=Paper=
{{Paper
|id=Vol-2030/HAICTA_2017_paper55
|storemode=property
|title=Identification of Free Amino Acids in Brewer’s Yeast after Heavy Metals Biosorption
|pdfUrl=https://ceur-ws.org/Vol-2030/HAICTA_2017_paper55.pdf
|volume=Vol-2030
|authors=Andreea Stănilă,Zorita Diaconeasa,Floricuta Ranga,Florinela Fetea
|dblpUrl=https://dblp.org/rec/conf/haicta/StanilaDRF17
}}
==Identification of Free Amino Acids in Brewer’s Yeast after Heavy Metals Biosorption==
https://ceur-ws.org/Vol-2030/HAICTA_2017_paper55.pdf
Identification of free Amino Acids in Brewer’s Yeast
after Heavy Metals Biosorption
Andreea Stănilă1, Zorita Diaconeasa1, Floricuta Ranga1, Florinela Fetea1
1
Faculty of Food Science and Technology, University of Agricultural Sciences and Veterinary
Medicine, 400372, 3-5 Manastur St., Cluj-Napoca, Romania,
e-mail: andreea.stanila@usamvcluj.ro
Abstract. Yeasts of genera Saccharomyces are efficient biosorbents for heavy
metal ions. The aim of this study was to identify if the free amino acids present
in brewer yeast are involved in metal biosorption due to their capacity to
coordinate metal ions. As biosorbent was used non-living brewer’s yeast type
Saccharomices cerevisae at 0.5% yeast dose. Copper, lead and zinc solution of
1mg/L concentrations were prepared using their salts. The experiments were
conducted at pH=6, which is the most appropriate for amino acids extraction.
The amino acids were identified by HPLC-DAD/-ESI-MS chromatography.
The experiments were conducted by mixing metals solution with yeast and
shaken at a constant speed of 120 rpm at 200C for 120 minute. The samples
were centrifuged at 2500 rpm for 15 minute and the pellet were analysed for
amino acids identification. The amino acids extraction from pellets was
performed using HCl 0,05M/deionized water (v/v) 1/1. The HPLC analysis
was performed on a Agilent 1200 system equipped with a binary pump
delivery system LC-20 AT, a degasser DGU-20 A3, diode array SPD-M20 A,
UV–VIS detector (DAD). Amino acids were identified using an EEZ:Faast Kit
for free amino acids, The amino acids identified by HPLC method were
glycine, glutamic acid, leucine, isoleucine, ornithine, lysine, histidine,
homophenylalanine, tyrosine, glutamine in control brewer yeast (before
biosorption) and their profile differs according with the metal ions types.
According with the peaks area there are differences in the presence of the
amino acids due to the possible coordination with copper, lead and zinc ions.
Keywords: Brewer yeast, amino acids, biosorption, heavy metals
1 Introduction
Biosorption can be defined as the selective sequestering of metal soluble species
that result in the immobilization of the metals by microbial cells. Biosorption is a
process with some unique characteristics. It can effectively sequester dissolved
metals from very dilute complex solutions with high efficiency. This makes
biosorption an ideal candidate for the treatment of high volume low concentration
complex waste-waters (Gadd, 1993; Wang, 2006). The selective sequestering of
metal soluble species that result in the immobilization of the metals by microbial
455
cells is defined as biosorption. It refers to physicochemical mechanisms of inactive
(i.e. non-metabolic) metal uptake by microbial biomass (Volesky 2001). Metal
sequestering by different parts of the cell can occur via various processes:
complexation, chelation, coordination, ion exchange, precipitation, reduction (Wang
2009, Alluri, 2007). Immobilization may be the result of more than one mechanism,
for example, metal complexation may be followed by metal reduction or metal
precipitation. These biopolymers, constituents of the cell wall and the other parts of
the cell possess functional groups that have a significant potential for metal binding
(Dakiky, 2002).
Metal uptake by non-living cells is mainly a passive biosorption and consists in an
adsorption of metal ions to the cells surface by interactions between metal and
functional groups displayed on the surface of the cells (Paknikar, 2003).
Brewer's yeast is made from a one-celled fungus called Saccharomyces cerevisiae
and is used to make beer. It also can be grown to make nutritional supplements. The
yeast biomass has been successfully used as biosorbent for removal of Ag, Au, Cd,
Co, Cr, Cu, Ni, Pb, U, Th and Zn from aqueous solution. Yeasts of genera
Saccharomyces, Candida, Pichia are efficient biosorbents for heavy metal ions. A
number of literatures have proved that S. cerevisiae can remove toxic metals, recover
precious metals and clean radionuclides from aqueous solutions to various extents
(Podgorskii, 2004; Tálos, 2009)
2 Materials and Methods
Saccharomices cerevisae biomass was supplied as a lyophilized by-product from
industrial ethanol production. Prior use as a biosorbent, the biomass was pretreated in
order to remove fine particles and to displace any metals already bound to the
sorption sites. The waste biomass as washed with deionized water by stirring
followed by centrifugation at 3000 rpm for 20 minutes. The supernatant was
discarded and the pellet was reslurried in deionized water. The procedure was
repeated for three times until the supernatant was clear. Metals solution were
prepared using the mixture of their salts CuSO4·5H2O, PbNO3·2H2O, ZnSO4 of
analytical reagent grade. The concentrations of metals ions established were 1 mg/L
for Cu2+, Zn2+, Pb2+ and were obtained by dissolving the appropriate salts in
deionized water.
Experimental procedure:
Metal ion binding experiments were performed by incubation of 25 mg biomass
(dry weight) with 50 ml mixture of metals ions-containing solution in 125-ml
Erlenmeyer flask on an orbital rotary shaker at 120 rpm for 120 minutes. The
experiment was conducted at pH=6 and was established by adjusting it with HCl
0.1M or NaOH 0.1M solutions. In order to identify the free amino acids from
biomass by HPLC chromatography the samples were centrifuged at 2500 rpm for 15
minutes, the supernatant was discarded and the pellets were analysed.
The free amino acids were extracted from the pellets using two types of solutions:
HCl 0,05M/deionized water (v/v) 1/1. HPLC analysis was performed on a Agilent
1200 system equipped with a binary pump delivery system LC-20 AT (Prominence),
456
a degasser DGU-20 A3 (Prominence), diode array SPD-M20 A, UV–VIS detector
(DAD). Amino acids (100 µl) from control brewer’s yeast and samples provided after
biosorption of metal ions were identified using an EZ:Faast Kit for free amino acids,
provided by Phenomenex (USA). The results are presented in the next table and
figures.
3 Results and Discussions
The absorption mechanisms of metals are different and depend on cellular
metabolism. In non-living brewer yeast this mechanism is one of physical-chemical
interaction between metal ions and some functional groups, especially amino and
carboxyl. The aim of this study was to establish the amino acids from brewer yeast
involvement in metal ions biosorption, as it is known that amino and carboxyl groups
could be responsible for complexation depending on pH intervals and metal:ligand
ratio. In the HPLC chromatograms these involvement could be observed by changing
the absorption intensities between amino acids from brewer yeast control and brewer
yeast charged with metal ions by biosorption.
The standard solutions of amino acids used for analysis were selected according with
the literature data regarding the presence of these compounds in brewer yeast
(Podgorskii, 2004). The main amino acids identified by HPLC method were glycine
(Gly), asparagine (ASN), glutamic acid (GLU), leucine (LEU), isoleucine (ILE),
ornithine (ORN), lysine (LYS), histidine (HYS), homophenylalanine (HPHE),
tyrosine (TYR), glutamine GLN), alanine (ALA), valine (VAL), triptophan (TRP),
phenylalanine (PHE), α-aminobutyric acid (ABA). The separation and identification
of amino acids were performed on brewer yeast uncharged with metal ions (control)
and brewer yeast charged with metal ions after their biosorption at different pH
values. The results are presented in the next table and figures. The profiles of
chromatograms are different between control brewer yeast and samples charged with
metal ions due to the absorption of copper, zinc and lead after incubation. It is
supposed that amino acids from brewer yeast has the capacity to coordinate these
metal ions and the complexes resulted have different retention times and peaks area
than free amino acids as it can be seen in the Table 1. From the data above it can be
presumed that amino acids are involved in metal coordination due to their capacity of
metal binding through carboxyl and amino groups. Another explanation regarding the
changes in amino acids profiles and content is that that it could be affected by
autolysis and fermentation conditions like time, temperature, pH, moisture content
(Cabuk, 2005).
457
Table 1. Amino acids identified in brewer yeast uncharged (control) and charged with metal
ions.
Nr crt Aa. Aa. Aa. Aa. Aa.
control control Cu biosorption Pb Zn
m/z area (area) biosorptio biosorptio
n n
(area) (area)
1 - - -
GLN/275 10704200
2 - 17414728 24690218
ASN/243 1,11E+08
3 34229200 62596676 86537960
GLY/204 2,93E+08
4 59079340 76963752 2,02E+08
ALA/218 4,8E+08
5 18151634 4,06E+08 2,96E+08
ORN/347 6,34E+08
6 2,16E+08 25244664 5,98E+08
PRO/244 8,99E+08
7 7473274 55393300 79203976
HIS/370 3,2E+08
8 - - -
VAL/246 1,29E+08
9 31089642 - 59059104
GLU/318 6,34E+08
10 - 23041980 25558244
TRP/333 76783688
11 16612316 38825024 87357976
LEU/260 3,63E+08
12 - 47576664 -
PHE/294 2,4E+08
13 6770684 8980013 25713988
ILE/260 68063784
14 - - -
ABA/232 23497502
15 26532408 94582776 2,33E+08
HPHE/189 2,83E+08
16 37071216 1,21E+08 2,67E+08
TYR/396 7,83E+08
The metal binding capacity differs from an amino acids to other: GLN, Val and AAA
which are present in uncharged brewer yeast sample disappear in the chromatograms
of charged brewer’s yeast with all metal ions; ASN and TRP have good affinity for
copper ions, GLU for lead ions, and PHE for copper and zinc ions. The others amino
acids present different affinity for metal ions according with their molecular structure
and the capacity to coordinate copper, lead and zinc. According to covalent index
Pb(II) ion is classified as a class b ion, while Cu(II) and Zn(II) are classified as
borderline ion, so the behavior of coordination is not full the same and the binding
capacity is different (Iqbal, 2004; Niebeer, 1973).
458
Fig. 1. HPLC chromatogram of amino acids extracted from control brewer’s yeast (A), amino
acids extracted from brewer’s yeast charged with copper ions (B), with lead ions (C), with zinc
ions (D).
4 Conclusions
This study represents a first step in elucidation of the absorption mechanism of metal
ions from aqueous solution by brewer yeast. Thus, the preliminary results lead to the
conclusion that free amino acids, above other constituents from yeast, could be
involved in metal biosorption. This process is strongly dependent on experimental
459
conditions, especially on pH level, as it is known that each metal ions have
characteristic pH limits for coordination with different organic ligands.
This study reveal the presence of 16 free amino acids in brewer’s yeast and
significant differences in their profile and content after biosorption of copper, lead
and zinc, which could be explained by the coordination of these ions by carboxyl and
amine groups of the ligands.
Acknowledgments. This work was supported by Romanian Ministry of Education
and Scientific Research UEFISCDI - PN-II-PT-PCCA-2013-4-0743
References
1. Alluri, HK., Ronda, SR., Setalluri, VS. (2007). Biosorption: an ecofriendly
alternative for heavy metal removal. African Journal of Biotechnology
6(25):2924-2931.
2. Cabuk, A., Akar, T., Tunali, S., Tabak, O., 2005. Biosorption characteristics of
Bacillus sp. ATS-2 immobilized in silica gel for removal of Pb(II). J.Hazard
Mater 136:317-323.
3. Dakiky, M., Khamis, M., Manassra, A., Mereb, M. (2002). Selective adsorption
of chromium (VI) in industrial wastewater using low-cost abundantly available
adsorbents. Adv. Environ. Res. 6: 533-540.
4. Gadd, GM. (1993). Interactions of fungi with toxic metals. Phytologist 124:25–60
5. Iqbal, M., Edyvean, R.G.J., 2004. Biosorption of lead, copper and zinc ions on
loofs sponge immobilized biomass of Phanerochaete chrysosporium. Miner.Eng.
17:217-223.
6. Paknikar, KM., Pethkar, AV., Puranik, PR. (2003). Bioremediation of
metalliferous Wastes and products using Inactivated Microbial Biomass. Indian J.
Biotechnol. 2: 426-443.
7. Podgorskii, VS., Kasatkina, TP., Lozovaia, OG. (2004). Yeasts-biosorbents of
heavy metals. Mikrobiol. Z 66:91-103.
8. Tálos, K., Páger, C., Tonk, S., Majdik, C., Kocsis, B., Kilár, F., Pernyeszi, T.
(2009). Cadmium biosorption on native Saccharomyces cerevisiae cells in
aqueous suspension. Acta Universitatis Sapientiae, Agriculture and Environment,
1: 20-30.
9. Volesky, B. (2001). Detoxification of metal-bearing effluents: biosorption for the
next century. Hydrometallurgy 59:203–16.
10. Wang, JL., Chen, C. (2006). Biosorption of heavy metals by Saccharomyces
cerevisiae: a review. Biotechnol Advances 24:427–51.
11. Wang, JL., Chen, C. (2009). Biosorbents for heavy metal removal and their
future. Biotechnology Advances 27:195-226.
460
12. Niebeer E., McBryde W.A., 1973. Free energy relationships in coordinate
chemistry (III): a comprehensive index to complex stability.
Can.J.Chem.51:2512-2524.
461