Bio-Science Department, Central Research
Institute of Electric Power Industry, Abiko City, Chiba 270-1194, Japan
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Cells immobilized by fixation into
inorganic carriers or entrapment in polymeric gel beads can serve as
biocatalysts within bioreactors and have been productively applied in
the pharmaceutical (24) and food industries (20)
as well as in the area of environmental protection. For example,
bioreactors used for wastewater treatment include a biofilm containing
bacterial cells fixed onto carriers (2, 9, 10, 27, 30) or
gel beads (6, 12, 14, 18, 19). In such arrangements, only
the surfaces of the carriers or beads have activity, because substrates
and oxygen for the immobilized cells are supplied only from the surface
(11, 16, 17, 23, 25, 31). Recently, however, improved
methods were described whereby the oxygen-rich surface of a gel was
used to mediate aerobic reactions, while the oxygen-poor interior was used for anaerobic reactions (7, 8, 13, 15, 26).
We have also reported on an immobilized cell bioreactor capable of both
aerobic and anaerobic reactions (28, 29). Instead of beads,
our bioreactor makes use of a tubular polymeric gel with an ammonia
oxidizer, Nitrosomonas europaea, and a denitrifier, Paracoccus denitrificans, trapped within. The immobilized
N. europaea at the external surface of the tube mediated
ammonia oxidation aerobically, while the immobilized P. denitrificans mediated anaerobic reduction of nitrite to nitrogen
on the inside of the tube. Ethanol serving as an electron donor for
denitrification flowed through the lumen of the tube. This
configuration was advantageous because it enabled our bioreactor to use
the ethanol effectively without having to add any ethanol directly to
wastewater. Unfortunately, since P. denitrificans is a
facultative anaerobe with both aerobic and anaerobic respiratory
enzymes (3, 32), it grew on both the inside and the external
surface of the tube, and the distributions of P. denitrificans and N. europaea overlapped
(29). P. denitrificans then competed with
N. europaea for oxygen, which suppressed the ammonia
oxidation rate by N. europaea. Here, we describe the use of
molecular hydrogen instead of ethanol as an electron donor for
denitrification. This substitution controlled the distribution of
P. denitrificans and avoided the competition for oxygen.
Bacterial strains and their immobilization.
The ammonia
oxidizer N. europaea IFO-14298 and the denitrifier P. denitrificans JCM-6892 were used in this study. As previously described (28), the bacteria were separately aerobically
cultured at 30°C and then coimmobilized with photocross-linkable
polymer PVA-SbQ (SPP-H-13; Toyo Gosei Kogyo Corp.). A glass tube was
used as a mold to form the polymeric gel containing the bacteria into a
tube (outside diameter, 12 mm; inside diameter, 5 mm; length, 125 mm).
Batch experiments for nitrogen removal.
Solutions of ammonia
or nitrate (200 ml) were treated with the tubular gel at 30°C while
stirring (100 rpm) (Fig. 1). The ammonia
solution contained (per liter) 0.472 g of
(NH4)2SO4, 0.2 g of
MgSO4 · 7H2O, 9 g of
Na2HPO4, 1.5 g of
KH2PO4, and 1 ml of solution containing trace
elements (1). The nitrate solution contained the same
components, except that the
(NH4)2SO4 was replaced with 0.722 g
of KNO3. Hydrogen gas (99.99%) serving as an electron donor for denitrification flowed through the lumen of the tube at a
rate of 360 ml/h. To acclimate the tube, it was exposed to the ammonia
solution for 96 h, after which the solution was renewed. This
process was repeated four times.
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FIG. 1.
Schematic diagram of a batch system with a tubular gel.
Artificial wastewater (200 ml) containing 100 mg of N/liter (as ammonia
or nitrate) was treated. The wastewater was agitated by stirring (100 rpm) at 30°C. Hydrogen gas serving as an electron donor for
denitrification flowed through the lumen of the tube (360 ml/h).
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Ammonia and nitrite in the solution were measured colorimetrically as
previously described (5). Nitrate concentrations were
determined using an ion-chromato analyzer (DX-AQ1120; Dionex Corp.) equipped with an IonPac AS12A column. Discharged gas from the
tube was collected and analyzed using a gas-chromato analyzer (CP-2002; Chrompack Corp.) equipped with a PoraPLOT-Q column and a
microthermal conductivity detector.
Fluorescent-antibody labeling.
Prior to and then after the
five batch experiments, sections of gel were cut away from the tube,
fixed, dehydrated, embedded in polyethylene glycol, and sliced using
the procedure of Hunik et al. (11). The sliced sections
were labeled for 45 min at 52°C in the dark with rabbit
anti-N. europaea-fluorescein isothiocyanate and with
rabbit anti-P. denitrificans-fluorescein
isothiocyanate. The labeled sections were then examined
under a fluorescent microscope as described previously
(28). The distributions of fluorescence within
the gels were analyzed in photomicrographs with an IPLab image analysis
system (Spectrum Signal Analytics Corp.).
Nitrogen removal by the tubular gel bioreactor.
When the
ammonia solution was aerobically treated with the tubular gel in a
batch system, the ammonia concentration in the solution decreased
linearly from 100 to 1.7 mg of N/liter within 72 h (Fig.
2A). The nitrite concentration in the
solution increased to 10.0 mg of N/liter within the first 48 h and
then decreased to 2.0 mg of N/liter during the next 48 h. Nitrate
was not detected (<0.05 mg of N/liter) at any time during the
experiment. When nitrate solution was treated in the same way, the
concentration of nitrate decreased from 100 to 8.3 mg of N/liter within
72 h (Fig. 2B). A small amount of nitrite (~3 mg of N/liter) but
no ammonia (<0.05 mg of N/liter) was detected. Nitrogen gas production was thought to accumulate in the lumen of the tube, since nitrous oxide
was detected within the lumen of the tube exposed to acetylene, which
is the inhibitor of N2O reductase (data not shown). Neither gas production nor leakage from the external surface of the tube was
observed during the experiments. To confirm the utilization of
hydrogen, the nitrate solution was treated with nitrogen (99.99%) flowing through the tube instead of hydrogen. Under these conditions, the nitrate concentration in the solution did not decrease, and neither
ammonia nor nitrite was detected in the solution (data not shown).
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FIG. 2.
Time-dependent changes of the nitrogen concentration in
wastewater containing ammonia (A) or nitrate (B) during the first batch
experiment. Data are expressed as means ± standard deviations
(SD) (n = 4).
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These results show that the tubular gel bioreactor can effectively
remove ammonia, nitrite, and nitrate by aerobic nitrification and
anaerobic denitrification with hydrogen as the electron donor and with
the produced N2 gas going out from the lumen of the tube. Our bioreactor did not depend on the denitrifying activity of N. europaea, which has been recently reported (4, 21, 22), since the N. europaea used in this study showed no
denitrifying activity (28). N. europaea showed no
growth in medium containing nitrite and nitrate with hydrogen (data not shown).
Acclimation of tubular gel.
To acclimate the tubular gels,
batch experiments were repeated five times. In the second experiment,
the ammonia concentration in the solution declined to <1 mg of N/liter
within 48 h; during the same period, nitrite increased to 36.0 mg
of N/liter and then decreased to <1 mg of N/liter over the following
24 h. In the third, fourth, and fifth batch experiments,
ammonia and nitrite disappeared within 24 and 72 h,
respectively. The rates of ammonia oxidation (ammonia to
nitrite) and nitrogen removal (ammonia to nitrogen gas) by the tube
were calculated on the basis of changes in the ammonia and
nitrite concentrations during the initial 12 h of the batch
experiments. During the first batch experiment, the rates were
calculated, respectively, to be 1.623 ± 0.194 and 1.469 ± 0.441 g of N/day/m2 of gel surface (Table
1). By the fifth experiment, these rates had increased to 6.886 ± 0.353 and 3.371 ± 0.239 g of
N/day/m2 of gel surface.
The nitrogen removal rate by the tubular gel was half the ammonia
oxidation rate (Table 1). This reflects, in part, the dimensions of the
tube: the internal surface area (denitrification zone) was less than
half that of the external surface area (nitrification zone). In
experiments using ethanol as the electron donor where denitrification
and nitrification zones were almost same size, the nitrogen removal
rate was 19% lower than the ammonia oxidation rate (Table
2). In an experiment using a plate-shaped
gel with external and internal surface areas of equal size, the
nitrogen removal rate was 22% lower than the ammonia oxidation rate.
The lower rate of nitrogen removal might be ascribed to the fact that denitrification requires prior nitrification and would therefore not
exceed the ammonia oxidation rate in an ammonia-rich solution. On the
other hand, we observed a nitrogen removal rate per unit area of
internal surface that was higher than the ammonia oxidation rate
(Table 2), indicating that when hydrogen is used, nitrogen removal
rates can approach ammonia oxidation rates.
Bacterial distribution within the tubular gel bioreactor.
The
distributions of N. europaea and P. denitrificans
within the tubular gel were microscopically investigated with
fluorescently-labeled antibodies. Before the batch experiments, small
colonies of N. europaea and P. denitrificans were
sparsely spread throughout the tube. After the fifth batch experiment,
colonies of N. europaea were concentrated within a region
extending from the external surface to a depth of 200 µm (Fig.
3A) and were not detected in the interior
of the tube (Fig. 3B); colony sizes were largest in regions closest to
the external surface and decreased in size as a function of distance
from the external surface. Conversely, after the batch experiments,
colonies of P. denitrificans were concentrated in a region
extending from the internal surface to a depth of 100 µm (Fig. 3D),
with colonies situated closer to the internal surface growing larger
than those situated at a greater depth. The size and number of those
colonies farthest from the internal surface did not increase during the
course of the experiments (Fig. 3C). To compare the distributions of
P. denitrificans and N. europaea more
quantitatively, their respective distribution densities were assessed
by analyzing the photomicrographs described above. Figure
4 shows that when hydrogen was used as
the electron donor, the distribution of P. denitrificans was
clearly separate from that of N. europaea.
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FIG. 3.
Fluorescence photomicrographs of N. europaea
(A and B) and P. denitrificans (C and D) in regions of a
tubular gel cut in cross section. Panels A and C show the external
surface of the tubular gel, whereas panels B and D show the internal
surface. The corresponding areas within the tubular gel are illustrated
in the center diagram.
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FIG. 4.
Distributions of N. europaea and P. denitrificans within a tubular gel. The solid and dotted curves
show the relative biomasses of N. europaea and P. denitrificans, respectively. These curves were determined by
analysis of the images in Fig. 3A and D.
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With hydrogen as the electron donor, ammonia was removed by the tubular
bioreactor just as efficiently as with ethanol and without
accumulating nitrite or nitrous oxide. Indeed, the ammonia oxidation
rate was as much as 25% higher than that seen with ethanol (Table 2).
What is more, when hydrogen served as the electron donor, the
distributions of P. denitrificans and N. europaea
were completely distinct from one another. Because hydrogen is only slightly soluble in water (0.9 mmol/liter at 0°C and 1 atm), its diffusion from the inner surface of the tube was limited, which restricted the distribution of P. denitrificans. This
resultant clear separation of the two bacterial strains and the
extinction of the competition for oxygen was most likely the reason for
the 25% increase in the ammonia oxidation rate.
In summary, we showed that the distributions of P. denitrificans and N. europaea could be separated within
a tubular gel bioreactor. Their separation effectively increased the
cooperation between nitrification and denitrification within our
bioreactor, and this approach should also be effective with a variety
of other bioreactors requiring combinations of aerobic and anaerobic reactions.
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