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OCT 30 1904
ENVIRONMENTAL INFLUENCES ON MICROBIAL POPULATIONS
AND ACTIVITY OF THE FOREST FLOOR*
by
Martin Witkamp
Radiation Ecology Section, Health Physics Division
Oak Ridge National Laboratory, Oak Ridge, Tennessee
Microbial populations are main agents in the breakdown of leaf
litter and subsequent mineralization and humus formation. Populations
.
."
-
and their activity have been reported for many different environments.
This paper will establish quantitative relationships of microflora and
microbial activity with each other, with environmental factors, and with
rates of litter breakdown based on some of my data from European and
American forests.
DIT
The principal method used to enumerate microbial populations was
the widely-used dilution plate method, so that the results can be com-
pared with those from similar studies. This method, which only reflects
the fast growing zymogenous section of the microflora, was augmented by
-
*
*
*
direct counts of fungal myceliuza on soil suspension slides.
These
.
RAZ
*
.
7
ty
a
counts are obtained by counting the crossings of mycelium with five
parallel lines in each microscopic field (Witkamp 1960).
Microbial activity of litter and soil was measured by rates of
00, evolution in closed boxes or in boxes without bottom on the forest
floor. The amount of 0.1 N KOH neutralized by absorption of co, in
boxes and bells of 1.75 dm each was measured by titration with 0.179 N HCI
2
SA
*Research sponsored by the U. S. Atomic Energy Commission under contract
with the Union Carbide Corporation.
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after addition of Baci,. These methods measured total soil respiration
.
by microflora, fauna, and roots combined. Rate of mycelial growth on
glass slides incubated in litter or soil gave a separate index for fungal
activity. The combined effect of microflora, fauna, and nonbiological
factors such as leaching and gravity on disappearance of litter was
measured as loss of weight by 10 to 30 g of leaves in 1 x 1 dm fiberglass
mesh litterbags exposed in the forest floor (Witkamp 1963, Witkamp and
Olson 1963).
Environmental effects on microbial populations
The main environmental factors influencing microbial populations
are the type of microbial substrate, moisture, temperature, exposure,
altitude, type of forest stand and vegetation, and the activity of the
litter fauna. The species of leaf was the most influential of the sub-
strate factors (Table 1). Mean counts differed significantly among all
species (P < 0.01) with the exception of fungal counts for redbud and
de
.
white oak. Relative differences among mean bacterial counts d various
leaf species were usually larger than among corresponding fungal counts.
Individual bacterial counts per gram of air dry litter varied from
8 x 10° on mulberry leaves to 4 x 103 on pine needles. Corresponding
fungal counts varied ten times less viz. from 24 x 20° to 26 x 103.
Populations tended to be positively correlated with annual average
moisture content of the leaves which, in a given climate, is a substrate
churacteristic influenced by porosity, cuticle, time of leaf drop, and
rate of decay. Population densities and c/N ratios of the leaf species
were usually negatively correlated. The low c/N ratios, stimulating
microbial growth, may result from a low content of microbially resistant
2
1.
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11
miny
25
29
.
- 3 -
compounds such as cellulose or lignin (Witkamp 1960), from a high per-
centage of usually readily decomposatle nitrogenous compounds, or from
Hote. The greater ability of bacteria to colonize nitrogenous substrates
and of fungi to decompose cellulose and lignin when available nitrogen .
18 low cause the bacteria / fungi ratios to be high on substrates with
low C/N ratios and vice versa (Table 1). Mean C/N ratios were 27.8,
31.9, and 33.2 for all fresh litter in comparable stands with calcareous
mll, acid mill, and mor forest floors respectively. Corresponding
bacteria /fungi ratios were 32, 8, and 4 (Witkamp 1960).
On the easily decomposable mulberry and redbud leaves the initially high
populations (Table 2) declined with time whereas on the more resistant
cak and pine litter the initially low population densities increased as
decomposition proceeded. Thus as the leaves decomposed and substrate com-
position and environmental conditions became more similar and stable, popu-
lation differences anong leaf species decreased. Simltaneous
fluctuations
of both fungal and bacterial populations also decreased under the influence
of moderated temperature and moisture conditions (Table 3).
Moisture contents of the leaves were not significantly correlated
with microbial populations of leaf litter, even though influence of moisture
on bacteria was greater than on fungi, which usually perform better at low
moisture levels than do bacteria (Table 4). Presumably, in the perhumid
climate of East Tennessee and in dead leaf tissue with high water holding
capacity, moisture was usually adequate to perpetuate the microflora of
litter. Marked seasonal moisture effects occurred in both bacterial and
fungal populations of surface mll and mor of oak stands in the Netherlands.
When moisture levels dropped below the wilting point microbial populations .
were greatly reduced. Similarly there was a highly significant correla-
tion (P < 0.01) of moisture with both mycelium density and fungal plate
counts in profiles of a Bandy soils series with limited water holding
capacity (18 - 32%).
. Temperature effects on microbial populations of litter with adequate
moisture and varying temperature were more pronounced than in the deeper
soil with lower moisture holding capacity and moderated temperature
fluctuations. Temperature was significantly (P < 0.05) and positively
correlated with fungal counts from litter (Table 4). This effect was
smaller on counts of the generally moisture responsive bacteria than on
the in general less moisture requiring fungi, presumably as a result of
the usually negative relationship between moisture and temperature in
the litter layer. High temperature causes rapid evaporation and thus
low moisture contents in the surface litter..
REMAR..
The influence of the type of forest stand on microbial population
was small as compared to the influence of leaf species. Bacterial counts
of various leaf species were significantly lower (P < 0.01) in coniferous
stands than in deciduous stands (Table 5), whereas fungal counts did not
differ significantly. Factors lowering the competative ability of bacteria
were presumably soluble agents such as bacterial inhibitors, or organic
acids which lowered the ph (Witkamp 1963).
Whether a slope was exposed to the north or to the south had no
significant effect on microbial populations by itself, but slope exposure
was significant (P < 0.01) when interacting with stand effects. Bacterial
counts from north slopes were nine times higher in hardwood than in coni-
ferous stands, whereas counts from south slopes did not differ. Pre-
sumably litter on south slopes of deciduous forests dried more frequently
than that on the north slopes. In the cooler con iferous forest litter
remained moist on both slope 8. Average moisture contents of leaves from
south slopes were 49 and 61% respectively. Those from litter from rorth
slopes were 60.5% in both types of stand. Consequently on north Blopes
Btand influence prevailed. This interaction between stand and exposure
was not significant for fungal counts, probably because moisture is not
as much a limiting factor for fungi as it 18 for bacteria (Witkamp 1963).
Both bacterial and fungal populations of leaf litter decreased
with increasing elevation. This increase was more significant (p < 0.05)
between 1040 and 2600 m elevation than between 260 and 2040 m (P >0.05).
Presumably the difference between counts at 1040 and 1600 m was mainly a
temperature effect, moisture conditions being adequate at both levels.
Between 260 and 1040 the temperature effect was counteracted by periodic
drying at the lower elevations with more evaporation and less precipi.
tation.
Effects of the soil on microbial populations are mainly results
of differences in humus content, moisture regimes, and pl. Within a
sandy soil series 18 annual averages of mycelium density showed high
positive simple correlations with humus (r = 0.90) and moisture (r = 0.92)
contents (Witkamp 1960). Mycelium length/fungal counts ratios were up
to 15 times higher under oak than under pine. High correlation of annual
means of fungal populations with bumus and moisture contents reflect
indispensibility of moisture and humus for fungal development as well as
a high positive correlation between humus and moisture content of sandy
series
Boils (r = 0.91, 0 = 18). In the same soils/both mycelium density
and colony plate counts decreased highly significantly(P < 0.01) with in-
creasing depth.
.
6
.
Effects of soil pH not only favored the competative ability of
bacteria and actinomycetes over that of fungi but also influenced
vegetation and soil fauna. The ground cover of a caloareous soil had
a higher litter production (75 8 overdry/m?) with low c/N ratio (27.8).
than vegetation on a comparable actú 2011 (12 is overdry/m², c/N - 34.1)
thus favoring bacterial breakdown in calcareous s011 (Witkamp 1960).
Saprophageous litter fauna and its consumption on calcareous s011
were hiyiler than on similar acid 8011 (van der Drift 1962). Etter
consumption greatly stimulated bacterial populations by raising på, break-
ing the cuticle, exposing cell contents and improving moisture conditions
(van der Drift and Witkamp 1960). Mixing of 11tter and feces with deeper
layers will also reduce evaporation and thus drying of the microbial
substrate.
.
Environmental effects of microbial activity
Microbial activity measured as rate of microbial respiration or
1088 of weight by the substrate is the resultant of the activity by all
individual microbes. Consequently in a given climate loss of weight
by litter 18 highly correlated with mean microbial density (Fig 1).
(Bacteria + 153 x Fungi) is an estimate for combined microbial densities
equating the breakdown potentials of all fungal counts with the 153 times
higher sum of all bacterial counts collected under the wide variety of
environmental conditions of the experiment (Witkamp 1964). Bacterial
counts taken throughout the year reflected microbial activity measured
28 respiration rate better than did fungal counts (Table 4). Presumably
bacterial counts are better indicators of microbial activity than fungi
because for bacteria metabolic activity is almost proportional to cell
division whereas for fungi formation of spores and conidia, which are the
main snurces of fungal colonies, often signify the decline of fungal growth.
-
.
.
.
- 7 .
Annual means of fungal counts are a better criterium for microbial activity
(Fig. 1) than the individual counts taken throughout the year (Table 4)
presumably because the annual means reflect a fungal population potential
without time dependency.
" The low correlation between respiration rates and mycelium growth
18 presumably the result of atypical moisture conditions on the s11des
caused by condensate and possibly reflected a lack of correlation
between litter moisture and mycelium growth (Table 4). Taus microbial
activity on a given substrate is well. correlated with bacterial density,
and over long periods also with fungal density.
I already discussed effects of environmental factors on microbial
density. Tous lt remains for me to discuss the effects of environmental
factors on activity of the individual microbes.
In different climates and seasons ir. the forest belt, temperature
18 the main controlling factor for microbial, activity. Presumably in
forest areas moisture is not limiting over long periods of time. Mois ture
effects however are superimposed on the annual activity cycles which pri-
marily parallels the temperature cycle. (Witkamp and van der Drift 1961).
A multiple correlation analysis for respiration rates with environmental
factors and microbial populations on a variety of stands and substrates
in East Tennessee showed that temperature, leaf moisture, bacterial den-
sity and age of the 11tter contributed 64%, 19%, 12% and 5% to the multiple
correlation respectively. Contributions by fungal densities and mycelium
growth were negligable. The regression for these data were R = 46.50 +
3.22 T + 26.86 MD + 11.39 log B - 0.64 W.
Where R = respiration in wé co, hr? 801
T = temperature in c
.8.
M/D = moisture content of the litter a6 moist weight/dry weight
B = number of bacterial colonies from 100 g of air dry litter
W = number of weeks since last leaf fall
Respiration rates based on this regression closely follow respiration
rates measured in the field throughout the year (Fig 2). Respiration
rates were also highly correlated with 1.088 of weight by litter (r = 0.91,
n = 12). Average co, production was 170 ml for each gram of litter decom-
posed but this figure was higher (250 ml) for mulberry and redbud leaves
with high populations of bacteria than for pine and beech leaves (130 ml)
with high fungal populations. This is in accord with the more efficient
use of substrate by fungi than by bacteria. ;
In summary it appears that in a given climate substrate 18 the
main controlling factor for microbial population; and that bacterial counts
are the best index for mean microbial activity. Microbial activity at any
given time can be successfully predicted on the basis of bacterial density,
temperature, moisture, and litter age.
In different climates of the forest zone temperature is the main
controlling factor for microbial populations as well as for their activity.
Moisture efects are superimposed on the temperature effects connected with
altitude, slope exposure, and season.
.-
irti
4
References
van der Drift, J. 1963. The disappearance of litter in mull and mor
in connection with weather conditions and the activity of the
macro-fauna. p. 125-133. In Doeksen and van der Drift Soil Organisms.
van der Drift, J. and M. Witkamp. 1960. The significance of the break-
dow of oak litter by Enoicyla pusilla Burm., Arch. Neer.l. de 2001.
13:486-492.
Witkamp, M. 1960. Seasonal fluctuations of the fungusflora in mull and
mor of an oak forest. Publ. Inst. Biol. Field Res., Arnhen, Netherl.
46:1-52.
Witkamp, M. 1963. Microbial populations of leaf litter in relation to
environmental conditions and decomposition, Ecology 44:370-377.
Witkamp, M. 1964. Decomposition of leaf litter in relation to environ-
mental conditions, microflora, and microbial respiration. In press.
Witkamp, M. and J. S. Olson. 1963. Breakdown of confined and non-con-
fined oak litter, Oikos 14:138-147.
Witkamp, M. and J. van der Drift. 1961. Breakdown of forest litter in
L
relation to environmental factors, Plant and Soil 15:295-311.
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Figure 1 -
Tennessee, USA.
first year of decay in three forest stands in East
with microbial estimates and respiration during the
Relationships of loss of weight by four leaf species
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UNCLASSIFIED
ORNL-DWG 64-2279
1400

CORRELATION COEFFICIENTS (r)
(BACTERIA+153xFUNGI) p = 0.97
WEIGHT BACTERIAL COUNTS r=0.92
LOSS FUNGAL COUNTS r = 0.84
(TOTAL CO2 EVOLVED r=0.91
n=12 IIIIII
MICROFLORA ( BACTERIA +153 x FUNGI) Mg4
A MULBERRY +
A REDBUD
• OAK
o PINE
HW=39.38+0.469(B+153F )
0
10
20
30 40 50 60 70
LOSS OF WEIGHT ( % yr)
80
90
100
AL
./4"
x
Figure 2 -
Mean measured ( ) and calculated (----) biweekly respiration
rates of four leaf species during the first year of decay in
three forest stands in East Tennessee, USA.
1
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UNCLASSIFIED
ORNL-DWG 64-2278
RESPIRATION (HCO2 g- hr-1)

21
N D
1960
J
1961
F
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Air
.
Table 1. Means of microflora and litter moisture, initial c/n zatio, and
loss of weight by litter of four leaf species in oak, pine, and
maple stands in East Tennessee, USA during the first year of decay
bacterial counts
fungal counts
(105/8 dry)
leaf moisture
(moist wt/dry wt)
leaf species
c/N
% 1088 of weight/yr
mulberry
2.7
redbud
286
white oak
pine
·
15
1.7
:.....
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inisiniai....
: Table 2 - Regressions for logarithms of bacterial and fungal populations of
mulberry, redbud, white oak and pine litter decomposing in oak,
pine, and maple stands in East Tennessee, USA.
-
Leaf Species
-
Mulberry
--
Redbud
66
Oak
Period in
number of dup-
byou licate counts
Bacteria
De
Fungi
Jan-Aug
-0.078
0.082
Jan-Nov
-0.012
+0.0067
Jan-Nov
66 +0.0337
10.009-
Jan-Nov
+0.0312
+0.032
no significant difference (P >0.1) according to t-test,
all other differences significant (p < 0.02)
Pine
-
-
-
--
-
-
--
Table 3. Mean coefficients of variability (V = 100 8/ X) for bacterial
and fungal counts, mycelium growth, and leaf moisture in three
successive periods during the first year of decay of wlberry,
redbud, white oeſ, and pine litter in oak, pine and maple stands
in rast Tannesson, USA
January 9 March 6 .
August; 14.
February 20 July 31 Novemier 6
27
36
20
· Bacterial counts (10%)
Fungal counts (10/8)
Mycelium growth (cm/cm2 veck)
Leaf moisture (8 moist/& dry)
----
-
--- -......
.....
..
Table 4.
Simple correlation coefficients for microbial estimates with moisture,
temperature, and respiration from redbud, white oak and pine litter de-
caying in oak, pine, and maple stands over the period January 1960 -
October 1961 in East Tennessee, USA.
Litter moisture
(wet weight/dry weight)
0.12
Litter
temperature Respiration
(c) (u1/8.hr)
0.35**
Bacterial counts (20g 10°/8 dry)
Fungal counts (10g 10%/8 dry)
Mycelium growth (mm/cm2. week)
0.12
0.16*
0.04
.
0.12
-0.09
0.35**
0.05
8 • 198
*P:<0.05
**p <0.01
Table 5. Mean microbial counts from white oak and beech leaves collected
from different stands and elevations during the four seasons in
East Tennessee, USA.
number of dupe
licate counts
bacterial count
(10678 dry)
fungal counts
(10°/8 dry)
Stand
bardwood
884
coniferous
215
elevation
1600 m
226
32
1040 m
260 m
931
DATE FILMED
12/21/64








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