamins, Vit. C, which is essential for the body, is present in high concentration in both juices (Caballero et al., 2003).

Summarizing the data from the literature overview, a number of papers have been continuously describing the enormous amount of bacteria developing in fruits and in fruit juices. Bacterial contamination of juices was reported by Leff and Feirer (2013), describing the main taxa colonizing the juices - Actinobacteria, Bacteroidetes, Firmicutes, and Proteobacteria phyla. In contrast to freshly squeezed juices which are abundant in bacteria, pasteurized juices are considered to be free of bacteria (Caballero at al., 2003; Enikova, 2010). Usually, fruit juices are contaminated by microorganisms which colonize the fruit surface. Contamination occurs during harvesting and post-harvest periods. However, it is also possible that fruits and juice are invaded by microorganisms during transportation and storage (Kamal et al. 2014).

To eliminate all pathogenic microorganisms and their spores from fruit juices already produced, industrial-scale pasteurization is used, described in details by Perutzzi et al. (2017). This process

prevents contamination of juices and ensures their long shelf life, killing a plethora of bacteria such as Salmonella, Shigella, E. coli, Acetobacter, Alycoclobacillus, Bacillus, Gluconobacter, Lactobacillus, Leuconostoc, Zymomonas. It also kills yeasts such as Candida, Pichia, Saccharomyces and Rhodotorula. Pasteurization neutralizes the commonly encountered filamentous fungi such as Penicillium, Byssochlamus, Fusarium, Botritis, Talaromyces, Aspergillus and Mucor. During the heating process, many members of molds such as Aspergillus, Eurotium, Cladosporium and Alternalia are inactivated (Caballero et al., 2003; Trbst et al., 2009; Kemal et al., 2014)

Traditionally, pasteurization has been considered as a potential method for destruction of all microorganisms and their spores. Recently, a number of reports have been published describing the presence of the heat-resistant spore-forming bacterium Alycyclobacillus even in pasteurized juices (Smit et al., 2011; Osopale et al., 2016). Available data about the characteristics of this bacterium caught our attention and prompted us to conduct this investigation.

Target microbial groups in our investigation are mesophilic aerobic bacteria and facultative anaerobes, yeasts and molds, diarrheagenic E. coli and coliform bacteria as well as the possible heat-resistant spore-forming bacteria which survived the pasteurization. All these microorganisms have caused serious outbreaks round the world in the recent decade (Montville et al., 2012; Montville et al., 2017).

According to the international standards, very low amounts of microorganisms like bacteria, yeasts, molds, E. coli and coliform bacteria are permissible in juices. Being rich in carbohydrates, juices are a perfect source of fast growing bacteria (Sun, 2009).

As regards the total number of microorganisms (Table 1 and Table 2), our results meet the standard limits (ISO 6887-2017) max. Fifty CFU/ml juices of 1 liter tetra pack were free of microorganisms. Five colonies per ml were found in two 250 ml Tetra pack juices, which are insignificant and comply with the standard.

Molds and yeast total number in the investigated 12 juice samples also did not divert from the standard. All samples were free of molds and yeasts, except two samples which were positive, but still within the standard permissible values. ISO 21527-1-2008 prescribes the upper limit for yeast and mold spores ranging from 5 to 10 CFU/ml. The

Fig. 7. Peptone water-enriched samples of apple juice (1 liter and 250 ml Tetra pack) manufactured by BBB Ltd.

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results obtained were 5 CFU/ml, indicating that both juices are safe products. The high toxicity of mycotoxins on human brain function has been well documented since the 1970s (Kramer et al., 1976; Rice et al., 1977) and has continued during the last two decades (Kotzekidou, 1997; Weinedborner, 2001), making the analysis performed an important one.

The presence of E. coli and coliform gut bacteria is permitted by the standard but at a very low concentration of 0.3-1 CFU/ml (ISO 21528-2-2017). All 12 samples were negative for E. coli presence. Serotype O157:H7 is one of the pathogenic E. coli which caused fatal outbreaks a few years ago in Germany. The body intoxication is due to the action of the highly toxic Vero- and Shiga toxins, synthesized by O157:H7 (Proft, 2013; Montville et al., 2017). As reported by Juneja and Sofos (2010), the very low infectious dose (100 cells) makes this strain one of the most potent pathogens transferred by fruits and vegetables.

Regarding the possible presence of heat-resistant bacteria and their spores in the enriched cultures of two apple juices, the lack of growth proved that the tested juices were of high microbiological quality, met the standards and could be considered microbiologically safe and suitable for use.Conclusions

Our study revealed that all 12 investigated pasteurized juices (6 apple juices and 6 orange juices) meet the national, European and international standards in terms of their microbiological quality. Juices were free of aerobic mesophilic bacteria, facultative anaerobes, yeasts and molds as well as of the diarrheagenic E. coli and coliform bacteria. No heat-resistant spore-forming bacteria had grown via the enrichment experiment.

AcknowledgementsThis work was funded by the Faculty of

Master Education (Grant No 15/2019) and Central Fund for Strategic Development, New Bulgarian University

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Tolerance of Yeast to Formic Acid during Ethanol Fermentation

Folake T. Afolabi*, Godwin E. Oduokpaha, Abiodun A. OniludeIndustrial Microbiology and Biotechnology Unit, Department of Microbiology, University of Ibadan, Nigeria

AbstractThis study was carried out to investigate the tolerance of yeasts isolated from some Nigerian traditional

fermented foods to formic acid during laboratory-scale fermentation of ethanol. A total of 27 yeast strains were isolated from burukutu, ogi, kunu and palm wine. The yeasts were

screened for formic acid tolerance using spot plate technique on two culture media. One strain was selected based on its ability to tolerate up to 15 mM concentration of formic acid on Yeast Extract Peptone Dextrose Agar and was further identified as Candida tropicalis strain IFM 63517. C. tropicalis was used for fermentation of ethanol with varying concentrations of formic acid, ethanol and residual glucose concentrations which were monitored at intervals. The total viable cell count was determined using plate count technique. The highest ethanol yield of 8.36% (v/v) with a residual glucose concentration of 0.33 g/L was obtained from 0 mM formic acid (control fermentation vessel) with a total viable cell count of 8.7x109 cfu/ml, while the lowest ethanol yield of 8.00% (v/v) with a residual glucose concentration of 0.14g/L was obtained from 15 mM concentration of formic acid with a total viable cell count of 6.1x109 cfu/ml. The yeast strain used in this work exhibited a high ethanol yield despite the presence of an inhibitory compound (formic acid) when comparing the ethanol yield at its tolerance threshold (15 mM of formic acid) to the control fermentation vessel without formic acid.Keywords: yeast, formic acid, ethanol fermentation, residual sugar, Candida tropicalis.

РезюмеНастоящата разработка е насочена към проучване толерантността на дрожди, изолирани от някои

традиционни нигерийски ферментирали храни към мравчена киселина по време на ферментация на ета-нол в лабораторни условия.

От бурукуту, оги, куну и палмово вино са изолирани общо 27 щама дрожди. Проучена е резистент-ността на тези изолати към мравчена киселина върху две агарови културални среди. Един от тест-щамо-вете, идентифициран като Candida tropicalis IFM 63517, показва резистентност към мравчена киселина в концентрация до 15 mM при култивиране върху среда с декстрозо-пептонен агар и дрождев екстракт. C. tropicalis се използва за ферментация на етанол при различни концентрации мравчена киселина, етанол и глюкоза, които се измерват на интервали. Общият брой на жизнеспособнте клетки се определя чрез бро-ене на образуваните колонии в агарова среда в петриеви блюда. Най-високият добив на етанол от 8.36% (обем/обем) с концентрация на остатъчна глюкоза 0.33 г/л се получава от 0 mM мравчена киселина (кон-тролен ферментационен съд) с общ брой жизнеспособни клетки 8.7x109 бр. клетки/мл, докато най-ниски-ят добив на етанол - 8.00% (обем/обем) с концентрация на остатъчна глюкоза от 0.14 г/л се получава от концентрация на мравчена киселина 15 mM с общ брой жизнени клетки 6.1x109 бр. клетки/мл. При срав-няване резултатите се установява, че щам C. tropicalis IFM 63517 показва висок добив на етанол, въпреки наличието на инхибиращото вещество (мравчена киселина) дори в условията на праговата толерантност на щама (15 mM), в с сравнение с този от контролния ферментор (без наличие на мравчена киселина).

* Corresponding author: [email protected], [email protected]

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IntroductionYeasts, mostly strains of Saccharomyces

cerevisiae, have been widely used for bioethanol production industrially, because of their high fermentative ability, ethanol tolerance and rapid growth under anaerobic conditions. Apart from Saccharomyces, other genera of yeasts such as Candida, Kluyveromyces, and Schizosaccharomyces have also been employed in the bioconversion of lignocellulosic substrates to bioethanol (Ivanova et al., 2011). These yeasts, however, are susceptible to inhibitory compounds present in lignocellulose-derived hydrolysates (Martin et al., 2002). One possible solution is to detoxify the hydrolysate to remove the inhibitors; however, this creates additional costs and a potential loss of sugar (Almeida et al., 2007). An alternative approach and long-term solution to overcome this problem is to either screen for high inhibitor tolerant yeast strains or create genetically modified strains with desired tolerance properties.

Inhibitory compounds capable of inhibiting fermenting yeast fall into specific groups such as weak acids, furan derivatives and phenolic compounds (Sun and Tao, 2010). The types of toxic compounds generated, and their concentrations in lignocellulosic hydrolysates, depend on both the raw material and the operational conditions employed for hydrolysis (Taherzadeh et al., 2000). Toxic compounds can act to stress fermentative organisms to a point beyond which the efficient utilization of sugars is possible, ultimately leading to reduced product formation (Palmqvist and Hahn-Hagerdal, 2000b). Formic acid (methanoic acid) is one of the weak acid inhibitors present in lignocellulosic hydrolysates, with a typical concentration of approximately 1.4 g/L (30 mM) (Almeida et al., 2007; Greetham et al., 2014). The majority of the investigators who have observed the presence of this acid in fermented liquids, however, believed it to play some role in the fermentations of sugar by yeast. The inhibitory effect of formic acid has been ascribed to both uncoupling and intracellular anion accumulation and the reduction of the uptake of aromatic amino acids (Verduyn et al., 1992; Pampulha and Loureiro, 2000; Almeida et al., 2007). Formic acid is more toxic to yeast strains than either acetic acid or levulinic acid (Almeida et al., 2007; Hasunuma et al., 2011a), due to a lower pKa value (3.75 at 20°C) than acetic (4.75 at 25°C) and levulinic acid (4.66 at 25°C).

The undissociated form of weak acids can diffuse from the fermentation medium across the

plasma membrane and dissociate due to higher intracellular pH, thus decreasing the cytosolic pH (Verduyn et al., 1992; Pampulha and Loureiro, 2000). The decrease in intracellular pH is compensated by the plasma membrane ATPase, which pumps protons out of the cell at the expense of ATP hydrolysis. Consequently, less ATP is available for biomass formation. Inhibition of cytochrome oxidase also increases the production of cytotoxic reactive oxygen species (ROS), leading to cell death due to damage in ROS in cell compartments (Richter et al., 1995). For industrial and commercial purposes, low concentrations of formic acid are widely used as one of the major ingredients of antiseptics. According to the intracellular anion accumulation theory, the anionic form of the acid is captured inside the cell and the undissociated acid will diffuse out of the cell until equilibrium is reached. Weak acids have also been shown to inhibit yeast growth by reducing the uptake of aromatic amino acids from the medium, probably as a consequence of strong inhibition of the enzyme permease (Almeida et al., 2007).

On the other hand, formic acid has been shown to be a toxic metabolite of methanol, and is a commonly used organic solvent that has been long known to be a selective human neurotoxin (Roe, 1955). Formic acid causes both metabolic acidosis and ocular toxicity by affecting the retina and optic nerve cells, ultimately leading to blindness, a common and permanent consequence of methanol intoxication (Roe, 1955). Formic acid has been demonstrated in vitro to induce mammalian cell death by inhibiting the activity of cytochrome oxidase, the terminal electron acceptor of the electron transport chain that is involved in ATP synthesis, resulting in depletion of ATP and subsequent cell death due to reduction of energy levels so that essential cell functions cannot be maintained (Nicholls, 1975; Nicholls et al., 1976). Antioxidants such as catalase and glutathione/glutathione peroxidase may play a role in the protection of ocular cells from formic acid toxicity (Treichel et al., 2004).This work, therefore, aimed at investigating the tolerance of yeasts isolated from some Nigerian traditional fermented foods to formic acid during laboratory-scale fermentation of ethanol. Materials and MethodsMicroorganisms

All yeast used for this work were isolated from various fermented beverages (palm wine, burukutu, kunu) purchased from Ibadan metropolis in Oyo State, using standard microbiological isolation

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procedures. Stock of each strain was stored in malt extract agar (MEA) slant at 4°C until required. Spot plate analysis

Progressive sub-culturing of each isolate from a lower formic acid concentration to a higher concentration (0, 5, 10, 15 and 20 mM) using both MEA and yeast extract peptone dextrose agar (YEPDA) was carried out for the selection of the most tolerant strain. Yeast inoculum was prepared by taking a loopful of stock culture to 10 mL of demineralized water and the optical density was compared with MacFarlan standard number 0.5 containing approximately 1.5x108 cells/mL of yeast culture then 5 μL samples of each dilution of the yeast cultures were spotted on MEA and YEPDA plates. The plates were incubated anaerobically at 30°C for 48 h and visible growth differences were recorded (Homann et al., 2005).Molecular identification of the tolerant yeast strain

PCR was carried out in a GeneAmp 9700 PCR System Thermalcycler (Applied Biosystem Inc., USA) with a PCR profile consisting of an initial denaturation at 94°C for 5 min; followed by a 30 cycles consisting of 94°C for 30 s, annealing of primer at 55°C and 72°C for 1.5 min and a final termination at 72°C for 10 min.

The amplified fragments were sequenced using a Genetic Analyzer 3130xl sequencer from Applied Bio systems using manufacturers’ manual while the sequencing kit used was that of BigDye terminator v3.1 cycle sequencing kit. BioEdit software and MEGA 6 were used for all genetic analyses.Lab-scale fermentation

YEPD broth with the addition of formic acid at 0, 5, 10, 15, 20 mM was used in the laboratory-scale fermentations. The pH of the media was adjusted to 4.5 using phosphoric acid under aseptic conditions. From the broth, 100 mL was transferred into mini fermentation vessels (FVs). One mL of the prepared inoculum size of the most tolerant yeast strain was aseptically transferred into each of the bottles. All bottles were incubated at 30°C with shaking at 200 rpm for 24 hours. Samples were collected at specific time intervals to determine the total cell count, and concentrations of glucose.Total viable cell count

One mL of appropriate dilution factor of the suspension of the tolerant yeast strain was plated out using the spread plate method for the determination of the total viable cell of the yeast strain. Plate count technique was employed for the total viable cell of the tolerant strain based on the number of colony forming.

HPLC analysisAt intervals of 24 and 48 h, 10 mL samples

from the fermentation broth were aseptically collected for determination of the residual glucose concentration using High Performance Liquid Chromatography according to the method of Falque-Lopez and Fernandez-Gomez (1996). Glucose concentration was determined using Agilent 1200 HPLC system composed of: Detector: Refractive Index Detector (RID); Column: Grace-Davison Prevail Carbohydrate ES 5μ column (150mm x 4.6mm); Mobile Phase: 75% Acetonitrile LC Grade: 25% deionised water; Injection volume: 5.0 μl; Flow rate: 1.0 mL/min, and temperature: 25°C.GC analysis

At intervals of 24 and 48 h, 10 mL samples from the fermentation broth were aseptically collected for the determination of the ethanol concentration using Gas Chromatography model HP 6890 Powered with HP ChemStation rev. A 09 01 (1206) software according to the method of Vianna and Elber (2001).

ResultsSpot plate characteristics (screening)

All twenty-seven isolates were screened using the spot plate technique with different concentrations of formic acid. Two media, namely MEA and YEPDA for yeast propagation, were used in the screening process to access the tolerance of the various isolates to formic acid (Table 1).

On MEA, all the twenty-seven (100%) isolates were able to grow at 0 mM of formic acid, 11 (40.74%) isolates were able to grow at 5 mM of formic acid, 6 (26.22%) isolates were able to grow at 10 mM of formic acid, none (0%) was able to grow at 15 and 20 mM of formic acid, respectively. Whereas, on YEPD all (100%) the isolates were able to grow at both 0 and 5 mM concentration of formic acid, 10 (37.04%) were able to grow at 10 mM concentration of formic acid, 1 (3.70 %) was able to grow at 15 mM concentration of formic acid and none was able to grow at 20 mM of formic acid concentration.

From the results obtained from the spot plate screening of the isolates, it was observed that YEPDA was the best medium that could support the growth of the isolates as it was able to support the growth of one isolate (P8) up to 15 mM concentration of formic acid. Molecular identity of the tolerant strain

Gene sequence from the characterized isolate showed 99% identity similar to Candida tropicalis

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strain IFM 63517 internal transcribed spacer 1, partial sequence; 5.8S ribosomal RNA gene and internal transcribed spacer 2, complete sequence; and large subunit ribosomal RNA gene partial sequence. Determination of glucose concentration

The residual glucose concentration from the various mini fermentation vessels containing varying concentrations of formic acid was monitored during the period of fermentation at 24 and 48 h, respectively, using HPLC. With 0 mM of formic acid, residual glucose concentration at 24 h of fermentation was 0.4±0.00 while at 48 h of fermentation there was a decrease in the residual

glucose concentration to 0.33±0.01. With 5mM of formic acid, residual glucose concentration at 24 h of fermentation was 0.19±0.01 while at 48 h of fermentation there was a decrease in the residual glucose concentration to 0.10±0.01. With 15 mM of formic acid, residual glucose concentration at 24 h of fermentation was 0.21±0.01 while at 48 h of fermentation; there was a decrease in the residual glucose concentration to 0.14±0.01, as shown in Table 2.

Table 2. Residual glucose concentration at 24 and 48 h of glucose fermentation by C. tropicalis strain IFM 63517

Formic acid (mM)

Residual glucose concentration (g/L)24 h 48 h

0 0.40±0.00a 0.33±0.01a

5 0.19±0.01b 0.10±0.01b

15 0.21±0.01b 0.14±0.01c

Note: Means (results of duplicate) with different superscript letter down the column are statistically significant at p≤0.05Ethanol yield of glucose fermentation by C. tropicalis strain IFM 63517 at 24 and 48 h

The total ethanol yield from the various mini fermentation vessels containing varying concen-trations of formic acid was monitored during the period of fermentation at 24 and 48 h, respectively, using gas chromatography.

With 0 mM of formic acid ethanol yield at 24 h of fermentation was 5.99±0.02 while at 48 h of fermentation, there was an increase in the etha-nol yield to 8.36±0.05. With 5 mM of formic acid, ethanol yield at 24 h of fermentation was 5.68±0.01 while at 48 h of fermentation there was an increase in the ethanol yield to 8.24±0.01. With 15 mM of formic acid, ethanol yield at 24 h of fermentation was 5.01±0.02 while at 48 h of fermentation there was an increase in the ethanol yield to 8.00±0.02, as shown in Table 3.

Table 3. Ethanol yield at 24 and 48 h of glucose fermentation by C. tropicalis strain IFM 63517

Formic acid (mM)

Ethanol yield (%v/v)24 h 48 h

0 5.99±0.02a 8.36±0.05a

5 5.68±0.01b 8.24±0.01b

15 5.01±0.02c 8.00±0.02c

Values are means of triplicate plate count

S/N Isolate code

Formic acid concentrations of (mM)MEA YEPDA

0 5 10 15 20 0 5 10 15 201 P1 + - - - - + + - - -2 P2 + - - - - + + - - -3 P3 + - - - - + + - - -4 P4 + - - - - + + - - -5 P6 + + + - - + + + - -6 P7 + + - - - + + + - -7 P8 + + + - - + + + + -8 P9 + - - - - + + + - -9 P10 + - - - - + + - - -

10 P11 + + - - - + + - - -11 K1 + - - - - + + - - -12 K4 + + + - - + + + - -13 K5 + + - - - + + - - -14 K6 + + + - - + + + - -15 K7 + - - - - + + - - -16 K8 + + + - - + + - - -17 K9 + + + - - + + + - -18 K10 + - - - - + + - - -18 K10 + - - - - + + - - -20 B1 + - - - - + + - - -21 B2 + - - - - + + + - -22 B3 + - - - - + + + - -23 O1 + - - - - + + - - -24 O2 + + - - - + + - - -25 O3 + - - - - + + + - -26 O4 + - - - - + + - - -27 O5 + - - - - + + - - -

Key: + = Growth; - = No growth

Table 1. Spot plate screening of isolates using different concentrations of formic acid (mM)

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Total viable cell count determinationThe total viable cell count of C. tropicalis

strain IFM 63517 used in the fermentation process was determined using the plate count technique at intervals of 12, 24, 36 and 48 h. With 0 mM of for-mic acid, there was a total cell count of 5.9x109, 6.8x109, 7.4x109 and 8.7x109 cfu/ml at 12, 24, 36 and 48 h of fermentation, respectively. This is shown in Table 4. With 5 mM of formic acid, there was a total cell count at 48 hours 4.2x109, 5.1x109, 6.6x109 and 6.9x109 cfu/ml at 12, 24, 36 and 48 h of fermentation, respectively, while with 15 mM of formic acid, there was a total cell count of 3.3 x109, 4.7 x109, 5.3 x109 and 6.1 x109 cfu/ml at 12, 24, 36 and 48 h of fermentation, respectively.

Table 4. Total viable cell count of C. tropicalis strain IFM 63517 throughout the period of glucose fermentation with various concentrations of formic acid

Time(h)

Formic acid concentration (mM)0 5 15

0 1.5x108 1.5x108 1.5x108

12 5.9x109 4.2x109 3.3x109

24 6.8x109 5.1x109 4.7x109

36 7.4x109 6.6x109 5.3x109

48 8.7x109 6.9x109 6.1x109

Values are means of triplicate plate count

DiscussionDespite the fact that yeasts are readily capa-

ble of utilizing monosaccharides as their energy source, this ability can easily be hampered by the presence of inhibitory substances such as formic acid, which is a typical weak organic acid formed as a by-product in the anaerobic breakdown of glu-cose by yeast. Yeast isolates used in this work were obtained from various indigenous fermented bev-erages of Nigeria (palm wine, ogi kunu, burukutu). Serial dilution and appropriate dilution factors of x103 and x106 of each of these beverages were plat-ed out for yeast isolation. According to Banwo et al. (2015), the ready availability of yeast in such food products is a result of the high sugar content hence leading to the addition of desirable flavour to fermented foods.

Spot plate screening of the 27 yeast isolates from various fermented foods using both MEA and YEPDA showed that the highest tolerance thresh-old of the isolates to formic acid on MEA was 10 mM, where 6 (26.22%) isolates were able to grow

whereas there was an increase in the tolerance threshold on YEPDA up to 15 mM of formic acid with 1 (3.70%) isolate being able to grow. This re-sult agrees with the work of Keating et al. (2006), who utilized Yeast Nitrogen Base agar (YNBA) and YEPDA for the spot plate screening of yeast isolate to various inhibitory weak organic acids and found that yeast could tolerate higher inhibitory concen-trations on YEPDA compared to YNBA or MEA, which are mere minimal media for yeast isolation with reasons being that YEPDA as an enriched me-dium may have a higher buffering capacity against weak organic acids, of which formic acid is an ex-ample. Similarly, Oshoma et al. (2015) subjected various strains of non-Saccharomyces cerevisiae to various concentrations of formic acid and ob-served that S. paradoxus DBVPG6466, S. kudria-vzeii IFO1802, S. arboricolus 2.3319 S. cerevisiae NCYC2592 exhibited tolerance to 35 mM and 20 mM formic acid on YPD and YNB media, respec-tively, while other strains did not grow, thus show-ing that yeast can tolerate higher concentrations of inhibitors in YEPD medium than in other minimal growth media.

Molecular characterization of the most toler-ant and selected yeast isolate revealed the isolate to have 99% identity to C. tropicalis strain IFM 63517. Similar results where Candida species were isolated and characterized from fermented foods such as kunu, pito, ogi and palm wine were report-ed in the works of Sanni and Lonner (1993); Ikpoh et al. (2013); Banwo et al. (2015).

Based on the results obtained from spot plate screening, C. tropicalis IFM 63517 strain was se-lected and tested for formic acid tolerance. Com-pared with the controlled fermentation vessel with-out formic acid where cell growth was unaffected by any inhibitory compound, cell growth of C. trop-icalis IFM 63517 strain at both 5 mM and 15 mM concentrations of formic was still maintained above 75%. This result conforms to what was obtained by Huang et al. (2011), who studied the inhibitory ef-fect of organic acids on strains of Saccharomyces species. He stated that in the fermentation of glu-cose with different concentrations of formic acid, it was observed that the cell concentration decreased with an increase in formic acid concentration in the fermentation vessels. The reasons behind this have not been well established but a potential rea-son could be a result of the diversion of metabolic energy (ATP) in the cell to pump out excess proton from the cytoplasm of the cell for a balanced os-motic pressure at the expense of cell biomass pro-

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duction and accumulation (Wikandari et al., 2010).At 24 h, there was a statistically significant

difference in the ethanol yield at 0, 5 and 15 mM concentrations of formic acid. The same was ob-served at 48 h, although there was a significantly higher ethanol yield at 48 h. In summary, there was more ethanol yield at 48 h and observations revealed that as the formic acid concentration in-creased, there was lesser yield of ethanol, thereby signifying the repressing effect of increased con-centration of formic acid on ethanol yield by the yeast isolate.

The overall glucose consumption of C. tropi-calis IFM 63517 was not affected by formic acid. At 24 h of fermentation, the highest residual glucose (0.40±0.00) was observed at 0 mM of formic acid while it was least (0.19±0.01) at 5 mM formic acid. At 48 h of fermentation, the same trend of residual glucose was observed (0.33±0.01) and (0.10±0.01) for both 0 mM and 5 mM concentrations of formic acid, respectively. A similar result was reported in yeast fermentations with acetic acid by Keating et al. (2006) and this supports the assumption that in the presence of formic acid, metabolic activity is increased for the pumping out of protons from the cell thus leading to quick utilization of available glucose in the fermentation wort. Conclusion

The yeast strain used in this work exhibited a high ethanol yield despite the presence of an in-hibitory compound (formic acid) when comparing the ethanol yield at its tolerance threshold (15 mM of formic acid) to the control fermentation vessel without formic acid.

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Acta Microbiologica Bulgarica

GUIDE FOR AUTHORS

The journal Acta Microbiologica Bulgarica is organ of the Bulgarian Society for Microbiology (Union of Scientists in Bulgaria). The journal is continuation of the edited till 1993 journal of the same name, cited in Index Medicus and Medline. The new edition is published two times per year.

Types of articles

The journal publishes editorials, original research works, research reports, reviews, short communications, letters to the editor, historical notes, etc from all areas of microbiology. The manuscripts should not represent research results which the authors have already published or submitted in other books or journals. The papers submitted for publication in Acta Microbiologica Bulgarica are peer-reviewed by two experts from the respective scientific field who remain anonymous to the authors.

Article structure

The papers are published in English, accompanied by a bilingual summary (English and Bulgarian). The papers should be typed with double-spacing (28-30 lines), on a white paper in A4 format, with margins of 3 cm.

The length of the original research paper, including the annexes (tables, figures, etc.) should not exceed a signature or printer’s sheet (30,000 signs, that is, 16 pages with 30 lines each) in Times New Roman 12. The length of shorter reports should not exceed seven pages.The submitted manuscript must contain the name/s and surname/s of the author/s, the name and address of the institution and or organisation where it was prepared. The name and address of the corresponding author should be noted. The abstract should not exceed 250 words and should represent briefly the goals, methods, main results (with numerical data) and basic conclusions of the research.The most essential six keywords must be added to the abstract. The manuscript contains the following sections: introduction, materials and methods, results, discussion, acknowledgements, references. The introduction must be concise with a clearly defined goal and with previous knowledge of the problem. The materials and methods ought to contain sufficient data to enable the reader to repeat the investigation without seeking additional information. The results should be presented briefly and clearly, and the discussion should explain the results.The measuring units and other technical data should be given according to the Sl-system. Illustrations (tables and figures) are submitted separately and their places in the text should be clearly indicated. Tables should be given in a separate sheet, numbered and above-entitled. The figures must be accompanied by legends in a separate sheet.

Reference style

The references used are cited in the manuscript as follows:• In the case of single author - the author’s surname and the year of publication (Petrov, 2012);• In the case of two authors - the authors surnames and the year of publication (Petrov and Vassileva,

2013);• In the case of more than two authors - the first author’s surname, at all., and the year of publication

(Christova et al., 2014).The references included in the section “References” of the manuscript should be arranged first alphabetically and then further sorted chronologically if necessary. More then one reference from the same author(s) in the same year must be identified by the letters “a”, “b”, “c”, etc. place after the year of publication.

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Examples: Reference to a journal publication:Georgiev, P., V. Simeonov, T. Ivanov (2010). Production of thermostables enzymes. Biotechnol. Biotec. Eq. 27: 231-238. Reference to a book:John, R., W. Villiam, G. Wilmington (2011). New approach for purification of enteroviral proteins, in: Peterson, K., A. Smith (Eds.), Methods in Proteinology. Elsevier, London, pp. 281-304.Journal names should be abbreviated according to the List of Title World Abbreviations: http://www.issn.org/services/online-services/access-to-the-ltwa/Manuscripts should be submitted to the Editorial Board of the journal Acta Microbiologica Bulgarica in electronic version of the paper to the following address: [email protected] publications in Acta Microbiologica Bulgarica are free of charge. The authors of the manuscripts need to cover the costs of printing collared illustrations. https://library.caltech.edu/reference/abbreviations/

The edition of Acta Microbiologica Bulgarica volume 35 issue 3 is financially supported

by The National Science Fund of Bulgaria

(grants KP 06 NP-13/2018 and KP 06 MNF/26/05.08.2019)

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