Progress of Cryogenics

and
Isotopes Separation
Volume 19, issue 1/2016

Contents:

Influence of the Thermal Energy Storage on the

*
Hybrid Heating Systems' Energy Profile

CRYOGENICS AND ISOTOPIC TECHNOLOGIES - I.C.S.I. RM. VALCEA

NATIONAL RESEARCH AND DEVELOPMENT INSTITUTE FOR
Synthesis and Characterization of Iodine Doped
Graphene Byan Uncatalyzed Reaction

Ammonia Removal from Aqueous Solution by Ion

Exchange Using Clinoptilolite and Verification of
the Process Through a Numerical Study

Numerical Analysis on The Hydrogen Generation

from Sodium Borohydride Hydrolysis

The Removal of Heavy Metals Using the Bulk Liquid

Membrane Technique

Macro- and Microelements as Possible Markers of

Quality and Authenticity for Fruits and Derived
Products

Assessing the Authenticity of Vinegar by Combined

2 13
H-SNIF-NMR and C-IRMSAnalysis

Identification of New Isolates (Micro-Organisms)

from Vineyard Epiphytic Microflora Using Maldi-
Tof Mass Spectrometry

Sono - Chemical Degradation of Organic Compounds

from Wastewater

Determination of Physico-Chemical Parameters and

Water Quality Index (WQI) of Olt River

CONPHYS Press
Râmnicu Vâlcea
 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

ISSN: 1582-2575

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

2
 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
Progress of Cryogenics and

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,
Isotopes Separation

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 2H-SNIF-NMR and
13
C-IRMS Analysis
Popescu Raluca, Botoran Oana, Costinel Diana, Ionete Roxana Elena 75
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 ................................................................ 99
Instructions for Authors ............................................................................ 111

3
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 Bărbulescu1*, Constanţa Mihai2, Mihaela Begea3, Mihai Frîncu2,

Liviu Grigorică4, Gabriel Roceanu5, Simona-Ioana Marinescu1, Răzvan
Teodorescu2, Radu Tamaian6,7,8*
1
Pharmacorp Innovation SRL, Bucharest, Romania; 2University of Agronomic Sciences
and Veterinary Medicine Bucharest, Romania; 3University Politehnica of Bucharest,
Romania; 4Bevitech SRL, Bucharest, Romania; 5Carl Reh Winery SRL, Bucharest,
Romania; 6National 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; 7University of Bucharest, Faculty of Physics, 3Nano-SAE
Research Centre, Bucharest-Măgurele, Romania; 8Biotech 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
83
 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.
84
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 106. 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 Scoring
Score Value
ID (alternative name) Consistency
2.265
M3 Bacillus cereus (A)
'green'
Candida pelliculosa (Pichia
2.197
M4 anomala) (A)
'green'
(Bărbulescu I. D. et al., 2015)
Kloeckera apiculata
2.351
M8 (Hanseniaspora uvarum) (A)
'green'
(Bărbulescu I. D. et al., 2015)
1.289
M10 not reliable identification (C)
'red'
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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 (C)
'red'
Candida pulcherrima
1.884
SH4 (Metschnikowia pulcherrima) (B)
'yellow'
(Bărbulescu I. D. et al., 2015)
1.266
SH6 not reliable identification (C)
'red'
1.782
CS1 Bacillus subtilis (B)
'yellow'
1.808
CS5 Bacillus mojavensis (B)
'yellow'
1.824
CS7 Bacillus mojavensis (B)
'yellow'
1.846
CS9 Bacillus vallismortis (B)
'yellow'
1.855
CS-Sol Bacillus subtilis (B)
'yellow'
1.81
Fz-CS Bacillus mojavensis (B)
'yellow'
1.786
CS Solb Anaerococcus sp. (B)
'yellow'
Candida pelliculosa
2.261
M4 (Pichia anomala) (A)
'green'
(Bărbulescu I. D. et al., 2015)
Candida pulcherrima 2.045
MCR2 (A)
(Metschnikowia pulcherrima) 'green'

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).
86
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
Comment
Pattern
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).

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

Figure 1. The acquired spectra of analyzed isolate SH6

Figure 2. The acquired spectra of analyzed isolate Mcr7

Figure 3. Macroscopic aspect of SH4 and Mcr7 colonies

88
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.

REFERENCES

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
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Crauwels S., Zhu B., Steensels J., Busschaert P., De Samblanx G., Marchal K.,
Willems K. A., Verstrepen K. J., Lievens B., 2014, Assessing
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EPIPHYTIC MICROFLORA USING MALDI-TOF MASS SPECTROMETRY
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ISSN: 1582-2575