PRODUCTION
OF DISSOLVED
ORGANIC
MATTER
FROM DEAD GREEN ALGAL
CELLS.
II. ANAEROBIC
MICROBIAL
DECOMPOSITIOW
Al&a
Department
Otsuki2 and Takahisa Hanya
of Chemistry,
Faculty of Science, Tokyo Metropolitan
Fukasawa, Setagaya, Tokyo, Japan
University,
ABSTnACT
At the 60th day of anaerobic decomposition
of dead cells of Scenedesmus sp. at 2OC,
the 30% of added algal cell carbon was transformed
into dissolved organic carbon ,and
20% mineralized;
50% remained as particulate
matter. On the other hand, 8% of the added
algal cell nitrogen was transformed
into dissolved organic nitrogen, 48% was mineralized,
and 44% remained in particulate form.
The dissolved organic compounds consisted mainly of lower fatty acids and yellowish
acidic substances.
Some proteinaceous
material
was found.
An’aerobic
decomposition
patterns are compared with those under aerobic conditions
and suggest the presence of
relatively high concentrations
of dissolved organic matter in anaerobic natural environments.
sition of dead plankton
provided
that
methane fermentation does not occur.
We present here the results of an cxpcrimental study of the production of DO,M by
microbial
decomposition
of dead green
algal cells under anaerobic conditions. The
cxpcrimcnts were carried out with high conccntrations of algal ccl1 material because it
provided anaerobic conditions easily and
made direct examination of dissolved organic products possible.
We wish to thank Drs. R. D. Hamilton
and R. G. Wetzel for critically reading the
manuscript.
INTRODUCTION
Although several studies have been made
of oxygen consumption by sediments (Hayes
and MacAulay 1959; Edwards and Rolley
1965; Pamatmat and Banse 1969), there arc
relatively few studies of the anaerobic decomposition of organic matter in natural
aquatic
environments
( Richards
1965;
Richards et al. 1965; Koyama and Tomino
1967; Adams and Richards 1968). These investigations dealt mainly with the transformation of nitrogen compounds and with
mineralization
rates of organic matter.
However, little is known of the production
of dissolved organic matter (DOM)
by
microbial decomposition of dead algal cells
under anaerobic conditions. Skopintzev ct
al. (1965) found no appreciable difference
between the production of dissolved organic carbon (DOC) by decomposition of
plankton under aerobic and anaerobic conditions,
The general nature of the anaerobic decomposition of organic matter
( Toericn and Hattingh 1969) suggests that
a considerable amount of DOM should be
produced by anaerobic microbial dccompol The work was supported in part by Atomic
Energy
Commission
Contract
AT( U-1)-1599,
COO-1599-43
and National
Science Foundation
Grant GB-15665.
2 Present address:
W. K. Kellogg
Biological
Station, Michigan State University,
Hickory Comers
49060.
LIMNOLOGY
AND
OCEANOGRA.I’IIY
MATERIALS
AND
METHODS
The DOM is defined as organic compounds which pass through a Milliporc HA
filter (mean pore size, 0.45 p). Scenecksmus
sp., grown in an artificial pond and cstimated at > 99% pure by microscopy, was
killed by freeze-drying.
The average elemental composition of alga was 51.9% C,
7.5% I-1, and 9.2% N on an ash-free basis,
and its ash content was 5.1% of the dry
weight ( Otsuki and Hanya 1972).
Anaerobic decomposition
of the algal
cells was begun at pH 7 in the apparatus
shown in Fig. 1. The medium consisted of
100 ml of l/30 M KHzPOd, 200 ml of l/30
M Na&lP04,
and 2,700 ml of distilled water.
Each 3,000 ml of the medium was incubated
258
MARCH
1972,
V. 17( 2)
ANAEROBIC
PRODUCTION
OF
DOM
D
H2
FIG.
water
concH2SOq Hz0
1. Schematic
tank controlled
diagram of the decomposition
by thermostat;
C-gas
burette;
at 20 * 0.X’ and 30 4 1C in two amber
bottles.
Microflora
were inoculated as follows :
100 ml of distilled water was added to a
conical beaker containing about 50 g of wet
mud from the dark, reductive portion of
mud collected from near the center of Lake
Haruna (Otsuki and Hanya 1967). After
bubbling the suspension with II2 (about
20 ml/min) for 48 hr at room tcmperaturc,
10 ml of supernatant solution was added to
the two bottles of media, from which dissolved oxygen was removed by purging
with I-12. After 48 hr, 15 g dry wt of the
algal cells were added, the I-I2 gas flow
stopped, and a gas burcttc set. Each sample
( about 150 ml) was thoroughly stirred bcfore sampling, the gas burette (C) taken
off from gas inlet ( D ), and the medium
bubbled with I-12 until sampling was over.
Particulate matter in the sample water was
collected by centrifugation at 9,006 rpm for
10 min at 5C. The supernatant solution was
adjusted to about pH 8 with 1 N NaOII
solution to prcvcnt the escape of volatile
organic acids and then passed through
Milliporc HA filters.
The DOC was determined by a modification of the method of Katz ct al. (1954) and
B
A-Amber
apparatus.
d-gas inlet.
5N KOH
decomposition
bottle;
B-
Tczuka (1964) using K&O8 as oxidant in
a Thunberg tube and followed by titration
with 0.05 N I-ICI after oxidation in a Thunberg tube. The precision was estimated to
be 10% for up to 10 mg C/liter in a lo-ml
sample volume. Dissolved organic nitrogen
(DON) was dctcrmined by the Kjeldahl
method, using HgSO4 as a catalyst (Amer.
Public Health Ass. 1960). The precision was
within 5% in the range up to 10 mg N/liter
in a 25-ml sample volume. The amount of
algal material mineralized during the incubation time was cstimatcd as the difference
bctwecn total organics at the start and the
sum of particulate organics and dissolved
organics at the sampling time. Carbohydrate in the dissolved organic product
was dctcrmined by a modification
of the
anthronc method of Morris ( 1948) and the
carbohydrate-c
was calculated using glucose as a standard. Particulate organic
matter was dried in a vacuum desiccator
over P20s. Organic carbon and nitrogen
were dctermincd by an clcmcntal analyzer
and by the micro-Kjcldahl
method. The
amount of bacterial organic matter in the
particulate
organic matter was not dctermincd, but most of it was algal cell
debris containing chlorophyll
degradation
260
AKIRA
OTSUKI
AND
products. All analysts wcrc done in duplicatc. Dctcction of lower fatty acids was
made by one-dimensional paper chromatography ( Block ct al. 1958). Organic acids
wcrc cxtractcd as follows. After acidifying
the sample water with 6 N IICl, the organic
acids wcrc extracted in ether in a scparatory
funnel three times and the pooled ether cxtracts washed twice with distilled water.
The washed extract was extracted with 20
ml of 0.1 N NI14011 solution and the ammonia solution containing organic acids concentratcd to 1 ml in a vacuum desiccator.
Ammonium
salts of organic acids were
spotted on chromatograph paper with a
Amino acids were dctcctcd
microsyringc.
by two-dimensional paper chromatography
after acid hydrolysis. The solvent systems
used were phenol: water (75:25 w/w)
as
the first solvent and n-butanol:acetic
acid:
water (4: 1: 1 v/v/v).
The infrared spcctrum was obtained on a spectrophotometer
by the KBr disk method.
Aerobic cleconaposition experiments
with the clissolvecl organic product
The media containing the DOM produced under anaerobic conditions were
diluted to about one-fourth and one-sixth
with water for the measurcmcnt of biological oxygen demand (Amer. Public Health
Ass. 1960) to give mineral nutrients .to
aerobic bacteria. Each 500-ml sample of
water was kept in an amber bottle at 20C
ancl acrated constantly with clean air. Surface water (50 ml) from Lake IIaruna was
used as an inoculum.
Sample water was
siphoned off at different intervals and DOC
TAKAIIISA
HANYA
..L---
-
I
Time (days)
IrIG.
content
tion,
3.
Changes in pII with time.
determined
after Millipore
filtra-
RESULTS
The concentration
of DOC incrcascd
rapidly during the first 30 days, lcvelcd
off, and then remained nearly constant bctwecn the 60th and 150th day ( Fig. 2).
After the 150th day it dccrcased slightly.
A higher tempcraturc incrcascd the initial
rate of decomposition, but after the 40th
day thcrc was no apprcciablc differcncc bctwecn 20 and 30C. The medium at 2OC
fell below pH 6 after the 15th day in spite
of buffering
by phosphate compounds
( Fig. 3). Paper chromatography showed a
prcdominancc of acetic acid and the prcsencc of propionic and formic acids; there
was also an unknown spot not transported
by the solvent system used. After the production of DOC, thcrc was no marked dccrease in its observed concentration.
This
indicates that mcthanc fcrmcntation did not
occur in spite of the presence of the lower
fatty acids. No gas evolution was actually
obscrvcd in the gas burcttc.
The concentration
of DON fluctuated
irregularly during the first 20 days and then
0
50
z
m
r’
\
,I0
Time
20%
/o--o-o-o
\o
% \
x/x-+--x-x
--
-
O-0
X-
30%
-L ~~
SO
1
100
Time
(days)
FIG. 2. Changes in concentration
organic carbon (mg/liter).
0
of dissolved
( days
I
150
I
200
1
FIG.
4. Changes in concentration
organic nitrogen (mg/liter).
of dissolved
ANAEROBIC
TABLE
1. The proportion
Carb-C/DOC
Time ( clays)
5
10
15
20
30
40
60
100
150
200
* Mainly
PRODUCTION
of carbohydrate-C
X 100
OF
and protein-c
Protein-C/DOC
in dissolved
3oc
2oc
3oc
15.5
9.1
5.9
4.8
4.9
4.8
4.1
4.1
3.7
90.5
31.1
14.6
16.9
17.1
14.7
13.6
12.5
11.5
11.4
26.5
18.0
16.0
7.4
ii
G:4
i::
organic
X 100
2oc
F78
415
4.5
3.5
4.1
261
DOM
product
Others *
;:2”
?I
s:o
10.1
2oc
3oc
59,s
78.6
77.4
78.4
80.8
82.9
83.4
84.7
83.5
67.6
77.2
79.1
87.8
87.3
88.7
89.6
88.2
87.0
83.5
organic acids.
rcmaincd nearly constant both at 20 and
at 30C ( Fig. 4). The amount produced at
20C was higher than that at 30C. Table 1
shows the proportion
of carbohydrate-C
and protein-C in the total DOC produced.
Protein-C was calculated by assuming a
conversion factor of
nitrogen-to-protein
6.25 and an avcragc carbon content of protcin of 53%. The proportion of carbohydratc-C was about 4-7% throughout
the
period of decomposition cxccpt for the first
10 days at 20C; the proportion of protein-C
was 7-16% at 30C and ll-17% between
the 15th and 200th day.
At 2OC, by the 60th day, about 30% of
the algal cell carbon was transformed into
DlOC and 20% was mincralizcd;
50% remaincd in particulate form ( Fig. 5 ) , About
8% of the algal cell nitrogen was trans-
formed into DON, 48% was mincralizcd to
NIIR, and 44% rcmaincd as particulate organic matter (Fig. 6). After the first 60
days, anaerobic decomposition
continued
very slowly. Similar patterns were obtained
at 30C with slightly higher rates of decomposition in the first stages.
Upon aerobic decomposition of the dissolved organic products, the concentration
of DOC rapidly decrcascd during the first
3 days and then remained nearly constant
(Fig. 7)) indicating that about 20% of the
anaerobic products arc not easily mincralizcd by bacteria. After the 7th day, organic
aggrcgatcs were formed which settled to
the bottom after the 15th day.
KINETICS
OF
ANAEROBIC
DECOMPOSITION
Figure 8 shows decomposition as a function of time, where [C] 0 is the initial conz
” 100
H
xx’c--x-x-x
30 x
g
0’
0”
Time
X
X
0
t 0;
.& 0
50
100
Time
(days
150
200
1
0111
0
50
100
150
200
(days)
l?
5. Proportion
of change in algal cell carbon at 20C. D-Dissolved
organic carbon; Mmineralized
carbon; R-residual
carbon as particulate matter.
FIG.
FIG. 6. Proportion
of change in algal cell
nitrogen at 20C. D-Dissolved
organic nitrogen;
M -mineralized
nitrogen; R-residual
nitrogen as
particulate matter,
AKIRA
262
OTSUKI
AND -.IAKAHlbA
-* I ---^ . HAN
--. _--1.
YA
2OOr
0.4
X
t
0.3
g
CI
0-2
loo0
i
0-I
q)(
‘X-XIX
0
o-o.o/--‘o
I
IO
I
I
I
I
1
20
30
40
50
60
Time
ccntration of alga1 cell carbon or nitrogen,
and X is the amount of organic carbon or
nitrogen which was decomposed, [C ] o - X
is actually measured as particulate organic
matter. These plots give lines that broke
at 10 days at 30C and at 35 days at 20C.
They do not intercept the origin, indicating that decomposition of the algal cells
began before they were added to the
medium. It is unlikely, however, that more
than 10% of the dry algal cells were decomposed during weighing. Since the algae
were killed by freczc-drying, some organic
components of the cells were made water
soluble and dissolved when the cells were
added to the medium (cf. Otsuki and Hanya
1972). This dissolution of ccl1 material
must not be regarded as microbial dccomposition.
Table 2 gives the overall rate constants
2. Rate constants (per day -F- standard
deviation, p = 0.05) of anaerobic decomposition.*
Period of decomposition:
OGk5 days at 20C; lo-40
days at 30C
TABLE
‘Carbon
Nitrogen
0’
0
2oc
3oc
0.0088 + 0.0067
0.0156 + 0.0015
0.0051 5 0.0041
0.0099 ?I 0.0040
* The rate constants obtained
from
determined by linear regression analysis.
the
slopes
were
20%
/O
/..P”
I
IO
I
20
I
I
30
40
Time (days)
I
50
I
60
(A)
( days)
FIG. 7. Aerobic decomposition
of the dissolved
organic product
(in mg/liter)
at 20C. X-Dissolved organic product at the 150th day incubated
at 20C; o-dissolved
organic product at the 200th
day incubated at 20C.
x--;-0-
2
/
,J
$fo
t
\
0’
0
I
30%
X-X
r
04
03
0.2
01
01
0
I
IO
I
20
I
I
30
40
Time (days)
I
I
50
60
(B)
as a function
of time.
FIG. 8. Decomposition
( A)-Algal
cell carbon; ( B)-nitrogen.
concentrations
of algal cell car[Cl 0-Initial
bon or nitrogen; X-amounts
of algal cell carbon
or nitrogen decomposed at a given time.
of decomposition obtained from the slopes
of Fig. 8. The rate of decomposition of
algal cell nitrogen is faster than that of
algal cell carbon in the initial stages, and
the rates depend on temperature. These rcsuits are in agreement with those observed
in the field by Koyama and Tomino ( 1967)
and arc similar to effects observed under
aerobic conditions
(Otsuki
and Hanya
1972 ) .
DISCUSSION
Otsuki and Hanya (1972) found that the
decomposition of algae under aerobic conditions resulted in the production of DOC
from about 7% of the aIga1 cell carbon at
the 30th day at 20C. The dissolved organic
ANAEROl3IC
PRODUCTION
product consisted mainly of a yellow unknown acidic material and of a proteinaccous substance.
The results of anaerobic decomposition
experiments indicated that about 60% of the
algal cell carbon decomposed was transformed into DOC, and 80% of this was
readily
mineralized
by bacteria under
aerobic conditions, The dissolved organic
products of anaerobic decomposition
of
algal cells consist mainly of organic acids
( Table 1). The yellow substances were not
extractable by ether from acidified medium
(below pH 3) but were extracted by
butanol. During extraction, insoluble organic matter concentrated at the interface
bctwecn solvents. After centrifugation and
vacuum-drying
it gave a white amorphous
solid, which, like that from the aerobic expcriments, was composed of amino acids
( glutamic
acid, asparatic acid, alaninc,
leucine plus iso-leucine, mcthioninc, phenylalaninc, valine, arginine, glycine, lysine,
serine, threonine ) and 12 unidentified
ninhydrin-positive
spots. This proteinaceous
material was detected in the medium after
incubation for 30,150, and 200 days.
About 20% of the DOC at the 150th and
the 200th day was not easily mineralized by
bacteria (Fig. 6). This amount is roughly
equivalent to the total amount of DOC
produced by aerobic decomposition of the
same green algal cells. The amount of DON
is also in agreement with that at the 30th
day in aerobic decomposition. These facts
suggest that regardless of aerobic or anaerobic conditions, microbial decomposition
of dead algal cells is accompanied by the
production of the refractory DOM, consisting mainly of acidic substances arising
from yellow and from protcinaccous material.
Two major differences appeared in the
patterns of anaerobic decomposition as compared to aerobic decomposition:
The production rate of DO’M increased more than
four times and the rate constant of decomposition of dead algal cell carbon and nitrogen dccrcascd to less than half.
Some anoxic marine environments show
no cvidcnce of large increases in DOC with
depth ( Richards 1965). Concentrations of
FIG.
ticulate
OF
263
DOM
9. Changes in C:N
matter with time.
atomic
ratio
of par-
DOC twice those in the epilimnion were
found in the anaerobic layer about 5 cm
from the bottom of a mesotrophic lake in
Japan (Mizutani,
Otsuki and Hanya, unpublished).
When the method of Menzel
and Vaccaro ( 1964) is applied to the determination of DOC in anaerobic waters,
there may be a loss of such volatile organic
acids as formic, acetic, and propionic, produccd by fermentation, during the bubbling
with Nz under acidic conditions to remove
inorganic carbon. Adams and Richards
( 1968) found that the amounts of DOM
extracted by petroleum ether and ethyl acctate were larger in anaerobic layers than in
oxygen-bearing layers in an anoxic fjord.
Atkinson and Richards ( 1967) suggested
that methane in the marine environment is
produced by anaerobic ferrncntation
of
fatty acids of low molecular weight. Our
results indicate that lower fatty acids, which
are converted into methane, can be supplied
by organic acid fermentation of algal cell
materials in natural environments.
A considerable part of the algal cells rcmaincd as particulate matter. Its C:N ratio
showed irregular changes in the first stage
of decomposition, but after the 50th day
remained nearly constant ( Fig. 9). The
infrared spectra of the particulate matter
wcrc almost identical to those of the original green algae throughout the period of
decomposition
under both aerobic and
anaerobic conditions. The amount of particulatc matter at the end of the experiments
was about 2030% of the algal cell carbon
and nitrogen added under both aerobic and
anaerobic conditions, an amount in good
agreement with those in other experimental
264
AKIRA
OTSUKI
AND
studies (Grill and Richards 1964; Skopintzcv
et al. 1965) carried out at much lower conccntrations of organic matter than ours.
This indicates that our experimental conditions were good for bacterial growth, but
the possibility remains that they arc responsible for some of the differcnccs obscrvcd.
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