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Progress of Cryogenics

Progress of Cryogenics

and

and

Isotopes Separation

Isotopes Separation

Volume 19, issue 1/2016

Contents:

Influence of the Thermal Energy Storage on the Hybrid Heating Systems' Energy Profile

*

Synthesis and Characterization of Iodine Doped Graphene Byan Uncatalyzed Reaction

Ammonia Removal from Aqueous Solution by Ion Exchange Using Clinoptilolite and Verification of the ProcessThrough a Numerical Study

Numerical Analysis on The Hydrogen Generation fromSodiumBorohydride Hydrolysis

The Removal of Heavy Metals Using the Bulk Liquid MembraneTechnique

Macro- and Microelements as Possible Markers of Quality and Authenticity for Fruits and Derived Products

Assessing the Authenticity of Vinegar by Combined

2

H-SNIF-NMR and

13

C-IRMSAnalysis

Identification of New Isolates (Micro-Organisms) from Vineyard Epiphytic Microflora Using Maldi- Tof Mass Spectrometry

Sono - Chemical Degradation of Organic Compounds fromWastewater

Determination of Physico-Chemical Parameters and Water Quality Index (WQI) of OltRiver

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NATIONAL R ESEARCH AND DEVELOPMENT INSTITUTE FOR

CRYOGENICS AND ISOTOPIC TECHNOLOGIES

NATIONAL R ESEARCH AND DEVELOPMENT INSTITUTE FOR CRYOGENICS AND ISOTOPIC TECHNOLOGIES
NATIONAL R ESEARCH AND DEVELOPMENT INSTITUTE FOR CRYOGENICS AND ISOTOPIC TECHNOLOGIES
NATIONAL R ESEARCH AND DEVELOPMENT INSTITUTE FOR CRYOGENICS AND ISOTOPIC TECHNOLOGIES
NATIONAL R ESEARCH AND DEVELOPMENT INSTITUTE FOR CRYOGENICS AND ISOTOPIC TECHNOLOGIES
NATIONAL R ESEARCH AND DEVELOPMENT INSTITUTE FOR CRYOGENICS AND ISOTOPIC TECHNOLOGIES

CONPHYS Press

CONPHYS Press

Râmnicu Vâlcea

R

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mnicu V

â

lcea

Volume 19, issue 1/2016

National Research and Development Institute for Cryogenics and Isotopic Technologies - ICSI Rm. Valcea -

Progress of Cryogenics and Isotopes Separation

CONPHYS Press

Rm. Valcea

Progress of Cryogenics and Isotopes Separation

EDITORIAL BOARD

Alexander ANDREEV

Russian Academy of Sciences, Moscow

Rei FERNANDES

Superior Technical Institute, Lisboa, Portugal

Sema Z. BAYKARA

Yildiz Technical University, Istanbul, Turkey

Ashok VASEASHTA

Institute for Advanced Sciences Convergence Northfield, VT and Arlington, USA

Marius PECULEA

Romanian Academy

Konstantin PETROV

Institute of Electrochemistry and Energy System of Bulgarian Academy of Sciences

Pegie COOL

University of Antwerp, Department of Chemistry Antwerp, Belgium

Progress of Cryogenics and Isotopes Separation, having a publication frequency of 2 issues per year, was founded in 1996, with the aim to emphasis the scientific achievement within a wide range of isotopes application. The journal is indexed in various reputed databases: Contemporary Science Association (CSA), EBSCO, ULRICH'S, SCIPIO, GALE, INSPEC, INIS Romania. The journal seeks to cover a wide range of issues regarding isotopic processes and isotopes application with particularly emphasize on interdisciplinary studies, thus making it attractive both to readers and to those submitting manuscripts. The main topic areas of the journal are: Isotopes Stable and Cryogenic Technologies; Nuclear Energy – Fission and Fusion; Energetic Technologies of Hydrogen; Science and Materials Engineering; Environmental, Life Quality and Food Safety.

The papers published in Progress of Cryogenics and Isotopes Separation should present novel results and have either theoretical significance or practical utility or both. They may be presented in the form of full articles or short communications.

Senior Editor: Ioan STEFANESCU - National Research and Development Institute for Cryogenics and Isotopic Technologies – ICSI Rm. Valcea, Romania Editor: Vasile STANCIU – National Research and Development Institute for Cryogenics and Isotopic Technologies – ICSI Rm. Valcea, Romania

Technical box:

Layout: Camelia ASPRITA Cover: Camelia ASPRITA

Printed at: CONPHYS Typography Rm. Valcea Phone no.: +40-250-731512, +40-250-733323 E-mail: conphys@conphys.ro

Edited by:

ISSN: 1582-2575

National Research and Development Institute for Cryogenics and Isotopic Technologies – ICSI Rm. Valcea

Progress of Cryogenics and Isotopes Separation

Volume 19, issue 1/2016

National Research and Development Institute for Cryogenics and Isotopic Technologies - ICSI Rm. Valcea -

Influence of the Thermal Energy Storage on the Hybrid Heating Systems' Energy Profile * Teodora Melania Șoimoșan, Raluca Andreea Felseghi ........................

5

Synthesis and Characterization of Iodine Doped Graphene Byan Uncatalyzed Reaction Adriana Marinoiu, Elena Carcadea, Irina Petreanu, Elena Marin, Bianca Sucea, Amalia Soare ..................................................................

19

Ammonia Removal from Aqueous Solution by Ion Exchange Using Clinoptilolite and Verification of the Process Through a Numerical Study

Claudia Cobzaru, Adriana Marinoiu, Gabriela Antoaneta Apostolescu, Ramona Elena Tataru-Farmus, Nicolae Apostolescu, Corina Cernatescu ...................................................................................

27

Numerical Analysis on the Hydrogen Generation from Sodium Borohydride Hydrolysis

Elena Carcadea, Mihai Varlam, Ioan Stefanescu, Adriana Marinoiu, Mircea Raceanu .......................................................................................

35

The Removal of Heavy Metals Using the Bulk Liquid Membrane Technique

Cristina Monica Mirea, Ioana Diaconu, Elena Ruse, Ecaterina Anca Serban, Dumitra Daniela Clej, George Alexandru Popa, Daniela Florentina Popa (Enache), Gheorghe Nechifor ..........................................

45

Macro- and Microelements as Possible Markers of Quality and Authenticity for Fruits and Derived Products

Roxana Elena Ionete, Oana Romina Dinca, Elisabeta Irina Geana, Diana Costinel ........................................................................................

55

Assessing the Authenticity of Vinegar by Combined 2 H-SNIF-NMR and 13 C-IRMS Analysis Popescu Raluca, Botoran Oana, Costinel Diana, Ionete Roxana Elena

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Identification of New Isolates (Micro-Organisms) from Vineyard Epiphytic Microflora Using Maldi-Tof Mass Spectrometry Iuliana Diana Bărbulescu, Constanţa Mihai, Mihaela Begea, Mihai Frîncu, Liviu Grigorică, Gabriel Roceanu, Simona-Ioana Marinescu, Răzvan Teodorescu, Radu Tamaian......................................................

83

Sono - Chemical Degradation of Organic Compounds from Wastewater

Mihaela Iordache, Ioan Iordache, Luisa Roxana Popescu, Dorin Schitea, Luoana Florentina Pascu ......................................................................

91

Determination of Physico-Chemical Parameters and Water Quality Index (WQI) of Olt River Claudia Sandru, Elena David ................................................................

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Instructions for Authors

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Progress of Cryogenics and Isotopes Separation

Volume 19, issue 1/2016

IDENTIFICATION OF NEW ISOLATES (MICRO-ORGANISMS) FROM VINEYARD EPIPHYTIC MICROFLORA USING MALDI-TOF MASS SPECTROMETRY

Iuliana Diana Bărbulescu 1* , Constanţa Mihai 2 , Mihaela Begea 3 , Mihai Frîncu 2 , Liviu Grigorică 4 , Gabriel Roceanu 5 , Simona-Ioana Marinescu 1 , Răzvan Teodorescu 2 , Radu Tamaian 6,7,8*

1 Pharmacorp Innovation SRL, Bucharest, Romania; 2 University of Agronomic Sciences and Veterinary Medicine Bucharest, Romania; 3 University Politehnica of Bucharest, Romania; 4 Bevitech SRL, Bucharest, Romania; 5 Carl Reh Winery SRL, Bucharest, Romania; 6 National Research and Development Institute for Cryogenics and Isotopic Technologies - ICSI Rm. Valcea, Uzinei Street no. 4, P.O. Box Râureni 7, 240050, Râmnicu Vâlcea, Romania; 7 University of Bucharest, Faculty of Physics, 3Nano-SAE Research Centre, Bucharest-Măgurele, Romania; 8 Biotech Corp SRL, 4th Uzinei Street, Râmnicu Vâlcea, Romania

ABSTRACT

This paper presents the identification of new isolates (micro-organisms) from mature grapes berries & leaves of specific grape varieties and soil from Oprișor area, using MALDI-TOF mass spectrometry. Red grape varieties taken in study were: Cabernet Sauvignon, two Merlot varieties and Shiraz. In the previous study we identified yeast by MALDI-TOF from this wine area. For each variety of red grapes, leaves and soil samples have been also studied. The main micro- organisms from soil were Bacillus sp. from Cabernet area. From Shiraz and Merlot grapes samples the MALDI-TOF score value was 1.26 respectively 1.552, indicating a not reliable identification using MALDI-TOF mass spectrometry. These samples have been also characterized from microscopic and macroscopic point of view. Part of the samples which did not give reliable results by MALDI-TOF mass spectrometry will be the object for the next studies for more precise identification using techniques of molecular taxonomy.

Keywords: MALDI-TOF mass spectrometry, wines, yeast, bacteria.

1. INTRODUCTION

1.1. Microorganims and wines

It is known that Romania has a wide variety of wines, of which more than 100 are old, native varieties.

* Corresponding authors: Diana Iuliana Barbulescu, E-mail:barbulescudia@yahoo.com and Radu Tamaian, E-mail: radu.tamaian@icsi.ro phone: 0250 732744, fax: 0250 732746

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IDENTIFICATION OF NEW ISOLATES (MICRO-ORGANISMS) FROM VINEYARD EPIPHYTIC MICROFLORA USING MALDI-TOF MASS SPECTROMETRY

Šuranská H. et al. (2016) have chosen some indigenous strains coming from organically treated grape berries, for their oenological properties, and these strains will be used as the starter culture, because the application of an indigenous S. cerevisiae strain can enhance the regional character of the wines. In the past decade we have witnessed an explosion in the number of studies that use comparative genomic analyses, between both strains and species, to precisely dissect biological processes (Nieduszynski C. A. and Liti G., 2011).

  • 1.2. Yeasts

Yeasts are unicellular microorganisms, and now more than 1000 kinds of yeasts are used in brewing, food, medicine and other industries (Zhaochunhai,

2012).

Samples of grapes, leaves and soil have been collected during the harvest of 2014 and have been used to identify and characterize the isolated wine yeast strains using a Bruker microflex™ LT/SH MALDI-TOF mass spectrometer with nitrogen laser (Bărbulescu I. D. et al., 2015). Brettanomyces genus, with Brettanomyces bruxellensis being the predominant species, is reported to be spoilage yeasts that produce flavors in beer and wine. The aroma characteristics of their spoilage-causing metabolites are typically described as some other unpleasant odors, resulting in wines and beers that are less preferred by consumers (Crauwels S. et al., 2014).

  • 1.3. Bacteria

Despite its importance in plant health and crop quality, the diversity of epiphytic bacteria on grape berries, leaves and bark, is still poorly described, as does the role of telluric bacteria in plant colonization (Martins G. et al., 2013). Rousseau S. and Donèche B. (2015) presented results which can contribute to control epiphytic micro-organisms in over ripened fruit of grapevine. A decrease in water availability induced growth inhibition of all microbial strains. Okolie et al. (2013) evaluated, by DNA based method using the 16S rDNA of the microbial community, a bacterial diversity and also studied the fermentation dynamics in palm wine. The results showed that bacterial diversity in the palm wine sample is dominated by Lactobacillus and Leuconostoc species.

2. MATERIALS AND METHODS

  • 2.1. Ethics statement

All the samples in this study were collected from Oprișor area private proprieties, and the owners of the vineyards gave the permission to do the study.

  • 2.2. Soil sampling

Soil samples from Shiraz, Cabernet and Merlot were collected in plastic

bags.

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Iuliana Diana Bărbulescu, Constanţa Mihai, Mihaela Begea, Mihai Frîncu, Liviu Grigorică, Gabriel Roceanu, Simona-Ioana Marinescu, Răzvan Teodorescu, Radu Tamaian

2.3. Sampling grapes and leaves

Red grape varieties taken in study were: Cabernet Sauvignon, 2 Merlot varieties and Shiraz. At each variety sampling point, approximately 250-500 g of undamaged grapes have been aseptically removed from several sterile bags. Colony isolation has been done in the microbiological safety cabinet. For each sample were carried out decimal dilutions until 10 6 . The samples were spread on different media (YPG yeast extract, glucose and peptone agarised media, Gelose media and Sabouraud media) and cultivated at static conditions on MEMMERT incubator chamber. The isolates were the subject of identification by MALDI-TOF mass spectrometry.

2.4. Identification and characterization of new isolates using MALDI- TOF mass spectrometry

The equipment used for identification was a Bruker microflex™ LT/SH MALDI-TOF mass spectrometer. Peptide mass fingerprinting using a Bruker microflex™ LT/SH MALDI- TOF mass spectrometer with nitrogen laser was performed in order to identify and characterize the isolated yeast and bacteria strains. Isolates were processed using multiple measurements in order to ensure that their true biological variability was acquired. The acquired spectra were processed with the MALDI Biotyper 3.0 software.

3. RESULTS AND DISCUSSIONS

After the spectra have been obtained, the MALDI Biotyper software classified most of the bacteria isolates as species included in order Bacillus, with members from the following families (Table 1-4):

Table 1

Sample

Identified organism

Score Value

Scoring

ID

(alternative name)

   

2.265

Consistency

M3

Bacillus cereus

'green'

( A )

 

Candida pelliculosa (Pichia

2.197

 

M4

anomala) (Bărbulescu I. D. et al., 2015)

'green'

( A )

 

Kloeckera apiculata

2.351

 

M8

(Hanseniaspora uvarum) (Bărbulescu I. D. et al., 2015)

'green'

( A )

   

1.289

 

M10

not reliable identification

'red'

( C )

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IDENTIFICATION OF NEW ISOLATES (MICRO-ORGANISMS) FROM VINEYARD EPIPHYTIC MICROFLORA USING MALDI-TOF MASS SPECTROMETRY

 
   

1.552

 

MCR7

not reliable identification

'red'

( C )

 

Candida pulcherrima

1.884

 

SH4

(Metschnikowia pulcherrima) (Bărbulescu I. D. et al., 2015)

'yellow'

( B )

   

1.266

 

SH6

not reliable identification

'red'

( C )

   

1.782

 

CS1

Bacillus subtilis

'yellow'

( B )

   

1.808

 

CS5

Bacillus mojavensis

'yellow'

( B )

   

1.824

 

CS7

Bacillus mojavensis

'yellow'

( B )

   

1.846

 

CS9

Bacillus vallismortis

'yellow'

( B )

   

1.855

 

CS-Sol

Bacillus subtilis

'yellow'

( B )

   

1.81

 

Fz-CS

Bacillus mojavensis

'yellow'

( B )

   

1.786

 

CS Solb

Anaerococcus sp.

'yellow'

( B )

 

Candida pelliculosa

2.261

 

M4

(Pichia anomala) (Bărbulescu I. D. et al., 2015)

'green'

( A )

 

Candida pulcherrima

2.045

 

MCR2

(Metschnikowia pulcherrima)

'green'

( A )

Table 2. Meaning of Score Values (explanations to Table no 1)

Range

Description

2.300

3.000

highly probable species identification

2.000

2.299

secure genus identification, probable species identification

1.700

1.999

probable genus identification

0.000

1.699

not reliable identification

Table 3. Meaning of Consistency Categories (A - C) (explanations to Table no 1)

Category

Description

A

Species Consistency: The best match was classified as 'green' (see above). Further 'green' matches are of the same species as the first one. Further 'yellow' matches are at least of the same genus as the first one.

B

Genus Consistency: The best match was classified as 'green' or 'yellow' (see above). Further 'green' or 'yellow' matches have at least the same genus as the first one. The conditions of species consistency are not fulfilled.

C

No Consistency: Neither species nor genus consistency, classified as 'red' (see above).

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Iuliana Diana Bărbulescu, Constanţa Mihai, Mihaela Begea, Mihai Frîncu, Liviu Grigorică, Gabriel Roceanu, Simona-Ioana Marinescu, Răzvan Teodorescu, Radu Tamaian

Table 4. Matching Hints (explanations to Table no 1)

Matched

 

Pattern

Comment

Bacillus

Bacillus anthracis, cereus, mycoides, pseudomycoides, thuringien-

cereus

sis and weihenstephanensis are closely related and members of the Bacillus cereus group. In particular Bacillus cereus spectra are very similar to spectra from Bacillus anthracis. Bacillus anthracis is not included in the IVD MALDI Biotyper database. For differentiation, an adequate identification method has to be selected by an experienced professional. The quality of spectra (score) depends on the degree of sporulation: Use fresh material.

Bacillus

Is a member of Bacillus subtilis group. The quality of spectra

subtilis

(score) depends on the degree of sporulation: Use fresh material.

Bacillus

The quality of spectra (score) depends on the degree of sporulation:

mojavensis

Use fresh material.

Bacillus

The quality of spectra (score) depends on the degree of sporulation:

vallismortis

Use fresh material.

Bacillus

The quality of spectra (score) depends on the degree of sporulation:

atrophaeus

Use fresh material.

Bărbulescu I. D. et al. (2015) identified a new yeast strain from Oprișor area from the 2014 harvest by peptide mass fingerprinting using a Bruker microflex™ LT/SH MALDI-TOF mass spectrometer with nitrogen laser. MALDI Biotyper software classified most of fungi isolates as species included in order Saccharomycetales: Merlot grapes M4 - Candida pelliculosa (Pichia anomala) from Phaffomycetaceae family; Merlot grapes M8 - Kloeckera apiculata (Hanseniaspora uvarum) from Saccharomycodaceae family. Merlot grapes Mcr2: Metschnikowia pulcherrima (Candida pulcherrima) from family Metschnikowiaceae. Šuranská H. et al. (2016) studied 120 isolates that have been identified and grouped by molecular approaches and 45 of the representative strains and they were tested for selected important oenological properties. Begea M. et al. (2015) isolate (SH8) was identified by Bărbulescu I. D. et al. (2015) as belonging to family Metschnikowiaceae. Based on the present study, the spectra for not reliable identification are show bellow (Figures 1-3):

The strains M10, Mcr7, SH4 and SH6 gave a not reliable identification. The isolates have been also studied using microscopic and macroscopic methods (results not presented in this paper).

87

IDENTIFICATION OF NEW ISOLATES (MICRO-ORGANISMS) FROM VINEYARD EPIPHYTIC MICROFLORA USING MALDI-TOF MASS SPECTROMETRY

IDENTIFICATION OF NEW ISOLATES (MICRO-ORGANISMS) FROM VINEYARD EPIPHYTIC MICROFLORA USING MALDI-TOF MASS SPECTROMETRY Figure 1. The

Figure 1. The acquired spectra of analyzed isolate SH6

IDENTIFICATION OF NEW ISOLATES (MICRO-ORGANISMS) FROM VINEYARD EPIPHYTIC MICROFLORA USING MALDI-TOF MASS SPECTROMETRY Figure 1. The

Figure 2. The acquired spectra of analyzed isolate Mcr7

IDENTIFICATION OF NEW ISOLATES (MICRO-ORGANISMS) FROM VINEYARD EPIPHYTIC MICROFLORA USING MALDI-TOF MASS SPECTROMETRY Figure 1. The
IDENTIFICATION OF NEW ISOLATES (MICRO-ORGANISMS) FROM VINEYARD EPIPHYTIC MICROFLORA USING MALDI-TOF MASS SPECTROMETRY Figure 1. The

Figure 3. Macroscopic aspect of SH4 and Mcr7 colonies

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Iuliana Diana Bărbulescu, Constanţa Mihai, Mihaela Begea, Mihai Frîncu, Liviu Grigorică, Gabriel Roceanu, Simona-Ioana Marinescu, Răzvan Teodorescu, Radu Tamaian

4. CONCLUSIONS

We isolated yeasts and bacteria strains from the Oprișor vineyard, one of the most famous vineyards in Romania. The MALDI Biotyper software classified most of isolates as species included in order Bacillus, with some particular strains that could not be matched with strains from database and were added as new entries (taxa). For a more precise identification, samples SH6 and MCR 7 will be the subject of DNA molecular analysis in the future study.

Acknowledgements. This work was financially supported by the national project 150/2014 – “Increasing the competitiveness of wine-growing products, applying bioconversion technologies”, in the frame of the Romanian national programme PARTNERSHIPS IN PRIORITY AREAS that is supported by the Romanian Executive Agency for Higher Education, Research, Development and Innovation Funding (UEFISCDI) and National project no. PN 16 36 04 02:

Advanced research on food origin authentication in order to implement innovative fingerprinting methodologies – Emphasis on fruit-based products. We would like to thank to the project partner Carl Reh for the permission to take samples from the Oprișor vineyard.

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Bărbulescu I. D., Mihai D., Begea M., Mudura R., Teodorescu R., Mihai C., Grigorică L., Roceanu G., Marinescu S. I., Tamaian R., 2015, A Case Study – Yeasts Identification in Order to Implement Terroir and Vineyard Management at Oprișor Winery Using the GIS Technology and a Proteomic Approach, in: Buzzini, P., Abbas, C., Tei, F. (Eds.), Book of Abstracts of 32nd International Specialized Symposium on Yeasts: “Yeast Biodiversity and Biotechnology in the Twenty-First Century.” University of Perugia, Perugia, p. 162.

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