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Applied and Environmental Microbiology, December 2000, p. 5155-5160, Vol. 66, No. 12
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Coliform Bacteria and Nitrogen Fixation in Pulp and
Paper Mill Effluent Treatment Systems
Francis
Gauthier,1,2
Josh D.
Neufeld,1,2
Brian T.
Driscoll,2 and
Frederick S.
Archibald1,2,*
Pulp and Paper Research Institute of Canada
(PAPRICAN), Pointe-Claire, Québec, Canada, H9R
3J9,1 and Department of Natural Resource
Sciences, McGill University, Ste-Anne-de-Bellevue, Québec,
Canada, H9X 3V92
Received 14 April 2000/Accepted 14 September 2000
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ABSTRACT |
The majority of pulp and paper mills now biotreat their combined
effluents using activated sludge. On the assumption that their
wood-based effluents have negligible fixed N, and that activated-sludge microorganisms will not fix significant N, these mills routinely spend
large amounts adding ammonia or urea to their aeration tanks (bioreactors) to permit normal biomass growth. N2 fixation
in seven Eastern Canadian pulp and paper mill effluent treatment systems was analyzed using acetylene reduction assays, quantitative nitrogenase (nifH) gene probing, and bacterial isolations.
In situ N2 fixation was undetectable in all seven
bioreactors but was present in six associated primary clarifiers. One
primary clarifier was studied in greater detail. Approximately 50% of all culturable cells in the clarifier contained nifH, of
which >90% were Klebsiella strains. All primary-clarifier
coliform bacteria growing on MacConkey agar were identified as
klebsiellas, and all those probed contained nifH. In
contrast, analysis of 48 random coliform isolates from other mill water
system locations showed that only 24 (50%) possessed the
nifH gene, and only 13 (27%) showed inducible
N2-fixing activity. Thus, all the pulp and paper mill
primary clarifiers tested appeared to be sites of active N2
fixation (0.87 to 4.90 mg of N liter
1 day1)
and a microbial community strongly biased toward this activity. This
may also explain why coliform bacteria, especially klebsiellas, are
indigenous in pulp and paper mill water systems.
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INTRODUCTION |
Good performance of activated-sludge
biotreatment systems depends on the concentrations of several key
nutrients, including bioavailable "fixed" nitrogen. Unlike
municipal sewage, pulp and paper mill wastewaters are typically rich in
carbohydrates but poor in fixed nitrogen, due to the high C/N ratio
typical of wood. Therefore, careful dosing of the raw effluent with
fixed N and P prior to biotreatment is essential. This is a substantial
expense. A ratio of 100:5:1 for bioavailable carbon (C) to nitrogen (N) to phosphorus (P) in the feed (raw effluent) is usually recommended. Excess fixed N is also undesirable, as it may result in fish-toxic free
ammonia reaching the receiving waters and eutrophication (19). Successive nitrification and denitrification of excess N may also cause "rising sludge" in the secondary clarifier due to
entrapment of N2 gas bubbles, resulting in biosolid losses to the receiving waters (16).
The biological fixation of N2 usually requires the
following conditions: (i) readily available carbohydrates as an energy source (5, 12), (ii) low fixed-nitrogen concentrations
(5), and (iii) absence or very low concentrations of
dissolved oxygen (DO) (5, 12-14). N2 fixation
has been reported from some pulp and paper mill aerated lagoons
(4, 8), which may have regions supplying these key
conditions. The N2 fixation in these lagoons was shown to
be capable of supplying the entire N requirements of the system,
corresponding to more than 600 kg of N day1
(8). In contrast, N2 fixation is unlikely to
occur in activated-sludge aeration tanks or secondary clarifiers of
pulp and paper mills because (i) the DO level typical of
activated-sludge operation is too high (usually 1 to 3 mg of
O2 liter1) for nitrogenase to function
(8, 21), (ii) nutrient nitrogen has already been added as
NH3 or urea, repressing N2 fixation (18), and (iii) levels of free sugars are usually very low
because of the intense competition for carbon and energy sources by the activated-sludge biomass (16).
A mill primary clarifier is an unmixed settlement tank or basin,
continuously removing suspended wood fibers and particles, and fillers
or coaters such as clay, starch, and calcium carbonate from combined,
pH-adjusted raw mill effluents. Primary clarifiers usually meet the
three criteria given above for the growth and activity of
N2-fixing microorganisms. Since their function is to
mechanically settle particles and fibers, there is no aeration (low
DO), and as supplemental fixed N has not been added yet in most mills,
the C/N ratio is likely to be high. Previous work on the ecology of
coliform bacteria in several pulp and paper mill effluent systems found
permanent coliform populations, with Klebsiella strains
predominant, in all primary clarifiers examined (11a).
Indeed, the presence of N2-fixing members of the
Enterobacteriaceae, including Klebsiella sp.
strains, in pulp and paper mill water systems has long been known
(2, 4, 6, 8, 9, 15, 18, 20-22).
In this study we used both acetylene reduction (AR) (an indirect
measurement of nitrogenase activity) and functional gene probing (for
the nifH nitrogenase gene) to detect N2-fixing
bacteria and N2 fixation in seven primary-clarifier and
bioreactor ecosystems. To measure the abundance and composition of
N2 fixers in one particular clarifier, we enumerated the
culturable bacterial community, the coliform bacteria, and we tested a
subsample of the resulting colonies for the nifH gene by
colony hybridization. We then identified a subset of those organisms
that tested positive using biochemical characterization. This is the
first report demonstrating N2-fixing activity by both
cultured isolates and active in situ populations in pulp and paper mill
primary clarifiers. The results also indicate the predominance of
the genus Klebsiella in these nitrogen-fixing communities.
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MATERIALS AND METHODS |
Sampling procedures.
Seven Eastern Canadian pulp and paper
mills were chosen in order to include a broad range of pulp and
papermaking processes and biotreatment system designs (Table
1). Over 17 months, samples from the
primary clarifiers and from the aeration tank biomass and liquor were
collected. The primary-clarifier samples were collected at several
depths using a 1.2-liter Kemmerer autosampler. Grab samples were
transported in 500-ml polypropylene bottles on ice. AR assays and
bacterial cultures were done within 4 and 24 h of sampling,
respectively.
Total counts, coliform isolates, and pure culture controls.
Total bacterial counts used triplicate serial dilutions and plating of
samples onto 1/5-strength Trypticase soy agar (TSA) plates (Difco)
supplemented with 1% (wt/vol) NaCl and 1% (wt/vol) glucose. Coliform
counts used triplicate dilution plating on both MacConkey and eosin
methylene blue agar (Difco). All plates were incubated at 37°C for 18 to 24 h. Total and fecal coliforms (TC and FC) were selected for
an enumerated as described previously (11a) using the
most-probable-number (MPN) methods recommended in Standard
Methods for the Examination of Water and Wastewater, sections
9221B and 9221E (1). Isolations were carried out as described previously (11a) using MacConkey agar. Strains
were identified using the standard API 20E biochemical test procedure (Biomérieux).
Control bacterial strains are listed in Table
2. Prior to DNA extraction,
Azoarcus tolulyticus was grown aerobically in modified
R2A
broth (ATCC culture medium 2120). All other pure (control)
cultures
(Table
2) were grown aerobically in 10 ml of nutrient
broth (Difco).
Flask cultures.
Coliform colonies isolated from MacConkey
agar were transferred successively onto "high-N"
glucose-thioglycolate agar plates and into 10-ml tubes of the
corresponding "N-free" broth containing, per liter, 10.0 g
of D-glucose, 6.3 g of
K2HPO4, 1.7 g of
NaH2PO4, 0.1 g of MgSO4, 0.008 g of Na2MoO4, 0.008 g of ferric citrate, 0.5 g of Na-thioglycolate, and 0.001 g of resazurin. The high-N broth also contained 0.2 g of yeast extract liter1
and 0.5 g of Casamino Acids liter1. In situ
N2 fixation activity was measured by the AR assay as described by Knowles et al. (18). Assays were initiated by
the removal of 5 ml of N2 and the addition of 5 ml of
acetylene (final concentration, 10% [vol/vol]), followed by
incubation at room temperature with shaking (120 rpm). To assay
N2 fixation by isolates, 1 ml of a "preenrichment"
culture growing on 9 ml of fresh "N-free" medium was pipetted into
50-ml flasks, and the assay was performed as for clarifier samples
(above). The AR assay was used essentially as described previously
(18).
nifH gene probe.
From the published sequence of
the Mo-Fe nitrogenase genes of Azotobacter chroococcum
(10, 17), we designed primers to amplify a portion of the
nifH gene from a purified plasmid (pER4) containing
nifHDK from A. chroococcum. The two primers,
designated NIFN3 (5'-ATCCACCACCACTCAGAACC) and NIFC3
(5'-ATAACGCCGAACTCCATCAG) amplified a 780-bp region of
nifH (sequence positions 285 to 1064; GenBank accession no.
M20568). To obtain labeled probe, we amplified the probe sequence by
PCR, incorporating digoxigenin (DIG)-labeled dUTP according to the
manufacturer's protocol (PCR DIG probe synthesis kit; Roche Molecular
Biochemicals). The amplified probe was purified after 1% agarose gel
electrophoresis (with an agarose gel DNA extraction kit from Roche
Molecular Biochemicals).
DNA extraction.
DNA was extracted from pure cultures using a
sodium dodecyl sulfate (SDS)-based method (3). Well-mixed
primary clarifier grab samples (500 ml) were blended in a Waring
blender (1 min), then filtered through cheesecloth and centrifuged
(10,000 × g, 10 min, 4°C). The pellet was
resuspended in 1 to 3 ml of lysing solution containing 100 mM Tris-HCl
(pH 8.0), 300 mM NaCl, 20 mM EDTA (pH 8.0), and 2% (wt/vol) SDS. One
milliliter of resuspended cells was placed in a 2-ml screw-cap tube
containing 1 g of zirconia-silica beads (diameter, 0.1 mm)
(Biospec Products, Bartlesville, Okla.) and 1 ml of phenol-chloroform
(1:1, vol/vol). The cells were lysed by 5 min in a beadbeater and then
centrifuged (2,000 × g, 15 min), and the aqueous
DNA-containing supernatant was repeatedly phenol-chloroform and
chloroform extracted. Phase separation was carried out at 10,000 × g for 15 min. The purified DNA was treated
with RNase, precipitated with isopropanol, washed with 70% ethanol,
dried, and resuspended in 200 to 500 µl of Tris-EDTA (TE) buffer.
Purified DNA was quantified on a 1% agarose gel using an AlphaImager
1200 with AlphaEase software (Alpha Innotech).
ATP extraction.
ATP was extracted from primary-clarifier and
activated-sludge samples using a trichloroacetic acid-EDTA-based ATP
release method and was measured using commercial luciferin-luciferase assay reagents (FL-AAM, FL-AAS, and FL-AAB; Sigma). Light production was measured using an LKB model 1250 luminometer.
Dot blot hybridization.
Purified DNA (1 µg) was alkali
transferred to Hybond N+ membranes (Amersham) using a dot
blot manifold. After UV cross-linking (Stratagene Stratalinker),
membranes were rinsed in 0.5 M Tris-HCl (pH 7.0) to ensure removal of
alkali, because the probe's DIG label is alkali labile. The
hybridization and wash procedures were modified from earlier methods
(25, 27). The membranes were prehybridized in roller bottles
(42°C, 4 h) in 50% formamide-5× SSPE (1× SSPE is 0.18 M
NaCl, 10 mM NaH2PO4, 1 mM EDTA [pH 7.7]-5× Denhardt's solution-0.2 mg of salmon sperm DNA ml1.
Denhardt's solution is, per liter, 0.2 g of Ficoll, 0.2 g of polyvinylpyrrolidone, and 0.2 g of bovine serum albumin.
Hybridization was carried out for 14 to 16 h at 42°C in 10 ml of
prehybridization solution with 10% (wt/vol) dextran sulfate and 10 ng
of denatured nifH probe ml1. The membrane was
washed in 2× SSC (1× SSC is 0.15 M NaCl plus 0.015 M sodium
citrate)-0.1% SDS (15 min at room temperature [RT]), 0.5×
SSC-0.1% SDS (15 min at RT), 0.1× SSC-0.1% SDS (15 min at RT), and
0.1× SSC-1% SDS (15 min at 42°C). Detection was carried out by
using nitroblue tetrazolium-5-bromo-4-chloro-3-indolylphosphate (NBT-BCIP) as a colorimetric substrate for alkaline phosphatase according to the manufacturer's protocols (Roche Molecular Biochemicals).
Colony hybridization.
Random colonies from both TSA and
MacConkey plates were chosen for hybridization analysis. These isolates
and pure culture controls were transferred to TSA plates. After 24 h at 37°C, colonies were transferred to Hybond N+
membranes (Amersham) and lysed (23). Hybridization with
nifH, washes, and detection were done as for the dot blots.
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RESULTS AND DISCUSSION |
Occurrence of N2 fixation in mill aeration tanks
(bioreactors).
Existing reports of N2 fixation in pulp
and paper mill treatment systems are limited to aerated lagoons
(4, 8) and laboratory-scale activated-sludge systems held at
abnormally low DO and fixed-N levels (11a, 20;
D. J. Gapes, N. M. Frost, T. A. Clark, P. H. Dare,
R. G. Hunter, and A. H. Slade, presented at the 6th IAWQ Symposium on Forest Industry Wastewaters, Tampere, Finland, 6 to 10 June, 1999). In this study, full-scale on-line activated-sludge systems
and primary clarifiers were examined for N2-fixing
activity. The biomass and suspended liquor from the activated-sludge
aeration tanks (bioreactors) of the seven mills described in Table 1
were sampled, and AR assays were run within 4 h. While the
nifH gene was detected in the samples, none showed any
N2 fixation activity (data not shown).
Occurrence of N2 fixation in mill primary
clarifiers.
In contrast, both the nifH gene and active
N2 fixation were present in all of the six mill primary
clarifiers tested (Fig. 1; Table
3). Since all dots contained the same
total DNA, the nifH probe signal amplitudes (Fig. 1)
indicate the relative proportion of N2-fixing genes in the
total population, not the absolute population size. The amount of
nifH gene probe that hybridized to these primary-clarifier DNA samples varied from very low to as much as that seen using control
DNA from pure cultures of known N2-fixing organisms. This suggests that a high proportion of the total microbial consortium in
the primary clarifier carries the nifH gene. We also
observed a general correlation between the nifH probe signal
intensity and the average in situ nitrogen-fixing activity (Table 3)
calculated from all sampling sites we tested in each clarifier (Fig.
1). The maximum N2 fixation rates observed in the six
primary clarifiers ranged between 0.87 and 4.90 mg of N
liter1 day1 (Table 3), comparable to the
maximum rates previously reported from an aerated lagoon system (5.5 mg
of N liter1 day1) (8).
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FIG. 1.
Dot blot hybridization of 1-µg DNA samples to the
nifH gene probe. Positive controls (from left to right) are
primary-clarifier isolates KPN3, KPG2, and KPG3, A. tolulyticus, Pseudomonas stutzeri JM300,
Sinorhizobium meliloti, and Rhodobacter
sphaeroides. Negative controls (from left to right): KPG1
(activated sludge isolate), Escherichia coli K-12, P. stutzeri Zobell, Paracoccus denitrificans,
Corynebacterium nephridii, Pseudomonas sp. strain
G-179, no DNA (NaOH) control. Seven primary-clarifier samples (see
Table 1 for mill treatment system characteristics) were taken from mill
B-1, nine from mill E, nine from mill F, eight from mill G, and two
from mill C. Samples were taken at various depths at different
locations across the clarifier diameter. The actual biochemical
N2-fixing activity indicated, in milligrams of N fixed per
liter per day, is an average of in situ activity measured at all
sampling points (±, 0 to 0.5; +, 0.5 to 1.0; ++, 1.0 to 1.5; +++, 1.5 to 2.0).
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The primary clarifiers, which are responsible for mechanical settling,
had, as expected, only a fraction of the biomass density
of the
corresponding activated-sludge-containing aeration tanks.
At a depth of
4 m, the mill C primary clarifier had 167 ng of
ATP
ml
1, while the mill C aeration tank contained 3,400 to
5,200 ng of
ATP ml
1.
Monitoring the mill C primary clarifier for 6 months indicated that
N
2 fixation probably occurs continually (Table
3). Because
the primary clarifiers are unmixed and thus highly heterogeneous,
the
amount of N
2 fixation (and biomass density) varies greatly
with the sampling site selected (Table
3). To see if there was
net
N
2 fixation in the primary clarifier, the fixed N
concentrations
of the mill C primary-clarifier input and output were
compared
(three sets of grab samples collected on three different
days).
The input effluent had on average a total Kjeldahl nitrogen
concentration
(TKN) of 3.8 ± 0.2 mg/liter, and the clarified
output effluent
had an average TKN of 4.8 ± 0.7 mg/liter,
indicating a substantial
increase in fixed N (about 120 kg, based on
120,000 m
3 of raw effluent clarified day
1),
despite the decrease in total C accompanying the removal of
settleable
solids by the primary clarifier. Because of the fluctuations
in the
operation of each mill and the large variations between
mills, these
can only be considered preliminary data for mill
C, and applicable only
to mill C. Since the mill measures the
C-to-N ratio of the 107 tonnes
of primary biosolids removed day
1 by this clarifier to be
650:1 to 800:1, it can be calculated
that the settled biosolids
decreased the output TKN by about 1.0
mg/liter, meaning that another
120 kg of N is produced day
1 in the primary clarifier of
mill C. The structure and bioavailability
of the N entering and exiting
the primary clarifiers are unknown.
After passage into the mill C
aeration tank, most of this N appears
to become part of the activated
sludge, as the mill adds ammonia
and ammonium pyrophosphate to a C/N/P
ratio of 100:4.0 to 4.5:0.8.
If all this deliberately added N is taken
up, the activated (secondary)
sludge should have a C/N ratio between
20:1 and 25:1. In fact,
the measured ratio is typically 9:1, strongly
suggesting that
a large amount of the N arriving from the primary
clarifier is
captured in the activated sludge. Conversely, the fact
that the
primary-clarifier diazotrophs fix N in the presence of an
influent
TKN of 3.8 mg/liter suggests that the N originating in the
mill
is not readily available to
them.
In most of the six primary clarifiers, higher N
2 fixation
rates were observed at greater sampling depths (Table
3 and Fig.
2). There are three likely reasons.
Firstly, ATP measurements
showed higher biomass concentrations in
deeper samples, which
contain more solids (Fig.
2). Secondly, because
nitrogenase is
O
2 sensitive, the low oxygen tension
observed in deeper samples
may allow more N
2 fixation per
unit of biomass (Table
3). Thirdly,
the settling of wood particulates
in primary clarifiers may also
improve carbohydrate availability at
greater depths, and readily
available carbohydrates are essential for
N
2 fixation, since a
large amount of ATP is required.
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FIG. 2.
Influence of depth on N2 fixation activities
(bars) and ATP concentrations (lines) of three primary clarifiers.
Sampling was performed at the middle of the clarifier radius except for
mill G (3 m from the center). N2 fixation is reported as
means of duplicate samples ± standard deviations. ATP
concentrations were not measured for the mill C primary clarifier.
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The rate of N
2 fixation was shown to be nutrient limited in
the mill C primary clarifier, at least in bottom sludge samples,
where
in situ fixation was highest (Fig.
3).
Added glucose and
acetate increased the N
2-fixing activity
more than sevenfold over
24 h. Although the
Enterobacteriaceae, particularly
Klebsiella pneumoniae, are metabolically versatile (
20), it is
likely that
the glucose was the major driver of increased
N
2 fixation. Bruce
and Clark (
4) observed that
the AR rate of
K. pneumoniae isolates
growing on Kraft mill
effluent in an aerated stabilization basin
(lagoon) was greatly
stimulated by 5 g of glucose liter
1. Thus, the
nutrient limitation that exists in the aeration tank
biomass
(
16) also occurs in the primary clarifier, at least
for
N
2 fixation.
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FIG. 3.
Effects of added nutrients on N2 fixation in
the mill C primary clarifier. The sample was obtained at a 5-m depth.
Glucose and sodium acetate (final concentrations, 0.05% [wt/vol])
were added. Values are means of duplicate samples ± standard
deviations. Error bars that are not visible are smaller than the
symbols.
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Importance of Klebsiella in the primary-clarifier
N2-fixing community.
Previously, we demonstrated that
coliform bacteria, predominantly Klebsiella strains, were
numerous in several of the primary clarifiers examined
(11a). To determine the dominant taxa in the
primary-clarifier N2-fixing community, nonselective (dilute TSA) agar plate counts were done. From the same plates 188 colonies, 94 each from samples A and B (Table 4), were
screened by colony hybridization. Of these isolates, 94 (50%) were
nifH positive. Identification of 20 randomly selected
nifH positive isolates revealed that 18 (90%) were
klebsiellas. Thus, N2-fixing coliform bacteria
belonging to the genus Klebsiella account for 45% of the
total bacterial population culturable on a nonselective medium. The
API 20E system we used for colony characterization classed all
the isolated klebsiellas as K. pneumoniae. However, previous work revealed that a high proportion of the K. pneumoniae
isolates found in pulp and paper mill ecosystems could be more
appropriately referred to as Klebsiella terrigena or
Klebsiella planticola due to their inability to produce gas
from lactose at 44.5°C (11a). The high numbers and
proportions of Klebsiella strains growing on the
nonselective TSA plates indicate a higher proportion of coliform
bacteria in primary-clarifier samples than was estimated by the
TSA/MacConkey plate count ratios (Table 4). Thus, a combination of gene
probing, API identification, and classical nonselective growth
techniques shows that, at least in this primary-clarifier system, the
MacConkey agar MPN counts seriously underestimate the total number of
coliform bacteria present.
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TABLE 4.
Occurrence of nifH-containing members among
total culturable bacteria and coliform bacteria from a selected
primary clarifier, and predominance of Klebsiella
spp. among N2-fixing bacteria isolated
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Colony hybridization of 188 MacConkey isolates showed that 98% of the
coliform population in the primary clarifier carried
nifH.
Of the 186
nifH-bearing isolates, 20 were randomly chosen,
and all (20 of 20) were identified as
Klebsiella strains
(Table
4). The seven mill systems surveyed here were all shown
previously
to support numerous coliform bacteria (
11a), but
this is the
first report demonstrating that primary clarifiers have
large
populations of bacteria actively fixing nitrogen in situ, and
even larger populations with the genetic potential for N
2
fixation.
Unlike previous results from a lagoon treatment system
(
4),
our findings indicate that N
2-fixing
Klebsiella strains are the
major diazotrophs in pulp and
paper mill water and treatment
systems.
Does the presence of the nifH gene correlate with
measured N2-fixing activity?
A total of 48 coliform
isolates from several mills were tested for the nifH gene
and their ability to reduce acetylene (Table 5). Of the 48 isolates, 13 (27%) showed
AR activity while 24 (50%) possessed the nifH gene. No
isolate testing negative for nifH could reduce acetylene,
and all strains reducing acetylene tested positive for nifH.
Comparable active-to-potential N2 fixation ratios were
observed with K. pneumoniae isolates, with 9 of 32 (28%)
and 18 of 32 (56%) displaying N2-fixing activity and the presence of the nifH gene, respectively. The percentage of
N2-fixing K. pneumoniae isolates among isolates
from the seven mill water systems (28%) is consistent with the
findings of a previous report in which 32% of klebsiella from several
ecosystems, including pulp mills, possessed N2-fixing
ability (18). The low proportion of nifH-positive
K. pneumoniae isolates (56%) seen in Table 5 compared with
the proportion in Table 4 (100%) is presumably due to the fact that
the coliform bacteria for which results are shown in Table 5 were
isolated from many places in the seven pulp mill water systems, not
just from primary clarifiers. Other evidence for the selection of
N2 fixers by the primary-clarifier environment is the
increase in the proportion of nifH-positive K. pneumoniae isolates, from 56% (18 of 32) for isolates from all
sampling locations (Table 5) to 83% (15 of 18) when only K. pneumoniae isolates from primary-clarifier samples are considered. In the primary clarifier, N2-fixing klebsiellas likely
possess selective growth advantages over non-N2-fixing
bacteria, such as the following: (i) raw mill process effluents
typically have high C/N ratios (2, 11a, 12, 18);
(ii) the temperatures in primary clarifiers can rise to 40°C, also
selecting for klebsiellas, since >90% of the strains from these seven
paper mill water systems are thermotolerant (grow at 44.5°C)
(11a); and (iii) the very low DO levels observed in primary
clarifiers (Table 3) presumably select for facultative bacteria, such
as coliforms (4, 21).
Conclusions.
(i) The combination of in situ and isolate
nifH gene probing and AR assays with classical microbial
enumeration, isolation, and identification was very effective in
elucidating the nature, magnitude, and microbiology of N2
fixation in mill water systems.
(ii)
Klebsiella strains actively fixing N
2 are
major components of pulp mill primary-clarifier microbial
communities. This
probably explains why coliform
bacteria are indigenous to pulp
and paper mill water systems
(
2,
11a).
(iii) N
2 fixation by klebsiellas and related coliform
bacteria is commonplace and continuous in these primary clarifiers and
may contribute significantly to the fixed N required by the
activated-sludge
biomass. It should also increase the value of the
combined dewatered
sludges as
fertilizers.
(iv) Every pulp and paper mill biotreatment system is unique. The
relative importance of the N
2 fixed by coliforms in each
primary clarifier, and whether it can be substantially increased
by
adjustments to the clarifier's mode of operation, remains to
be
shown.
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ACKNOWLEDGMENTS |
We thank the staff of the seven mills investigated for their
cooperation. The suggestions of R. Knowles and technical support of F. Young are appreciated. We also thank B. Ward and R. Ye for supplying us
with pure culture controls. Y. Chan is thanked for his generous
donation of plasmid pER4.
This research was funded in part by the Pulp and Paper Research
Institute of Canada (Paprican) and by support for F. Gauthier and
J. D. Neufeld from the Natural Sciences and Engineering Research Council (NSERC) of Canada.
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FOOTNOTES |
*
Corresponding author. Mailing address: PAPRICAN, 570 St. John's Blvd., Pointe-Claire, Québec, Canada, H9R 3J9. Phone:
(514) 630-4100. Fax: (514) 630-4134. E-mail:
farchibald{at}paprican.ca.
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REFERENCES |
| 1.
|
American Public Health Association.
1998.
Standard methods for the examination of water and wastewater, 20th ed.
American Public Health Association, Washington, D.C.
|
| 2.
|
Archibald, F. S.
2000.
The presence of coliform bacteria in Canadian pulp and paper mill water systems a cause for concern?
Water Qual. Res. J. Can.
35:1-22.
|
| 3.
|
Ausubel, F. M.,
R. Brent,
R. E. Kingston,
D. D. Moore,
J. G. Seidman,
J. A. Smith, and K. Struhl (ed.).
1994.
Current protocols in molecular biology.
John Wiley & Sons, Inc., New York, N.Y.
|
| 4.
|
Bruce, M. E., and T. A. Clark.
1994.
Klebsiella and nitrogen fixation in pulp and paper mill effluents and treatment systems.
Appita J.
47:231-237.
|
| 5.
|
Cannon, M.,
S. Hill,
E. Kavanaugh, and F. Cannon.
1985.
A molecular genetic study of nif expression in Klebsiella pneumoniae at the level of transcription, translation and nitrogenase activity.
Mol. Gen. Genet.
198:198-206[CrossRef][Medline].
|
| 6.
|
Caplenas, N. R.,
M. S. Kanarek, and A. P. Dufour.
1981.
Source and extent of Klebsiella pneumoniae in the paper industry.
Appl. Environ. Microbiol.
42:779-785[Abstract/Free Full Text].
|
| 7.
|
Carlson, C. A., and J. L. Ingraham.
1983.
Comparison of denitrification by Pseudomonas stutzeri, Pseudomonas aeruginosa, and Paracoccus denitrificans.
Appl. Environ. Microbiol.
45:1247-1253[Abstract/Free Full Text].
|
| 8.
|
Clark, T. A.,
P. H. Dare, and M. E. Bruce.
1997.
Nitrogen fixation in an aerated stabilization basin treating bleached kraft mill wastewater.
Water Environ. Res.
69:1039-1046[CrossRef].
|
| 9.
|
Duncan, D. W., and W. E. Razzell.
1972.
Klebsiella biotypes among coliforms isolated from forest environments and farm produce.
Appl. Microbiol.
24:933-938[Medline].
|
| 10.
|
Fallik, E.,
Y.-K. Chan, and R. Robson.
1991.
Detection of alternative nitrogenases in aerobic gram-negative nitrogen-fixing bacteria.
J. Bacteriol.
173:365-371[Abstract/Free Full Text].
|
| 11.
|
Finn, T. M.,
B. Kunkel,
G. D. Vos, and X. E. R. Signer.
1986.
Second symbiotic megaplasmid in Rhizobium meliloti carrying exopolysaccharide and thiamine synthesis genes.
J. Bacteriol.
167:66-72[Abstract/Free Full Text].
|
| 11a.
| Gauthier, F., and F. S. Archibald. The ecology of
"fecal indicator" bacteria commonly found in pulp and paper mill
water systems. Water Res., in press.
|
| 12.
|
Hardy, R. W. F.,
R. C. Burns, and R. D. Holsten.
1973.
Applications of the acetylene-ethylene assay for measurement of nitrogen fixation.
Soil Biol. Biochem.
5:47-81.
|
| 13.
|
Hill, S.
1988.
How is nitrogenase regulated by oxygen?
FEMS Microbiol. Rev.
54:111-130[CrossRef].
|
| 14.
|
Hill, S.,
G. L. Turner, and F. J. Bergensen.
1984.
Synthesis and activity of nitrogenase in Klebsiella pneumoniae exposed to low concentrations of oxygen.
J. Gen. Microbiol.
130:1061-1067[Abstract/Free Full Text].
|
| 15.
|
Huntley, B. E.,
A. C. Jones, and V. J. Cabelli.
1976.
Klebsiella densities in waters receiving wood pulp effluents.
J. Water Pollut. Control Fed.
48:1766-1771[Medline].
|
| 16.
|
Jenkins, D.,
M. G. Richard, and G. T. Daigger.
1993.
Manual on the causes and control of activated sludge bulking and foaming, 2nd ed.
Lewis Publishers, Chelsea, Mich.
|
| 17.
|
Jones, R. L.,
P. Woodley,
A. B. Zinoni, and R. L. Robson.
1993.
The nifH gene encoding the Fe protein of the molybdenum nitrogenase from Azotobacter chroococcum.
Gene
123:145-146[CrossRef][Medline].
|
| 18.
|
Knowles, R.,
R. Neufeld, and S. Simpson.
1974.
Acetylene reduction (nitrogen fixation) by pulp and paper mill effluents and by Klebsiella isolated from effluents and environmental situations.
Appl. Microbiol.
28:608-613[Medline].
|
| 19.
|
Möbius, C. H.
1991.
Nitrogen and phosphorus limits for nutrient deficient industrial wastewaters.
Water Sci. Technol.
24:259-267.
|
| 20.
|
Neilson, A. H., and A.-S. Allard.
1985.
Acetylene reduction (N2-fixation) by Enterobacteriaceae isolated from industrial wastewaters and biological treatment systems.
Appl. Microbiol. Biotechnol.
23:67-74.
|
| 21.
|
Neilson, A. H., and L. Sparell.
1976.
Acetylene reduction (nitrogen fixation) by Enterobacteriaceae isolated from paper mill process waters.
Appl. Environ. Microbiol.
32:197-205[Abstract/Free Full Text].
|
| 22.
|
Niemela, S. I., and P. Vaatanen.
1982.
Survival in lake water of Klebsiella pneumoniae discharged by a paper mill.
Appl. Environ. Microbiol.
44:264-269[Abstract/Free Full Text].
|
| 23.
|
Sambrook, J.,
E. F. Fritsch, and T. Maniatis.
1989.
Molecular cloning: a laboratory manual, 2nd ed.
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
|
| 24.
|
Satoh, T.,
Y. Hoshino, and H. Kitamura.
1976.
Rhodopseudomonas sphaeroides forma sp. denitrificans, a denitrifying strain as a subspecies of Rhodopseudomonas sphaeroides.
Arch. Microbiol.
108:265-269[CrossRef][Medline].
|
| 25.
|
Smith, G. B., and J. M. Tiedje.
1992.
Isolation and characterization of a nitrite reductase gene and its use as a probe for denitrifying bacteria.
Appl. Environ. Microbiol.
58:376-384[Abstract/Free Full Text].
|
| 26.
|
Vincent, J. M.
1941.
Serological properties of the root-nodule bacteria. I. Strains of Rhizobium meliloti.
Proc. Linn. Soc. N. S. W.
66:145-154.
|
| 27.
|
Ye, R. W.,
M. R. Fries,
S. G. Bezborodnikov,
B. A. Averill, and J. M. Tiedje.
1993.
Characterization of the structural gene encoding a copper-containing nitrite reductase and homology of this gene to DNA of other denitrifiers.
Appl. Environ. Microbiol.
59:250-254[Abstract/Free Full Text].
|
| 28.
|
Zhou, J.,
M. R. Fries,
J. C. Chee-Sandford, and J. M. Tiedje.
1995.
Phylogenetic analyses of a new group of denitrifiers capable of anaerobic growth on toluene and description of Azoarcus tolulyticus sp. nov.
Int. J. Syst. Bacteriol.
45:500-506[Abstract/Free Full Text].
|
Applied and Environmental Microbiology, December 2000, p. 5155-5160, Vol. 66, No. 12
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Copyright © 2000, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
