Invertase production and molasses decolourization by cold-adapted filamentous fungus Cladosporium herbarum ER-25 in non-sterile molasses medium

Invertase production and molasses decolourization by cold-adapted filamentous fungus Cladosporium herbarum ER-25 in non-sterile molasses medium

Accepted Manuscript Title: Invertase production and molasses decolourization by cold-adapted filamentous fungus Cladosporium herbarum ER-25 in non-ste...

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Accepted Manuscript Title: Invertase production and molasses decolourization by cold-adapted filamentous fungus Cladosporium herbarum ER-25 in non-sterile molasses medium Author: Mesut Taskin Serkan Ortucu Yagmur Unver Ozden Canli Tasar Mustafa Ozdemir Haluk Caglar Kaymak PII: DOI: Reference:

S0957-5820(16)30145-8 http://dx.doi.org/doi:10.1016/j.psep.2016.07.006 PSEP 833

To appear in:

Process Safety and Environment Protection

Received date: Revised date: Accepted date:

27-10-2015 6-7-2016 12-7-2016

Please cite this article as: Taskin, M., Ortucu, S., Unver, Y., Tasar, O.C., Ozdemir, M., Kaymak, H.C.,Invertase production and molasses decolourization by cold-adapted filamentous fungus Cladosporium herbarum ER-25 in nonsterile molasses medium, Process Safety and Environment Protection (2016), http://dx.doi.org/10.1016/j.psep.2016.07.006 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Highlights 1.

A new fungal strain with high invertase activity could be isolated.

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A new non-sterile culture process for invertase production was designed.

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This process could be also applied for molasses decolourization.

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This process may reduce energy consumption and worlkoad.

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Invertase production and molasses decolourization by cold-adapted filamentous fungus

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Cladosporium herbarum ER-25 in non-sterile molasses medium

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Mesut Taskin,

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Serkan Ortucu,

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Yagmur Unver,

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Ozden Canli Tasar, 4Mustafa

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Ozdemir, 5Haluk Caglar Kaymak

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Erzurum, Turkey

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University, Erzurum, Turkey

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Adiyaman, Turkey

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Department of Biology, Science Faculty, Ataturk University, Erzurum, Turkey

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Department of Horticulture, Faculty of Agriculture, Atatürk University, Erzurum, Turkey

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Department of Molecular Biology and Genetics, Science Faculty, Erzurum Technical

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Adiyaman University, Central Research Laboratory Application and Research Centre,

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Department of Molecular Biology and Genetics, Science Faculty, Ataturk University,

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*Corresponding author:

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Mesut Taskin

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Tel.: +90 442 231 44 03

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Fax: +90 442 236 0948

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E-mail: [email protected]

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Running head: Cultivation of a cold-adapted fungus in non-sterile molasses medium

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Abstract

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This study was undertaken to remove the coloring compounds of molasses as well as produce

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extracellular (exo) invertase in sterile and non-sterile molasses medium by using cold-adapted

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filamentous fungus Cladosporium herbarum ER-25. It was determined that a combination of

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low culture pH (5.5), temperature (20 °C) and high molasses concentration (6%) could

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completely prevent undesired bacterial contamination during the cultivation of C. herbarum

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ER-25. Under the optimized non-sterile culture conditions, the maximum invertase activity

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(36.1 U/mL) was attained after 72 h. On the other hand, the fungus could remove toxical dark

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brown pigments (melanoidins) in non-sterilized molasses medium through biodegradation and

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bioadsorption mechanisms. A color removal rate of 64.8 % in non-sterile medium could be

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achieved at the end of 144-h cultivation period. It was found that laccase and manganese

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peroxidase were responsible for biodegradation. No ligninase activity was detected for the

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fungus during the cultivation. Maximum laccase (4.6 U/mL) and manganese peroxidase (3.5

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U/mL) activities could be reached after 120 h. Higher invertase activity and color removal

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rate were achieved in non-sterilized medium compared to sterilized one. This is the first report

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on invertase production from cold-adapted microorganisms under non-sterile culture

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conditions. As an additional contribution, use of cold-adapted fungi for molasses

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decolourization was investigated for the first time in the present study.

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Key words: Cladosporium herbarum ER-25; cold-adapted; invertase; non-sterile molasses;

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decolourization

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1. Introduction

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Invertase, known as beta fructofuranosidase (EC 3.2.1.26), plays a catalytic role in the

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conversion of sucrose into glucose and fructose (Rubio & Navarro, 2006). The hydrolysis of

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sucrose by invertase produces invert syrup, which contains glucose and fructose at equimolar

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concentrations. The invert syrup is used in food and beverage industries as a humectant in the

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prepration of candies, noncrystallizing creams, jams and artificial honey (Taskin et al.,

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2013a).

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So far, a number of reports on invertase production by thermophilic or mesophilic

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microorganisms have been published (Kotwal et al., 1999; Rubio et al., 2002; Shaheen et al.,

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2008, Canli et al., 2011; Taskin et al., 2013a). On the other hand, limited studies have been

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performed on the invertase production potential of cold-adapted microorganisms (Skowronek

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et al., 2003; Turkiewicz et al., 2006). However, to our knowledge, no attempt has been done

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on invertase production by cold-adapted microorganisms under non-sterile culture conditions.

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Cold-adapted (psychrophilic and psychrotrophic) microorganisms are distinguished

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from mesophiles by their ability to grow at low temperatures. The application of cold-adapted

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microorganisms in the temperature range of 0–20 °C prevents the risk of microbial

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contamination (Skowronek et al., 2003). If the temperature of culture medium is adjusted to a

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low value preventing the growth of mesophilic and thermophilic countarparts, cold-adapted

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microorganisms may be cultivated for invertase production in this medium under non-sterile

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culture condition.. Especially significant energy saving may be possible, when production

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media with large volume are directly used without sterilization. Another important property of

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cold-adapted microorganims is that these microorganims are capable of producing the cold-

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active enzymes such as amylase, lipase, protease, xylanase, cellulase, chitinase, pectinase,

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citrate synthase and DNA ligase (Joshi & Satyanarayana, 2013). In this regard, performing of

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invertase production under non-sterile culture conditions as well as exploring of cold-active

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invertase producing microorganisms may make significant contributions to biotechnological

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studies. Molasses is a co-product of sugar production from sugar beet or sugar cane. Sugar beet

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molasses is a solution of sugar, organic and inorganic matter in water with a dry substance of

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74-77% (w/w). Total sugars (mainly sucrose) of sugar beet molasses constitute approximately

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47-48% (w/w) (Kalogiannis et al., 2003). Up to now, several attempts have been made to

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produce industrially valuable substances using molasses, which is preferred due its low cost

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and high sugar content (Ergun & Mutlu, 2000; Kalogiannis et al., 2003; Taskin et al., 2012;

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Taskin et al., 2013b). Furthermore, it has been reported that molasses can be used as an

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invertase production susbtrate due to its high sucrose content (Veana et al., 2014). But, there

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is no study on use of molasses as an invertase production substrate for cold-adapted

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microorganisms. On the other hand, molasses is reported to contain dark brown pigments as

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known melanoidins. Its discharge in the soil inhibits seed germination, decrease soil alkalinity

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as well as manganese availability. In aquatic system, it blocks the sunlight penetration and

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photosynthesis (Yadav and Chandra, 2012). Therefore, removal of melanoidins from

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molasses based distillery wastewater is critically important for the environmental safety. In

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this context, it has been shown that biological treatment of molasses can be performed by

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using certain fungi and bacteria, which have the ability to remove color compounds including

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melanoidins (El-Barbary et al., 2009; Yadav and Chandra, 2012). However, no study has been

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performed on molasses decolourization potential of cold-adapted fungi. Therefore, the present study was designed to assess the potential of locally isolated

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cold-adapted filamentous fungus Cladosporium herbarum ER-25 to remove color compounds

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as well as produce invertase, laccase, manganase peroxidase and ligninase in non-sterile

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molasses medium.

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2. Materials and Methods

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2.1. Isolation, screening and identification of invertase-producer fungi

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Isolation of invertase producer fungi was performed from different soil samples collected

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around Erzurum city (Turkey) during winter in years 2014 and 2015. The isolation

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experiments were performed on petri dishes containing sterile molasses agar medium. This

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medium was prepared by dissolving 1.5 g (NH4)2 SO4, 1 g KH2PO4, 0.5 g MgS04, 0.2 g

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CaCl2, 0.2 g NaCl, 0.03 g FeSO4 and 20 g agar in one liter of 3% molasses (pH 4.0). The

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petri dishes after inoculation were incubated at 4 °C for 15 days. Filamentous fungi grown on

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agar medium were selected, sub-cultured and purified.

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For the screening procedure, the isolates were separately subjected to sporulation process

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on standard potato dextrose agar (PDA) slants at 15 °C. After a sporulation period of 168 h,

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10 mL of sterile saline water (0.9% NaCl) was added to the culture slant of each isolate, and

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the slant was then vortexed. The final concentration of each spore suspension was adjusted to

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106 spores/mL with sterile –saline water. Two milliliter of spore suspension for each isolate

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was directly transferred into a 250-mL erlenmayer flask containing 100 mL of the non-sterile

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molasses broth medium. This medium did not contain agar in contrast to the isolation

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medium. The flasks were cultured at 15 °C in a shaking incubator (ZHWY-200B, Zhicheng

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Analytical Co., Shanghai, China) at 150 rpm. After a cultivation period of 96 h, the cultures

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were centrifuged and the obtained cell-free supernatants were used for determination of

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extracellular (exo) invertase activity.

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The isolate with the highest exo-invertase activity was used for the subsequent

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experiments. The identification of the isolate was performed according to sequence analysis

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of conserved sequences in 5.8S rDNA and 28S rDNA. For this purpose, obtained sequences

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were compared to all known sequences in the Genbank by use of BLASTN 2.2.26. program

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(Zhang et al., 2000) and then deposited into GenBank with access number KP939079.1.

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2.2. Optimization of non-sterile culture conditions for invertase production and color

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removal

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The optimization experiments for exo-invertase production were carried out in 250 mL flasks

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containing 100 mL of non-sterile molasses broth medium. In the case of screening

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experiments, two milliliters of spore suspension for each isolate were used as inoculation of

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broth medium. The preliminary experiments were performed to determine the most suitable

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culture temperature (5, 10, 15, 20, 25, 30 and 35 °C). Following this, different initial pHs (pH

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4-7), molasses concentrations (1-8%) and incubation times (with 12-h intervals up to 168 h)

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were studied, respectively.

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While non-sterile culture conditions were prepared, the molasses medium and

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apparatuses were not sterilized. After the molasses medium was prepared in a beaker, it was

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transferred into the non-sterile flasks. Flasks were not sterilized and directly inoculated with

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the spore suspension of the fungus. Furthermore, the flasks were not covered with cotton

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plugs during the cultivation. Namely the media and growth flasks were open to the

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environment. To determine the degree of possible contamination in the medium, 0.1 mL

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sample taken from the culture medium was spread on a glass slide and then examined by

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using an Olympus BX51 microscope (Taskin et al., 2015).

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2.3. Analytical methods

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For the determination of exo-invertase activities and biomass concentrations, the culture

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medium of each fungus was centrifuged at 5000 rpm for 5 min at the end of cultivation

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period. After centrifugation, mycelia obtained were washed three times with distilled water to

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remove the residual medium components and then dried at 80 °C to a constant weight for the

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determination of biomass concentration.

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The obtained supernatants were used as an exo-invertase source. The invertase activity

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was assayed by measuring the reducing sugars released from sucrose. To do this, 0.1 mL of

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enzyme solution was mixed with 0.9 mL of 0.1 mol/L sodium acetate buffer (pH 5.5)

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containing 3 % sucrose (w/v). The mixture was placed in glass test tubes and incubated at 35

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°C for 10 min. Following this, the mixture was placed in a boiling water bath for 5 min to stop

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the reaction and allowed to cool at room temperature. The amount of liberated glucose was

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measured using the 3,5-dinitrosalicylic acid (DNS) method (Miller 1959). Absorbances were

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assayed at 550 nm using a spectrophotometer (UV Mini-1240, Shimadzu, Japan). The

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standard curve was prepared for DNS using fructose and glucose at equimolar concentrations.

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Exo- invertase activity is defined as the amount of enzyme required for the hydrolysis of 1

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μmol of sucrose per minute.

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For the determination of color removal rate, 5 mL sample taken from the culture was

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firstly centrifuged at 5000 rpm for 5 min and the color intensity of the solution was measured

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at 475 nm with a spectrophotometer against the control (non-inoculated medium). The

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decolourization yield was expressed as the degree of decrease in the absorbance at 475 nm

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against the initial absorbance at the same wavelength (Kumar and Chandra; 2006; Seyis and

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Subasioglu, 2009; Bharagava et al., 2009; Yadav and Chandra 2012). Percentage (%)of color

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removal in liquid medium was determined by using the following formula. % color removal=

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(Initial absorbance − observed absorbance) / (Initial absorbance × 100).

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During color removal, laccase (Lac) activity was determined using ABTS as the substrate

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(Forootanfar et al., 2000) . The reaction mixture contained 0.5 mL ABTS (5 mM) dissolved in

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100 mM acetate buffer (pH = 4.5) and 0.5 mL of culture supernatant (after diluted) followed

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by incubation at 37°C and 120 rpm. Oxidation of ABTS was monitored by an increase in

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absorbance at 420 nm (ε420 = 36,000/M cm). One unit Lac activity was defined as the amount

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of enzyme required to oxidize 1 μmol of ABTS/min. Manganese peroxidase (MnP) activity

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was determined at 468 nm using dimethoxyphenol (DMP) as the substrate (Field et al.,

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1993). Lignin peroxidase (LiP) activity was determined by the oxidation of veratryl alcohol at

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310 nm (Tien and Kirk, 1988). In order to elucidate whether bioadsorption mechanism played or not a role in color

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removal, mycelial biomass was subjected to alkaline extraction process (Jiranuntipon et al.,

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2008). For this, mycelial biomass after centrifugation was resuspended with equal volume of

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0.1 M NaOH in a tube of 50 mL. The tube was vortexed to release more color-substances

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from mycelial biomass. Optical density of NaOH-extractable color substances after

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centrifugation was measured at 475 nm (0.1 M NaOH solution was used as control). The

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vortex process was continued until the optical density of NaOH-extractable color substances

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became constant at 475 nm (about 5 min).

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2.4. Statistical analysis

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Each experiment was repeated at least three times in two replicates. The analysis of variance

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was conducted using one-way ANOVA test using SPSS 13.0 for Microsoft Windows, and

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means were compared by Duncan test at the 0.05 level of confidence.

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3. Results and Discussion

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3.1. Isolation of invertase producer cold-adapted fungus

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Microorganisms are widely used for the production of industrially valuable substances such as

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enzyme and organic acid (Taskin et al., 2012; Taskin, 2013; Taskin et al., 2013b). Especially

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cold-adapted microorganisms are very important for biotechnological applications, since their

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cold-adapted proteins and enzymes find many application fields (Skowronek et al., 2003;

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Joshi and Satyanarayana, 2013).

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It is well known that microorganisms can be cultivated under sterile or non-sterile

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culture conditions for producing industrially valuable substances and/or for the treatment of

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wastewater effluents. Especially non-sterile culture technique draws a lot of attention, since it

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can be effectively applied in the industrial scale without the sterilization process that increase

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energy and time consumption as well as workload. This technique is based on a strategy,

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which makes the target test microorganism more dominant population in the growth medium

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by eliminating or restricting undesired contaminants. For this purpose, environmental and

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nutritional factors are designed according to the growth requirements of the target test

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microorganism. For example, thermophilic microorganisms are chosen to prevent the

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contamination of their mesophilic counterparts when lactic acid production is carried out

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under non-sterile conditions (Qin et al., 2009). Similarly, it has been reported that when

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fermentation is performed at low temperatures, most undesired mesophilic contaminants can

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be prevented or limited (Margesin et al., 2002). Tao et al. (2005) adjusted the pH of

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fermentation broth to 4.5 during ethanol production by an acid tolerant Zymomonas mobilis

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mutant under non-sterilized conditions, in order to avoid undesired contaminations.

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Santamauro et al (2014) reported that when a combination of low temperature and restricted

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nutrient availability preventing undesired contaminants was used during the cultivation, lipid

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production from the yeast Metschnikowia pulcherrima could be performed under-non sterile

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culture conditions. Taskin et al (2015) informed that a cold-adapted strain of yeast Yarrowia

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lipolytica could be cultivated in non-sterile whey medium for the production of lipid-rich

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biomass.

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As mentioned above, using a microorganism that thrives at low pH and temperature of

culture media may be a potential solution when a non-sterile culture process is designed.

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The present isolation experiments showed that a total of 44 strains of cold-adapted

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filamentous fungi could grow on molasses agar medium. The isolates were then screened for

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their ability to produce exo-invertase in molasses broth medium. Because, exo-enzymes have

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some advantages compared to their intra-counterparts.

For example, purification and

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downstream process of intra-enzymes are difficult and expensive, and also cause significant

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loss of time. Conversely, purification of exo- enzymes from culture broth is easier. Also, exo-

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enzymes are generally more stable than their intra- counterparts due to the presence of sulfide

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bonds (Cheetham 1995; Bali 2003). Furthermore, exo-enzymes can be produced at large

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amounts by using immobilized cells.

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The results elucidated that although all of thirty-five isolates could grow in the molasses

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broth medium, none of them showed exo-invertase activity. This finding indicated that intra-

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invertases of thirty-five isolates played role in the utilization of molasses sucrose as carbon

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source. Although the rest nine isolates had exo- invertase activity, some (ER-3, -15 and -17)

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of them showed poor growth performance in molasses broth medium. Of total 44 fungal

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isolates, the isolate ER-25 exhibited the maximum exo-invertase activity (17.7 U/mL) (Table

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1). Furthermore, the maximum color removal (72.4 %) was achieved using the isolate ER-25.

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As a result, the subsequent experiments were performed with the isolate ER-25. This isolate

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was identified as Cladosporium herbarum.

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The experiments also showed that higher biomass concentrations were attained for the

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isolates ER-14 and -39; however, exo-invertase activities of both isolates were relatively

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lower. This result was due to probably the conversion of invert sugars to organic acids rather

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than mycelial biomass. This finding is in accordance with some earlier reports showing that

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invertase activity is not strictly correlated with mycelial biomass concentration (Chaudhuri et

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al., 1999; Ikram-Ul-Haq & Ali, 2007).

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Table 1

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3.2. Design of non-sterile culture conditions for invertae production

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The preliminary optimization experiments showed that no bacterial contamination occurred at

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5 -20 °C, when the medium pH and molasses concentration were adjusted to 4.0 and 3%,

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respectively. Whereas, level of bacterial contamination was little at 25 °C and moderate at 30

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and 35 °C. Relatively higher fungal biomass and invertase activity were reached at the temperature

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of 20 °C (Table 2). Temperatures above 20 °C significantly inhibited fungal growth and exo-

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invertase activity. Besdies, no fungal growth and exo-invertase activity were observed at 35

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°C. Based these results, it was expected that there might be an invertase activity at 35 °C,

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since unwanted bacteria could grow at this temperature by using molasses sucrose as carbon

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source. But, it was seen that there was no exo-invertase activity in the medium at 35 °C.

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Therefore, effective use of molasses sucrose as carbon source at 35 °C could be attributed to

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the presence of intra-invertases in bacterial contaminants.

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It was also concluded from the present results that cold-adapted Cladosporium

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herbarum ER-25 had a psychrotolerant character. This is because that obligate psychrophiles

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are not able to grow at temperatures above 20 °C, whereas facultative psychrophiles

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(psychrotolerant) can tolerate temperatures above 20 °C (Rossi et al. 2009). Taking into

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account of these results, the subsequent experiments were carried out at the temperature of 20

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°C, which resulted in the maximum biomass and exo-invertase activity.

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Table 2

The experiments exhibited that there were no detectable signs of bacterial

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contamination at the pH values ≤ 4.5 when the temperature was stable at 20 °C. But, low

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fungal growth and exo-invertase activities were determined at the pH values of ≤ 4.5. Degree

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of bacterial contamination was little at pH 5.0 and 5.5, whereas it was moderate at pH 6.0. At

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pH values ≥ 6.5, there was significant bacterial contamination but a poor fungal growth and

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low exo-invertase activity. As seen from the Table 3, the maximum biomass concentration

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and exo-invertase activity could be reached at pH 5.5. These results demonstrated that even if

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a combined application of low culture pH (5.5) and temperature (20 °C) resulted in higher

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exo-invertase activity, it was not enough to completely prevent undesired bacterial

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contaminants Table 3

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Selecting a suitable nutrient medium for commercial production of microbial products is

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usually considered as a major aspect for improvement (Tinoi et al., 2005). Molasses is a good

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substrate for microorganisms because it contains sugars (mainly sucrose), nitrogenous

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compounds and vitamins. Molasses is also reported to contain macro, micro and trace

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elements such as phosphorus (P), sulfur (S), magnesium (Mg), iron (Fe), zinc (Zn), copper

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(Cu) and manganese (Mn) (Van der Poel et al., 1998; Survase et al., 2007; Yilmaztekin et al.,

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2008). Most of these elements are essential for microbial activities. For instance, P

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participates in the structure of nucleic acids, phospholipids, adenosine triphosphate (ATP),

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many cofactors and some proteins. S is required for synthesis of the amino acids cysteine and

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methionine, the vitamins thiamine and biotin and some carbohydrates. Mg acts as a cofactor

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for many enzymes and complexes with ATP (Prescott et al., 2002). K is required for the

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activities of many enzymes involved in protein synthesis. K also plays role in maintenance of

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osmotic balance in cells. Fe participates in the structure of cytochromes and functions a

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cofactor for enzymes. Mn aids many enzymes to catalyze the transfer of phosphate groups

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(Prescott et al., 2002; Mara and Horan, 2003). Considering the its low cost and rich nutritional

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composition, sugar beet molasses was selected as an exo-invertase production substrate in the

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present study.

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The most suitable concentration of molasses for the fungal growth and exo-invertase

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production was found to be 5%. A molasses concentration of 6% caused a little decrease in

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biomass concentration and exo-invertase activity. But, a significant inhibitory effect on both

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of them was observed at the molasses concentrations of ≥ 7%.

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There was a little bacterial contamination at the molasses concentrations of ≤ 5%.

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Conversely, undesired bacterial contamination could be completely prevented at the molasses

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concentrations of ≥ 6% (Table 4). This inhibitory effect on fungal and bacterial growth might

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be attributed to the presence of some toxic compounds inside molasses. Namely the amount

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of toxic compounds increased in the medium when the molasses concentration was increased.

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Finally, these toxic compounds might exert more inhibitory effect on fungal and bacterial

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growth. This assumption should have not been so surprising, since a previously published

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study demonstrated that molasses contained some phenolic compounds possessing

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antibacterial effect (Takara et al., 2007).

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The experiments showed that there was no significant difference between exo-invertase

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activities obtained at the molasses concentrations of 5 and 6%. Therefore, the subsequent

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experiments were performed at the molasses concentration of 6%, where undesired bacterial

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contamination could be completely prevented.

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Table 4

An appropriate incubation period is of critical importance for invertase synthesis, as

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prolonged incubation can decrease the enzyme production (Ikram-ul-Haq et al., 2005;

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Shankar

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production ceased with 36.1 U/mL enzyme activity after 72 h, the fungal growth continued up

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to 120th h (Figure 1). That is to say even if molasses sucrose was completely exhausted in the

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medium, the biomass concentration continued to increase between 72 and 120 h. This

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situation could be attributed to the utilization as carbon source of invert sugars (glucose and

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fructose), which were generated by invertase. Another reason of this increase might be due to

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the utilization as carbon source of phenolic compounds and/or melanoidins inside molasses,

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as reported in the previous study (Yadav and Chandra, 2012).

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& Mulimani, 2007). The results demonstrated that although exo-invertase

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The experiments also showed that the enzyme activity was stable between 72 and 96 h.

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This situation can be explained by the fact that the excessive invert sugar production in

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growth media causes glucose-induced feedback repression of invertase (Ikram-ul-Haq et al.,

332

2005). Besides, another reason might be probably the depletion of sucrose, which is a

333

substrate of invertase. On the other hand, it was determined that an incubation time over 96 h

334

gave rise to reductions in invertase activity, probably due to loss of enzyme stability.

335

ip t

331

3.3. Decolourization of molasses under non-sterile culture conditions

337

Dark brown pigments (melanoidins) of molasses can cause important environmental

338

problems, when they are directly discharged to soil and aquatic systems without pretreatment.

339

To solve this problem, fungi and bacteria can be used for the

340

containing molasses (El-Barbary et al., 2009; Seyis and Subasioglu, 2009; Yadav and

341

Chandra, 2012).

us

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336

M

an

treatment of effluents

The present study was maninly focused on producing invertase in non-sterile molasses

343

medium by using the cold adapted fungus. However, potential of this fungus to remove color

344

compounds of molasses under non sterile culture conditions was also studied to some extent.

te

d

342

The experiments demonstrated that the color removal potential of the fungus was low

346

within the first 24 h of cultivation in the molasses medium; however, it became high after

347

24nd h. Furthermore, it became clear that although the fungal growth ceased after 120 h, the

348

fungus continued to decolorize molasses medium (Figure 1). For example, color removal rate

349

reached to 64.8 % after a 144 h cultivation period. It is widely reported that living biomass of

350

fungi uses biodegradation and/or bioadsorption mechanisms in decolourization of molasses

351

and textile dyes (Fu and Viraraghavan, 2001; Pant and Adholeya, 2007). Especially it has

352

been documented that biodegradative abilities of fungi are depended on their extracellular

353

ligninoloytic enzymes such as laccases (Lac), lignin peroxidases (LiP) and manganese

354

peroxidases (MnP) (Sarnthima et al., 2009; Ballaminut et al., 2014; Kachlishvili et al.,

355

2014).). Hence, we decided to analyze the activities of three enzymes in order to elucidate

Ac ce p

345

Page 15 of 32

16 356

whether biodegradation mechanism became effective or not in color removal. As seen from Figure 1, there were significant increases in activities of MnP and Lac

358

between 24-72 h. Although activities of MnP and Lac increased between 72 and 120 h, these

359

increases were at low levels. Furthermore, activities of two enzymes did not show an increase

360

after 120 h. In brief, Lac and MnP of C. herbarum were detected simultaneously at the 24th h

361

and both enzymes showed a maximum activity peak at the 120th h. These results implied that

362

the fungus produced Lac and MnP in simultaneous manner. The similar trend was also

363

reported in the previous studies for Lac and MnP activities of some fungi such as

364

Cladosporium cladosporoides, Ceriporiopsis subvermispora, Cerrena unicolor, Lentinus

365

crinitus and Lentinus polychrous (Sarnthima et al., 2009; Babič et al., 2012; Ballaminut et al.,

366

2014; Ji et al., 2014; Kachlishvili et al., 2014). Conversely, some investigators reported that

367

Lac and MnP of some fungi and bacteria were produced in sequential manner (Arakaki et al.,

368

2011; Bettin et al., 2013; Yadav and Chandra, 2013). For example, Bettin et al (2011)

369

reported that Lac of Pleurotus sajor-caju was detected on the second day, showing an activity

370

peak on the sixth day and a decrease in activity after seven days. But, they reported that MnP

371

activity of this fungus was detected only on the fourth, ninth and 15th days. Yadav and

372

Chandra (2013) noted that when a mixed culture of different bacteria was used for

373

decolourization, the maximum MnP activity in the culture was reached after 96 h but the

374

maximum Lac activity after 120 h.

cr

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M

d

te

Ac ce p

375

ip t

357

In contrast to Lac and MnP, no LiP activity was detected in the medium during a

376

cultivation period of 168 h. Although too low increases in MnP and Lac activities occured

377

between 72 and 120 h, approximately 23% of total color removal (64.8 %) was achieved in

378

this time range. Hence, color removal before 120 h could be ascribed to not only

379

biodegradation (enzymatic activities) but also bioadsorption mechanism (hyphal uptake

380

mechanism). When the mycelial biomass obtained within the first 120 h was extracted with

Page 16 of 32

17

NaOH solution, brown color-compounds adsorbed by mycelial biomass passed into this

382

alkaline solution again. The experiments indicated that even if MnP and Lac activities

383

decreased after 120 h, the fungus continued to decolorize molasses medium between 120 and

384

144 h. Therefore, it was assumed that the color removal observed after 120th h might be

385

associated with mainly bioadsorption mechanism. When optical density of NaOH-extractable

386

color substance was assayed, it was seen that a mycelial biomass of 144 h adsorbed more

387

brown color-compounds (about 3%) than a mycelial biomass of 120 h. This value was very

388

closed to difference (3.3%) between color removal rates (respectively 61.5% and 64.8 %),

389

which were recorded in the molasses medium at 120 and 144 h. Considering the present

390

results, it was concluded that both biodegradation and bioadsorption potential of the fungus

391

became effective during color removal in molasses medium.

M

an

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cr

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381

392

Figure 1

3.4. Invertase production and molasses decolourization under sterile culture conditions

394

To compare the invertase production and color removal in sterilized and non-sterilized

395

molasses, the experiments were also conducted in non-sterilized molasses medium at the same

396

optimal culture conditions. Figure 1 and 2 show that lower invertase activity (29.4 U/mL),

397

biomass concentration (7.2 g/L) and color removal rate (61.3 %) were attained in sterilized

398

molasses medium. Some investigators have reported that sugar and nitrogen sources inside

399

molasses may be partially lost during sterilization process (Tao et al., 2005; Kopsahelis et al.,

400

2012). In this regard, we assumed that obtaining of lower exo-invertase activity in sterilized

401

molasses medium might be mainly due to partial decomposition of sucrose. Similarly,

402

obtaining of less biomass might be due to a decrease in concentration of sugar and/or nitrogen

403

sources, which are required for the fungal growth. Decrease in adsorption rate of color

404

compounds in the non-sterile medium might be due to probably the decreasing in biomass

405

concentration.

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Page 17 of 32

18 406

Figure 2 4. Conclusions

408

This study proved that a combination of low pH (5.5) and temperature (20 °C) as well as high

409

molasses concentration (6%) could make the cells of cold-adapted filamentous fungus

410

Cladosporium herbarum ER-25 more dominant population in non-sterile molasses medium.

411

Non-sterile molasses medium and culture equipments could be directly used for production of

412

three extracellular enzymes (invertase, laccase and manganase peroxidase). Furthermore, the

413

fungus showed a high potential (64.8%) to remove color compounds in non-sterile molasses

414

medium by using biodegradation and bioadsorption mechanisms. When ambient temperature

415

of environments such as laboratories, bioreactors and waste treatment plants becomes in the

416

range of 10-20 °C, cultivation media may not require any sterilization process since the

417

fungus can grow in this temperature range. Especially countries having cold climate may use

418

this process without temperature control. Furthermore, immobilized cells of this fungus may

419

be used for continuous enzyme production and/or molasses treatment in non-sterile medium.

te

d

M

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407

420

Compliance with Ethical Standards

422

The manuscript does not contain experiments involving Human Participants and/or Animals.

423

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421

424

Conflict of Interest

425

The authors declare that they have no conflict of interest. The authors alone are responsible

426

for the content and writing of the paper.

427 428 429 430

Page 18 of 32

19

References

432

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Bali, R., 2003. Isolation of extracellular fungal pectinolytic enzymes and extraction of pectic

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distillery spent wash (BMDS) and their degradation by manganese peroxidase and

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laccase producing bacterial strains. J. Env. Biol. 34, 755.

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Beet Molasses by Williopsis saturnus var. Saturnus. J. Inst. Brew. 114, 34-38.

592

Zhang, Z., Schwartz, S., Wagner, L., Miller, W., 2000. Greedy Algorithm for Aligning DNA

595

d

te

594

Sequences. J. Comput. Biol. 7, 203–214.

Ac ce p

593

M

590

Page 25 of 32

26

BC (g/L)

EIA (U/mL)

CR (%)

ER-3

3.7±0.10f

15.4±0.22e

17.5

ER-14

5.6±0.15a

11.8±0.10h

21.2

ER-15

3.8±0.10f

13.1±0.15f

31.2

ER-17

3.8±0.10f

16.4±0.15c

40.6

ER-21

4.1±0.17e

11.3±0.15i

ER-25

4.9±0.21c

17.7±0.26a

ER-33

4.6±0.10d

16.7±0.20b

62.1

ER-34

5.3±0.15b

15.9±0.25d

57.4

ER-39

5.4±0.20b

12.3±0.10g

58.0

cr

ip t

IC

an

Table 1. Screening of potential invertase producing fungi strains

42.3

us

72.4

M

595

All values are mean ± standard error of six determinations (n = 6). Alphabet letters with the

597

same letters in the same column are not significantly different at p≤0.05. IC, isolate code; BC,

598

biomass concentration; EIA, extracellular invertase activity and CR, color removal. Screening

599

conditons: Temperature = 15 °C, incubation time = 96 h, pH=4.0 and molasses concentration

600

= 3%.

602 603 604 605

te

Ac ce p

601

d

596

606 607 608 609

Page 26 of 32

27

611

ER25 under non-sterile culture conditions BC (g/L)

EIA (U/mL)

CD

5

1.9±0.10e

5.8±0.20f

-

10

4.1±0.10c

13.3±0.30d

-

15

4.9±0.15a

17.7±0.40b

-

20

5.1±0.20a

18.4±0.35a

25

4.6±0.10b

16.2±0.35c

30

3.2±0.15d

9.1±0.10e

++

35

NG

ND

++

cr

T (°C)

ip t

Table 2. Effect of temperature on invertase production from Cladosporium herbarum

an

610

-

us

+

All values are mean ± standard error of six determinations (n = 6). Alphabet letters with the

613

same letters in the same column are not significantly different at p≤0.05. T, temperature; BC,

614

biomass concentration; EIA, extracellular invertase activity, NG, no growth; ND, not

615

determined and CD, contamination degree. (-) = no contamination, (+) = low (little)

616

contamination, (++) = moderate contamination and (+++) = high contamination. Culture

617

parameters: incubation time = 96 h, pH=4.0 and molasses concentration = 3%.

619 620 621 622 623

d

te

Ac ce p

618

M

612

624 625 626 627 628

Page 27 of 32

28

Table 3. Effect of initial pH on invertase production from Cladosporium herbarum ER25

630

under non-sterile culture conditions BC (g/L)

EIA (U/mL)

CD

4.0

4.1±0.20e

14.3±0.20e

-

4.5

4.8±0.20c

17.3±0.15c

-

5.0

5.1±0.15b

18.4±0.30b

+

5.5

5.9±0.10a

19.2±0.20a

6.0

4.6±0.10d

16.4±0.40d

6.5

3.7±0.15f

13.1±0.10f

+++

7.0

2.1±0.25g

7.5±0.40g

+++

cr

ip t

pH

an

629

+

us

++

All values are mean ± standard error of six determinations (n = 6). Alphabet letters with the

632

same letters in the same column are not significantly different at p≤0.05. T, temperature; BC,

633

biomass concentration; EIA, extracellular invertase activity and CD, contamination degree. (-)

634

= no contamination, (+) = low (little) contamination, (++) = moderate contamination and

635

(+++) = high contamination. Culture parameters: temperature= 20 °C, incubation time = 96 h

636

and molasses concentration = 3%.

638 639 640 641

d

te

Ac ce p

637

M

631

642 643

Page 28 of 32

29

Table 4. Effect of molasses concentration on invertase production from Cladosporium

644

herbarum ER25 under non-sterile culture conditions BC (g/L)

EIA (U/mL)

CD

1

3.4±0.20f

6.9±0.20g

+

2

4.6±0.20e

13.6±0.35f

3

5.9±0.40d

19.2±0.40d

4

6.8±0.40bc

28.4±0.60c

5

7.5±0.45a

36.4±0.50a

6

7.3±0.20ab

36.1±0.55a

-

7

6.6±0.20c

35.1±0.40b

-

8

5.1±0.25e

14.4±0.15e

-

ip t

MC (%)

an

643

+

+ +

M

us

cr

+

All values are mean ± standard error of six determinations (n = 6). Alphabet letters with the

646

same letters in the same column are not significantly different at p≤0.05. BC, biomass

647

concentration; EIA, extracellular invertase activity; MC, molasses concentration and CD,

648

contamination degree. (-) = no contamination, (+) = low (little) contamination, (++) =

649

moderate contamination and (+++) = high contamination. Culture parameters: temperature =

650

20 °C, initial pH=5.5 and incubation time = 96 h.

652 653

te

Ac ce p

651

d

645

Page 29 of 32

30

Figure legends

654

Figure 1. Effect of incubation time on invertase production and color removal potential

655

of Cladosporium herbarum ER-25 under non-sterile culture conditions. CR, color

656

removal: Lac, laccase; MnP, manganese peroxidase; BC, biomass concentration and EIA,

657

extracellular invertase activity. Culture parameters: temperature = 20 °C, initial pH=5.5 and

658

molasses concentration = 6%.

cr

ip t

653

659

Figure 2. Effect of incubation time on invertase production and color removal potential

661

of Cladosporium herbarum ER-25 under sterile culture conditions. CR, color removal:

662

BC, biomass concentration and EIA, extracellular invertase activity. Culture parameters:

663

temperature = 20 °C, initial pH=5.5 and molasses concentration = 6%.

an

us

660

M

664

Ac ce p

te

d

665

Page 30 of 32

31 665

Ac ce p

te

d

M

an

us

cr

ip t

Figure 1

666 667

Page 31 of 32

32 667

Figure 2

668

671

Ac ce p

670

te

d

M

an

us

cr

ip t

669

Page 32 of 32