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February 2014, Volume 5, No.1
International Journal of Chemical and Environmental Engineering
Development of a rapid, effective method for
seeding biofiltration systems using alginate beadimmobilized cells
Wan Li Lowa, *; Corby Leea; Matt Wilkesa; Clive Robertsb , David J. Hillb
a
Odour Services International Limited, Cannock, United Kingdom
Faculty of Science and Engineering, Department of Biology, Chemistry and Forensic Science,
University of Wolverhampton, Wolverhampton, United Kingdom
*Corresponding author E-mail:
[email protected]
b
Abstract:
The antisocial and health problems associated with odours from waste handling sites has led to the design of specialized biofiltration
systems which use microorganisms to metabolize malodorous compounds to less malodorous compounds. In order to reduce the prolonged start-up process of a biofilter, such systems are often seeded with selected microorganisms to facilitate rapid biofilm formation.
In order to ease application, these microorganisms can be immobilized by entrapment within three dimensional polymer matrixes such
as alginate beads. The bead structure, slow biodegradability and good diffusion properties of beads made with alginate serve as a
simple protective layer which limits the exposure of microorganisms to unsuitable conditions. In addition, the beads can also
incorporate nutrients which will support the initial cell survival whilst they acclimatize to the new environment. Over time, the
biodegradable bead slowly loses its structural stability, thereby releasing entrapped cells to colonize the biofilter media. This research
investigates the development of freeze-dried alginate-immobilized cell beads into a commercially viable method to seed biofiltration
systems. The process by which alginate immobilized cells progressively colonizes, leading to biofilm formation within the structure of
the biofilter media will be illustrated by microbiological analyses combined with scanning electron microscopy. The cell
immobilisation and freeze drying methodology necessary for increasing the shelf life of the beads, whilst maintaining cell viability,
will also be described.
Keywords: Biofilter; odour control; alginate bead-immobilized; microorganisms.
1. Introduction
Odorous emissions may arise from various sources such
as manufacturing, petrochemical and food industries,
waste treatment plants as well as agricultural activities
[6]. Conventional methods commonly used to treat the
odorous emissions are via methods such as chemical
scrubbing, incineration, thermal oxidation and adsorption.
Compounds typically categorized as air pollution gases
are: volatile organic compounds (VOC – includes a wide
range of aliphatic, aromatic and chlorinated
hydrocarbons), sulphurous compounds (organic sulphur,
hydrogen sulphide), ketones, aldehydes, lower molecular
weight fatty acids, ammonia and amines [2, 6]. However,
not all of the compounds are normally associated with
odour problems. Compounds which are associated with
odorous air pollutants are: ammonia (NH3), volatile
organic
compounds
(VOC),
sulphur-containing
compounds such as hydrogen sulfide (H2S), sulfides (S2-)
and mercaptans (R-SH) [6]. Most of these compounds are
classified as irritants and some may be hazardous to
health at higher exposure [6, 8].
Biofiltration systems are gaining popularity as the choice
of odour control system with the modernization of
biotechnology. Such systems use microorganisms to
metabolize/neutralize the malodorous compounds, thus
rendering them non-odorous [6]. In addition, neutralizing
the odorous compound can potentially reduce the
exposure risk to health hazardous compounds, such as
H2S. Depending on the type of biofilter media used, the
population colonizing it can develop over time from the
indigenous microorganisms present via natural selection
[6]. Alternatively, suitable inocula can be introduced into
the biofilter to promote the initial colonization and
enhance the performance of the biofilter. Immobilization
of the cells within a three dimensional polymer matrix,
such as alginate, can be advantageous to the microbial
inocula. The cells benefit from a temporary protection
against any potentially degenerative changes in the new
environment, promote a higher localized cell loading and
prevent high dilution rates or inoculum wash out due to
the continuous irrigation process [5]. The degradable
nature of the alginates will also contribute to a slow
Development of a rapid, effective method for seeding biofiltration systems using alginate bead-immobilized cells
release mechanism which allows the encapsulated
microorganisms to be released from the matrix to
successfully colonize, attach and form a biofilm on the
biofilter media.
This research describes the development of alginate beadimmobilized cells for the rapid seeding of biofilters to
reduce the start-up process.
surface of the pumice. The pumice samples were
examined for biofilm formation using the scanning
electron microscope (SEM).
2.4 Scanning electron microscopy
An incubated sample of pumice was treated and prepared
according to an adapted standard method of preparing
SEM samples [5]. Small samples of pumice were rinsed
three times with ¼ strength Ringers solution to remove
any free-unattached cells. The samples were post-fixed by
soaking in a 2.5 % v/v gluteraldehyde in phosphate
buffered saline for 1 hour. Following that, the samples
were treated for 15 minutes using each solution from a
series of alcohol solutions (30 %, 50 %, 70 %, 90 % and
100 % v/v). Samples were subjected to overnight freeze
drying. The samples were mounted on scanning electron
microscopy stubs and coated with gold particles prior to
analysis.
2. Methods and Materials
2.1 Preparation of alginate bead-immobilized cells
A culture of Paracoccus sp. was grown in tryptone soy
broth (TSB) (30.0 g/L) for 24 hours in a 37 °C orbital
shaker. Aseptically, 150 mL of culture was added to
700 mL of sterile 2.0 % w/v alginate solution. The
alginate bead-immobilized cell beads were prepared by
allowing the alginate-cell mixture to dispense at the rate
of one drop per second into 1000 mL of sterile calcium
chloride (2.0 % w/v) on a slow shaking platform set at
50 rpm. The beads were left to cross-link in the calcium
solution for approximately 2 hours to form hard gel beads
and subsequently the excess calcium solution was drained.
The beads were then kept in a sterile container at 4 °C.
2.5 Freeze-drying alginate bead-immobilized cells
The prepared beads were soaked in sterile 20 % w/v
skimmed milk solution for 2 hours. The beads were
drained into metal mesh trays and kept in the freezer
(-20 °C) for 48 hours. The metal mesh trays containing
the beads (approximately 500 g) were subjected to freeze
drying for 24 hours. The freeze dried beads were
subsequently stored in an air tight container. The beads
can be rehydrated (cells resuscitated) by soaking a sample
of freeze dried beads in excess TSB for 48 hours.
2.2 Cell viability
Cell viability was determined by tenfold serial dilution of
500 µL of culture in 4.5 mL of ¼ strength Ringers
solution using aseptic technique. Samples (20 µL) of
dilutions 10-1 to 10-8 were aseptically plated on sterile
tryptone soy agar (TSA) (37.0 g/L) using the Miles and
Misra technique. The number of colony forming units per
mL (CFU/mL) was determined after incubating the agar
plates overnight at 37 °C.
Similarly, viability of the cells from the beads was
determined by serially diluting a sample prepared by
aseptically dissolving approximately 1 g of beads into
9.0 mL of 0.5 M phosphate buffer solution. The number
of colonies formed after incubating the TSA plates
determined the CFU per gram (CFU/g) of beads.
The effects of storage at room temperature (25 °C) and in
the fridge/cold room (4 °C) on the viability of the alginate
bead-immobilized cells over time were also determined
by sampling the beads for viability count at specific time
intervals.
3. Results and Discussion
Characterization of the beads produced using the method
described showed that the average bead diameter formed
was between 3.0 - 4.0 mm in diameter with an average
weight of between 0.02 – 0.03 g. Overnight cultures
reached at least 1 x 109 CFU/mL of Paracoccus cells and
upon immobilization, the viability was shown to attain at
least 1 x 108 CFU/g of beads. Figure 1 show the viability
of the immobilized cells when the beads were stored in an
incubator at room temperature (25 °C) and in the fridge or
cold room (4 °C) over time.
2.3 Colonization of immobilized cells on biofilter media
A sterile sample of pumice media (approximately 10 g)
was incubated in a petri dish containing either (i) 20.0 mL
of overnight culture of Paracoccus cell suspension, or (ii)
20.0 g of alginate bead-immobilized cells. This created a
condition whereby the pumice (i) was totally immersed in
the overnight culture, or (ii) the surface of the pumice was
covered with alginate beads. Sterile TSB (5 mL) were
added into each petri dish to provide some nutrient and
moisture to the cells. The petri dishes were incubated in a
static incubator for 72 hours at 37 °C. Post incubation, the
pumice media was rinsed three times with ¼ strength
Ringers solution to remove any cells unattached to the
Figure 1. Viability of alginate bead-immobilized cells stored in room
temperature and fridge/cold room over time.
30
Development of a rapid, effective method for seeding biofiltration systems using alginate bead-immobilized cells
The results indicate that the viability of the Paracoccus
cells undergo some changes over time (up to four-fold)
depending on storage conditions. At 12 days post-storage,
the alginate bead-immobilized cells stored at 25 °C
showed an increase in cell numbers, when compared to
those stored at 4 °C. In addition, at 36 days post-storage,
another increase in cell numbers was observed. The
mesophilic Paracoccus cells exhibit the ability to grow at
temperatures between 30 – 37 oC. At 25 °C, the cells can
adapt, grow to increase in numbers and undergo normal
cell growth cycles. However, at 4 °C this adaptation is
compromised by the effects of temperature on normal cell
growth. This results in only a slight change in cell
viability of the beads stored at 4 °C over the period of
50 days.
The micro-environment within the beads
encapsulates some nutrients, limits cell exposure to
external stress and toxic conditions whilst allowing the
cells to proliferate [7]. This enables the cells to maintain
viability over a longer period of time.
During the storage period, fungal populations were
occasionally observed to contaminate and grow on the
bead structures after 2 weeks of storage (results not
shown). In order to minimize the cost of mass-producing
alginate bead-immobilized cells suitable for seeding
biofilters the production was conducted only in a clean
(non-sterile) environment. Minor contaminations during
the process can thus occer which can lead to the
contaminant growth and bead spoilage.
In addition to maintaining cell viability, the ability of
cells to attach to the surface of pumice and form a biofilm
also plays an important role in the performance of a
biofilter. Fig. 2 (a and b) show the SEM photomicrograph
of pumice surface after 72 hours of incubation with
planktonic cells and alginate bead-immobilized cells. The
photomicrographs shown are typical representatives of
results obtained from a several pumice samples. Results
from Fig. 2 (a and b) indicate that the alginate
bead-immobilized cells can attach and colonize the
pumice surface more effectively when compare to the
planktonic cells. Limited microbial cells were observed
on the surface of the pumice incubated with planktonic
cells after 72 hours of incubation. This may be due to the
nature of the alginate-immobilization of the cells which
allowed cells to be concentrated locally [1], and not easily
“washed” away by the surrounding liquid media [5],
hence extending the capacity for surface-attachment.
Denser cell attachment was observed on the surface of the
pumice after prolonged incubation for up to two weeks
for both samples (results not shown). This indicates that
cell attachment was possible when incubated with a
suspension of planktonic cells but that strong attachment
required for colonization takes longer to achieve.
Figure 2a. SEM photomicrograph of pumice surface after
incubation in a suspension of planktonic cells for 72 hours
Figure 2b. SEM photomicrograph of pumice surface after
incubation in alginate bead-immobilized cells for 72 hours
The efficiency of cell attachment can be compromised by
the weak attachment formed on the surface of the pumice
which increases the risks of inoculums being washed off.
In order to maintain a stable attachment on a surface,
microbial cells need to produce and excrete a non-specific
adhesive extrapolysaccharide (EPS) substance for the
formation of biofilms [3]. Thus in an attempt to seed a
biofilter with liquid cultures, an initial re-circulatory
irrigation routine within the system can be beneficial to
minimize the potential loss of inoculums due to “washout” and allow longer cell-surface contact time to
promote better cell attachment. In contrast, results
indicate that alginate bead-immobilized cells can be an
advantageous technique for seeding biofilters as it allows
a more rapid rate of colonization and reduces the “washout” effect when irrigation (re-circulatory or noncirculatory) is applied to the system.
In addition, it is possible that prior to biofilm formation,
the surface chemistry of the pumice may be modulated for
its suitability for microbial colonization [3]. Thus, the
localized availability of nutrient leachate from the beads
onto the surface of the pumice may play a part in
increasing the rate of colonization.
31
Development of a rapid, effective method for seeding biofiltration systems using alginate bead-immobilized cells
The alginate beads were also subjected to freeze drying
in the attempt to preserve the cells and alginate bead due
to its degradable nature [4] and high risk of contamination
when stored over a long period of time. The freeze drying
process shrinks the bead size whilst removing at least
91 % of the initial bead weight. The viability and
rehydration results obtained are summarized in Table 1.
Trial 1
Initial viability in beads upon
production (CFU/g)
Freeze-dried bead weight (g)
Rehydrated bead weight (g)
Viability of beads upon rehydration
(CFU/g)
2.27 x 10
4. Conclusion
Results from this research show the feasibility of
producing viable alginate bead-immobilized cells suitable
for promoting rapid colonization on pumice stone.
Enhanced rate of colonization can be beneficial in
optimizing the activity of the biofilter thereby maximizing
its performance within a short time frame. The freezedrying technique described may be suitable for the
development of ready-to-go freeze dried alginate
bead-immobilized cell inocula to be used as an effective
and rapid biofilter re-seeding methodology.
Trial 2
8
2.27 x 108
2.38
14.18
2.34
9.70
4.36 x 107
1.06 x 108
ACKNOWLEDGMENT
Table 1. Summary of cell viability and weight of freeze dried beads
rehydrated in TSB for 48 hours
The authors are grateful to the Technology Strategy
Board, U.K. for the funding to fulfil this research.
The results in Table 1 indicate the feasibility of using
freeze-drying as a method of preserving both the alginate
bead as well as the viability of the cells to reduce the loss
of beads due to contamination.
During the rehydration process, the freeze dried beads
were fully restored to their original bead size. The process
of making large amounts of beads requires a lot of preproduction preparation, in addition to the actual time
required for beads production. Long production times for
inocula that have short shelf-lives are problematic when
required at short notice to re-seed a biofilter. Under such
circumstances, the availability of stocks of freeze dried
beads for re-seeding can help to minimize biofilter
downtime, provide rapid re-inoculation to revive and
optimize biofilter performance.
Currently, work is ongoing to determine the stability and
shelf-life of the freeze dried beads as well as the cell
viability when stored for longer periods of time (up to
12 months).
The increasing variation in processing and manufacturing
industries in modern society pose new challenges to air
pollution control. Traditional odour control system
technologies may not cope with this complicated mixture
of contaminants from modern industrial processes.
Suitable strategies to develop rapid and effective methods
for seeding and maintaining biofilters play an important
role in enhancing their performance. Effective strategies
need to be implemented at the design and initial planning
stage based on the “best-fit” concept, whereby it will not
only resolve the odour issue but will also keep within the
budgetary and legislative constraints. Further research is
currently underway to incorporate the growth of other
suitable microorganisms in order to create an adaptable
mixed culture of inoculums for the treatment of more
complex odours coming from modern industrial
processes.
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