FEMS Microbiology Ecology 85 (1991) 9-14
1991 Federation of European Microbiological Societies 0168-6496/91/$03.50
ADONIS 016864969100052M
9
0
FEMSEC 00307
Humic acid relieves pH-inhibition of nitrification
in continuous-flow columns
’,
M.J. Bazin A. Rutili
’
I,
’
A. Gaines a n d J.M. Lynch
Microbial Physiology Research Group, King’s College, Campden Hill Road, London, Department of Chemical Engineering,
Imperial College, Prince Consort Road London, and A FRC Institute o/ Horticultural Research, Worthing Road, Littlehampton, U.K.
’
Received 18 June 1990
Revision received 21 September 1990
Accepted 22 September 1990
Key words: Nitrification; Humic acid; pH; Continuous culture
1. SUMMARY
Humic acids obtained from a commercial source
and isolated from mushroom wheatstraw compost
inhibited nitrification by Nitrosornonas europaea
and Nitrobacter agilis in a continuous-flow column at concentrations in excess of 100 pg cm-’.
Lowering the pH of the input medium also reduced the rate of nitrification but in the presence
of the compost, and to a lesser extent the commercial humic acid, pH-inhibition was relieved. With
the commercial humic acid, this effect can be
explained in terms of the buffering capacity of the
extract.
2. INTRODUCTION
Autotrophic nitrification is the consecutive
oxidation of ammonium to nitrite and nitrate by
bacteria such as Nitrosornonas and Nitrobacter.
Correspondence to: M.J. Bazin. Microbial Physiology Research
Group, King’s College, Campden Hill Road, London W8 7AH,
U.K.
Recently it has been a subject of environmental
concern because it is associated with loss of
nitrogen from the soil by leaching, eutrophcation
of lakes and rivers, death of freshwater fish and
contamination of drinking water resulting in
blue-baby syndrome and, possibly, gastric cancer
[l].Nitrification is inhibited by pH below a value
of 7.5 [2]. We tested the effect of p H and humic
acid, the generic term for a mixture of compounds
extractable from the humus fraction of soil by
alkaline solutions, on nitrification in column reactors. Continuous-flow column systems have the
advantage of being stable to environmental perturbation and are thermodynamically open [3];
that is to say, in common with most ecosystems,
they exchange material and energy with the external environment [4].
3. MATERIALS AND METHODS
3.1. Bacterial strains and growth conditions
Nitrosornonas europaea and Nitrobacter agilis
were kindly provided by Dr. J. Prosser, Department of Genetics and Microbiology, University of
Aberdeen. Routine maintenance of the strains.
10
humic acids were characterised by the following
methods:
media and column design and operation were as
described by Bazin et al. [5]. Columns, containing
the two species of nitrifiers supported on small
glass beads, were supplied at fixed rates from the
top with nutrient solution. The input nutrient
contained 106 p g cm-3 NHZ-N. The effluent
from the bottom of the columns contained the
products of nitrification (nitrite and nitrate) and
unused ammonium. Analysis of this solution provided a quantitative estimate of the nitrifying
capacity of the system and the effect of adding
humic acids to the influent or changing its pH.
Effluent from the column was collected in a refrigerated fraction collector maintained at 4’ C to
inhibit further bacterial growth and metabolism.
3.2. Effluent analysis
NH:-N, NO;-N and NO;-N concentrations
in the effluent from the column were estimated in
a non-segmented continuous-flow analyser by the
Centre for Research in Analytical Chemistry and
Instrumentation, King’s College. Nitrifying activity was expressed as a percentage of the input
NHZ-N converted. For fixed rate of addition of
nutrient, this figure is proportional to the rate of
nitrification. The pH of the effluent was also
measured.
3.3. Humic acids
A commercial humic acid (AHA), originating
from peat, was obtained from the Aldrich Biochemical Company. Humic acid (MHA) was extracted from mushroom wheatstraw compost using
the method of Schnitzer and Skinner [6]. The
extract was purified by reprecipitating twice in 0.1
M NaOH and dissolving in Na,C03 at pH 12.
The residue was washed with distilled water and
vacuum dried. Dried, powdered samples of both
(i) Elemental analysis using a Perkin-Elmer 240
CHN analyzer, undertaken at the Microanalytical Laboratory, University College, University of London.
(ii) I3C CPMAS (cross polarisation with magic
angle spinning), TOSS (total suppression of
spinning side bonds) NMR (nuclear magnetic
resonance) analysis at the University of
London’s dedicated laboratory.
(iii) Infrared spectroscopy, using the KBr disc
technique in a Perkin Elmer FTIR model
1700 spectrophotometer.
(iv) Fluorescence spectroscopy of dilute aqueous
solutions, employing a Perkin Elmer LH5 fluorospectrophotometer.
(v) Titration of acid groups.
3.4. p H
The effect of pH was tested by adjusting the
pH of the influent medium with 0.1 M HCl or 7
M Na,CO,.
4. RESULTS
4.1. Humic acid analysis
The results obtained ..om elemental analysis
and titration of acid groups of the humic acids are
given in Table 1. Clearly, MHA contains more
nitrogen and less oxygen than AHA and the acidic
oxygen groups are distributed differently in the
two extracts.
13C NMR spectra of the two humic acids are
shown in Fig. 1 which indicates rather different
chemical properties for the two acids. The inter-
Table 1
Elemental analysis and acid group titration of humic acids
Humic
acid
%C
%H
%N
SO
MHA
AHA
53.2
4.9
4.05
4.0
0.6
a
53.0
Calculated by difference.
mM g-I dry weight
Molecular
formula
COOH
OH
31.9
C,(mHll I0,JN6
0.94
42.3
C,COH920WN
0.69
0.95
I .o
a
11
A
w
fbl
,
300
200
loo
I
0
-100
PPM
Fig. 1. ”C CPMAS TOSS N M R spectra for (a) MHA and (b)
AHA.
pretations of these spectra are based on those
suggested by Snape et al. [7] and was aided by
‘dipolar dephased’ spectra which distinguished
CH, from CH, and CH groups and substituted
from protonated aromatic carbon atoms.
The AHA spectrum shows absorption due to
carbonyl, probably carboxyl (approx. 171 ppm),
aromatic (approx. 133 ppm) and aliphatic (particularly at approx. 29 ppm) carbon atoms. In
fact, some 12, 29 and 59% of the carbon atoms
respectively were in these classifications. Approximately 66% of the aromatic carbon atoms
were substituted by carbon or oxygen atoms. The
major aliphatic absorption at 29.3 ppm was clearly
due to CH, groups in alkyl chains. Dipolar dephased spectra showed absorption at 27.1 and
15.4 ppm to be due to methyl groups present at
lower concentrations than the methylene groups.
Absorption at 27.1 ppm may be attributed to
methyl substituents on alicyclic and aromatic
structures whilst absorption at 75.4 ppm is generally attributed to methyl groups in ethyl substituents.
The MHA spectrum also showed absorption in
the carbonyl, aromatic and aliphatic regions, but
the proportions of carbon atoms attributable to
these groups were 8, 50 and 42% respectively. That
is, perhaps surprisingly, the ratio of aromatic to
aliphatic material was higher in this acid. Moreover, the aliphatic moiety in MHA was quite
different to that in AHA. There was little evidence
of long alkyl chains (i.e. little absorption around
29 ppm) and, in fact, the dominant ‘aliphatic’
absorption at 56.4 ppm was probably due to OCH,
substituents on aromatic rings, which accounted
for almost 30% of the carbon atoms present. Dipolar dephasing spectra showed methyl groups in
the 30.5 and 22.8 ppm regions, probably indicating branching of small aliphatic chains. In the
aromatic region, the intensity of a resolved peak
centred at approx. 147 ppm suggests that most of
the aromatic rings were phenolic or substituted by
methoxy groups. There were comparatively few
aromatic carbon atoms substituted by carbon
atoms and the overall spectrum is similar to that
of an oxidised lignin containing structures such as
those shown in Fig. 2.
Infra red spectra were consistent with the results indicated by 13C NMR analysis. There was
marked absorption by both aliphatic (2950-2800,
1450-1400 and approx. 1370 cm-’) and aromatic
(1600 cm-’) material as well as by carboxyl groups
(1700 cm-’). In the AHA relative absorption intensities by aliphatic C-H stretching vibrations
suggested that more CH, groups were present
than CH, groups, in agreement with the I3CNMR
analysis. Absorption in the 900-700 cm-’ region
suggests several substitution patterns to have been
present. Few rings had less than three substituents. A complex pattern of absorption in the
1700-1600 cm-’ region of the MHA infra red
spectrum could have been due to amide or amino
acid vibrations. Broad absorption centred at 840
cm-’ suggests only one or two isolated hydrogen
R
R
Fig. 2. Suggested lignin-like structure contained in MHA. R
contains COOH.
12
+Ammonium-N
-
*
-0- Ammonium
In the
Nmte-N
presence
Nitrate-N
of AHA
U Nitrite
Ammonium-N
+Nitrite-N
In
+Nitrare
the
presence
of MHA
Nitrate-N
100
-E
.
100
(Y
Fc
--i
0
80-
0
c
1
C
60-
$
4
-
60
c
01
5
E
I
40
t
z 20
0
20
5 4
0
0
100
200
300
400
500
6 0
6 6
7 2
7 8
8 4
I
0
Fig. 4. Effect of changing the pH of the input medium to a
nitrification column.
Humic acid concentraton ("0 c ~ n - ~ )
Fig. 3. Effect of humic acids on nitrification in a continuous
flow column as estimated from effluent analysis.
acids. MHA appears to have a higher proportion
of polyaromatic structures.
atoms to be present on the aromatic rings of
MHA, consistent with the lignin-type structure
shown in Fig. 2.
Solutions of both humic acids fluoresced in the
UV-visible regions of the spectrum. Excitation
and emission occurred at wavelengths which were
characteristic of the presence of mono, di- and
polyaromatic systems. Fluorescence intensities
varied markedly with the concentration of the
solution and it was clear that radiative and nonradiative energy transfer occurred. Table 2 shows
the relative emission intensities obtained at concentrations so dilute that emission was proportional to concentration. The table indicates that a
variety of aromatics were present in both humic
4.2. Ejfect of hurnic acids and p H on nitrification
At a nutrient input rate of 1.4 cm3 h-', approximately 96% of the ammonium was converted
to nitrate after the column had come to steady
state. As in previous studies [5,8], at steady states,
input nitrogen was conserved as NH:, NO; and
NO;, indicating negligible nitrogen maintenance
requirement and no chemical transformation of
NO; as reported by Boudot and Chone (91. At
concentrations of humic acid up to 100 p g c m P 3
in the input medium, little effect on nitrification
was observed. At higher concentrations, the process was inhibited in the manner shown in Fig. 3
and significant amounts of nitrite appeared in the
Table 2
Relative emission intensities of humic acids analysed by fluorescence spectroscopy at an excitation wavelength of 254 nm
length
-
275 nm
(monoaromatic)
310 nm
(diaromatic)
375 nm
(polyaromatic)
440 nm
(polyaromatic)
MHA
AHA
1
I
1.3
0.5
2.6
0.7
5.4
1 .I
Wave-
13
-
+Ammonlum (+ MHAI
5. DISCUSSION
d- Nmate (+ MHA)
Ammonium ( + AHA)
Nltraie I + AHA)
The interaction between humic acids and microorganisms appears not to have been clearly
defined. Hassett et al. [lo] reported that they had
a bactericidal effect on Staphylococcus aweus and
Serratia marcescens while Visser [ I l l found that
they stimulated growth of a wide variety of microbes isolated from soil. Our results indicate clearly
that nitrification is inhibited by the humic acids
we employed at concentrations in excess of 100 pg
cm- However, at this concentration they relieve,
at least to some extent, the inhibition of nitrification at low pH. In the presence of AHA, this relief
was less pronounced. The similarity between
nitrification activity as a function of effluent pH
in the presence and absence of AHA indicates that
this humic acid might act as a buffer in the
system. Such an explanation is not applicable to
the effect of MHA, however, because even at
reduced effluent pH, nitrification still occurred at
a comparatively high rate. The analysis of the two
humic acids revealed significant differences be-
’.
Ol
5 4
.
.
,’
6 0
6 6
I
7 2
.
I
7 8
I
I
8 4
1
I
9 0
PH
Fig. 5. Effect of changing the pH of the input medium to a
nitrification column in the presence of humic acids (100 p g
cm-’). In all cases, very little nitrite appeared in the effluent
solution.
effluent solution. The response of the system to
both types of humic acid was similar.
Reducing the pH of the input medium to the
column and allowing the concentration of nitrate
in the effluent to stabilize had the effect illustrated
in Fig. 4. Between pH 9 and 5.4 there was an
approximately linear decrease in the rate of nitrate
production and increase in the appearance of ammonium. Very little nitrite appeared in the effluent. When humic acid at a concentration of 100
pg cm-3 was added to the medium entering the
system, changing the pH of the input had the
effect illustrated in Fig. 5. MHA appeared to
relieve the inhibitory action of pH. Even at pH 5.4
nearly 90% of the ammonium was converted to
nitrate, while in the absence of humic acid less
than 60%conversion took place. AHA had a similar effect but the extent of the amelioration was
less.
Nitrification as a function of effluent p H is
shown in Fig. 6. The relationship between nitrogen output and pH in the presence and absence of
AHA was practically identical. However, in the
presence of MHA, considerably less inhibition
occurred.
0
Ammonium
Nitrate
0 Ammonium (+ MHA)
0 Nitrate ( + MHA)
A Ammonium (+ AHA)
A Nitrate (+ AHA)
4.5
5.5
6.5
7‘5
8.5
Output pH
Fig. 6. Nitnfication in a column reactor as a function of
effluent pH in the presence and absence of humic acids (100
pg cm-’). Negligible amounts of nitrite appeared in all samples.
14
tween them, especially with respect to nitrogen
content. Lignin is probably a major precursor of
humic acids and analysis of the ‘young’ material
extracted from mushroom compost revealed that
it is dominantly an oxidised lignin, unlike the
commercial material extracted from more ‘mature’
peat. However, this analysis could be misleading
as it is possible that a dominant component of the
MHA was aromatic material of microbial origin.
Although AHA apparently has a buffering capacity and MHA additionally relieves the effect of
low pH on nitrification, the chemical bases of
these phenomena are unclear at present. The consequences of t h s observation may be of environmental significance. It might be assumed that one
of the few advantages of acid rain is that it results
in a decrease in the autotrophic conversion of
ammonium to nitrate and thereby a decrease in
nitrate pollution. Our results indicate that in the
presence of some naturally occurring soil constituents, this might not be the case.
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