Engineering, 2012, 5, 118-120
doi:10.4236/eng.2012.410B030 Published Online October 2012 (http://www.SciRP.org/journal/eng)
The Poly(vinyl alcohol)-Immobilized Photobacteria for
Toxicology Monitoring
Kristina A. Alenina, Leyla E.Aleskerova, Polina B.Kascheyeva, Anvar D. Ismailov
Dept. of Microbiology, Biological Faculty, Moscow State University, Moscow, Russia
Received 2012
ABSTRACT
The present work is undertaken to studies the important factors affecting the stability of a light emission at immobilized photobacte-
ria, and application PVAGs luminous biosensors for biomonitoring of toxicants. The intensity and stability of a light emission is
competently controlled by: 1) intensity and persistens of a luminescent cycle using bacterial strain; 2) type of the carrier and the
composition of the gel-formation medium; 3) freeze-thawing procedures; 4) physical and chemical conditions of storage and applica-
tion.
Keywords: Component; Bioluminescen ce; Photobacter ia; Immobilization; Po ly (Vinyl Alcohol)
1. Introduction
The luminous bacteria are widely used as a biosensor for bio-
monitoring of toxicants [1-3]. The application of photobacteria
is defined by high sensitivity of bioluminescence to a wide
spectrum of toxic substances, and a fast responsibility of light
reaction. Bioluminescent activity reflects of a cell metabolism
to chemical compounds with toxic action, including the heavy
metals, aliphatic and aromatic hydrocarbons, phenols and a
number of other xenobiotics. The emission value serves as the
quantitative indicator of the general and specific toxicity of the
sa mp l e .
Light-emitting bacteria can be used for toxicity monitoring
both using the free and immobilized forms. The immobilization
of photobacteria allows to raise stability of light emission by
biosensors, what is could form the basis of a cost-effective,
real-time system for detecting environmental pollutions [4,5].
Many diver se gel mat rices has be en pro posed as po ssible carri-
ers for immobilisation of luminous bacteria, In this case, either
natural: agar, agarose, alginate, carrageenan, gelatin, collagen,
or synthetic (polyacrilates, polyurethanes, polyethers) bio- po-
limers has b een used as the gel-forming agents [6,7].
Taking into account that a luminescence activity extremely
sensitive to temperature influence, in quality gel-forming agent
is chosen poly (vinyl alcohol) (PVA). The crioPVA gel forma-
tion proceeds at negative temperatures. This carrier on struc-
tural, physical and chemical properties possess a number of
advantages in comparison with other carriers. PVA gels have
very high micro-and macro-porosities which provide favored
conditions for the non hindered mass transfer of substrates and
toxicants. Thermo-stability of cryoPVA gels exceeds that of
other commonly employed thermo-reversible gel carriers, such
as agar, agarose, ets. PVAGs are highly resistant to biological
degradation and practically insensitive to media composition,
— solutes, buffers, pH. PVA itself is a biologically compatible,
nontoxic, readily available low-cost polymer. Rheological pa-
rameters of cryoPVAGs are quite stable and allow for this car-
rier to by used under variety biomonitoring conditions [6,7].
The aim of this study was the analyses of the factors affect-
ing the stability of a light emission at PVA-immobilized pho-
tobacteria and application PVAGs luminous biosensors for
biom onitoring of tox ic a nt s .
2. Materials and Methods
Bacterial strains, media and culture grow conditions. The
strains used in this study were Photobacterium phosphoreum
strain №331 MC MSU
Immobilization procedures. The immobilization was car-
ried out according to a technique [6, 9]. Liquid biomass were
mixed with 13% solution of poly(vinyl alcohol), (Mm – 48000
Da), Gel solution were prepared at 24h in 1) the GM media; 2)
Na-phosphat buffer + 2% NaCl, pH 7.6, and 3) 3% NaCl. The
0.2 ml gel-cell solution pour out on 96-hole planshet, and
transferred to the freezing chamber (20°C) for gel formation
with simultaneous inclusion of cells in a polymeric matrix. The
final cell co n centration in granules about 1–5×107 cells/granule.
Prio r to each measure ment s sampl es were d e frosted at 4 °C over
24 h and placed on incubation media, and stored at 4°С. The
procedures of cryogenic gel-formation and cell reactivation in
detail s ar e described in [9].
Measurement of light intensity and соncentration of cells.
A bioluminescence registered on luminometer 1250 LKB-
Wallac and expressed in relative units, or absolute values of
photons output according the standard [10]. The cell contain
carried out under maintenance АТР using a bioluminescent
method with firefly luciferase [11]
3. Results
CryoPVAGs-cell immobilization.
The intensity and duration of light output by the isolated
cells is defined not only by natural emission properties of
strains and composition of incubation mixture. Except energet-

K. A. ALENINA ET AL.
119
ically substrates, the luminescence is complexly controls by the
ion ic force, temperat ure and рН.
It is obvious, that the composition of gel-formation media
should influence on cells stability in the carrier and metabolic
activity first of all on stages of gel formation. For a choice of
the optimal media composition and conditions, three difference
mi xtu r e s (G M -medium with and without pepton, and 3% NaCl)
has been used for gel formation, and the kinetics of light emis-
sion after “freezing/thawing” procedure was carried out. Crite-
ria were: initial intensity of a luminescence of granules, the
general integrated excitation of photons in the course of storage
of preparations to residual level of a luminescence less than
0.1% from initial, and the rate of decay.
The developed technology cryogenic gel formation has
allowed to keep survival of luminous bacteria in the carrier,
practically on 100%, without introduction additional crioprotecting
agents and procedures of a light induction. Specific
bioluminescent activity was restored to level of activity of free
cells (~105 photons/sec. per cell). At storage at –80°C
bioluminescent activity remained without changes in a current
of 2 years. The d etect ed l evel o f light emission of psychrophilic
strains at 4°С — over 1 month, at 20°С — 3 d ays . Th e analysis
of the specific activity of cells after a freezing/thawing
procedure and stabilization of light emission at 4°С (24 hours
from the beginning the heating stage), have shown, that most
effective gel for matio n mixt ur e is th e cu lture gr owin g media. In
this case the immobilized bacteria kept practically 100% level
of luminescent activity. The exception of peptone causes
decrease in specific luminescent activity on 1–1.5 order. The
most essential recession of a luminescence (in ~103 times) was
observed when the gels formation process has come with only
3% NaCl.
Thus the composition of the gel formation media, not incu-
bation mixture, makes the basic impact on intensity and dura-
tion of bioluminescence of immobilized preparations. Concentra-
tion of the carrier (5, 7, 10%) does not change emission and
kinetics parameters. The analysis of stability of PVA-
immobilized cells of photobacteria at storage at low
temperatures spent with use of the preparations formed in
growth culture media. It is established, that at –80°C remained
practically 10 0% level of b ioluminescent activity of granules (2
Figure 1. The time depende nce of a specific bioluminescent activity
during incubation at 4°С in 3% NaCl immobili zed (1) and fr ee (2)
cells of photobacteria.
years of supervision), at –20°C activity falling on 20% in a
current of year was observed, however residual activity and in
this case was on much higher (more, than on two order) level,
than residual activity of the preparations stored at 4°С. As
essential distinctions in inhibition kinetics is not revealed, the
time parameters postulated to free cells are chosen for the
toxicity analysis with granules.
The optimized conditions of reception and storage
immobilized cells formed a basis of use of luminescent
granules as biosensor controls. The analysis of inhibition
kinetics from free and immobilazed cells has shown similar
time profiles, testifying to absence serious diffusion restrictions
of a gel material and the form of granules for all types of
molecules. Supervision logically follows from structural
characteristics of the matrix having macropores. As essential
distinctions in inhibition kinetics is not revealed, the time
parameters postulated to free cells are chosen for the toxicity
analysis with granules. The table illustrates the immobilized
cells bioluminescence inhibition by various classes of toxins.
4. Discussion
For photobacteria, at which temperature optimum of a lumi-
nescence 15–25°С (depending on a strain), temperature influ-
ence is critical. Most often used in immobilization photobacte-
ria agar, agarose and alginate gels are formed in a range of
posi tive temperatures t hat is capab le to have negati ve influen ce
on luminescence activity of cells directly during immobilization
procedure. Essential value for viability of sea photobacteria
plays salt structure of environment. Optimum concentration for
light activity of a cells of 2–6% of chloride sodium. The stabil-
ity of Ca- and Sr-alginate gels at storage an d app licatio n is al so
influen ced by partial rep lacement of Ca+2 and Sr+2 by Na+ ions
[8]. Glycerol has been used as a necessary component for im-
mobi li s a ti on proces s and to m or e e f fe ctiv e stor a ge .
PVA-cryogels on structural properties [6,7] possess a
number of the characteristics having basic value for light
emission of photobacteria.
Toxicant Bioluminescence response,
detection range, mg/l
5 min 15 min
Phenol 100–600 100–400
Сu2+ 5–40 1–8
Zn2+ 10–60 0.5–4
Hg2+ 0.1–0.6 0.05–0.10
Pentachlorophenol 0.2–2.0 0.05–0.4
2,4-Dichlorophen oxyacetic Acid 1.0–10.0 0.5–10.0
2,4,5-Thrichlorophenoxyacetic Acid 0.5–4.0 1.0–8.0
The basic advantage consists that gel formation process

K. A. ALENINA ET AL.
120
proceeds at negative temperatures. The PVA-gels possess high
thermo stability in a wide range of positive temperatures up to
80°С, that creates possibility of use of a bioreactor in different
temperature modes. Physical and chemical para meters of carrier
slightly depend on structure of the environment of formation of
gel, in particular salt, which has great value for bioluminescen t
activity of bacteria. The PVA-gels has note been damage by
microorganisms. Essentially, that PVA it is nontoxic in relation
to the in clud ed pho tob acteri a. Conseq uen tly, th ese gel material s
can be considered as very promising carrier for use in photo-
bacteria entrap ment technologies
The results presented testify the long-term stability and high
intensity of a luminescence with PVA-Gs immobilized psyc-
hrophilic strains of photobacteria. It is established, that the
major importance fo r s tability of a lumin es cence has a choice of
photobacteria strain and of the gel formation mixture. A com-
position of incubation medium, рН, and especially temperature,
have complex an effect as on intensity and duration of a lumi-
nescence. Results suggests that the most stability of light
output by immobilized preparations can be achieved with incu-
bation at relative lo w (no more then 20°C) temper ature in al ka-
line (pH 8.5) mixtures.
High survival of cells, with preservation of specific activity
of light emission, at level of the free cells, presented to the
given work, reflect advantages cryogenic immobilization
photobacteria in PVA carrier and application PVAGs-lum
biosensors for biodetection of toxicants
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