THE EFFECT OF KRAFT PULP MILL EFFLUENTS ON THE FILAMENTOUS MARINE FUNGI WITH PARTICULAR REFERENCE TO ZALERION MARITIMUM (LINDER) ANASTASIOU Leslie Marian churchland B. A., University of British columbia, 1966 A THESIS SUBMITTED I N PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in the Department of Biological Sciences 0 Leslie Marian Churchland 1971 Simon Fraser university June, 1971
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THE EFFECT O F KRAFT PULP M I L L EFFLUENTS ON THE
FILAMENTOUS MARINE FUNGI WITH PARTICULAR REFERENCE
TO ZALERION MARITIMUM (LINDER) ANASTASIOU
L e s l i e M a r i a n churchland
B . A . , U n i v e r s i t y of B r i t i s h c o l u m b i a , 1966
A T H E S I S SUBMITTED I N PARTIAL FULFILLMENT O F THE
REQUIREMENTS FOR THE DEGREE O F
MASTER O F S C I E N C E
i n the D e p a r t m e n t
of
B i o l o g i c a l Sciences
0 L e s l i e M a r i a n C h u r c h l a n d 1 9 7 1
S i m o n Fraser u n i v e r s i t y
June, 1971
Title of Thesis: The effect of K r a f t pulp TI! 3 i F f f l u e n t s on the f llamentous marine C l l i l c ~ ~ 1 11 particular reference to zalerlon -- marl t L T ~ -- - m [ Lliider) Anastasiou
Examining Committee :
Chairman: Dr. B.P. Beirne h
Supervisor
L.J. Albri ht P
- L.D. Druehl
YW .Ffis't'i n External Examiner
Assistant Professor Simon Fraser University, British Columbia
Date Approved: fL.'*lr / 2, /97 /
The Effect of Kraft Pulp Mill Effluents on the
Filamentous Marine Fungi with Particular Reference to
.Zalerion maritimum (Linder) Anastasiou
ABSTRACT
Field studies near a Kraft pulp mill and at con-
trol stations in Howe Sound, ~ritish Columbia showed that
Kraft pulp mill effluents affected the species composition
of marine fungi. Phycomycetes were isolated as frequently
at the pulp mill as at the control stations, but Ascomy-
cetes were isolated less frequently at the pulp mill.
Certain members of the ~ungi ~mperfecti such as Mono-
dictys pelaqica (Johnson) Jones and Phialophora melinii
(~annfeldt) Conant were isolated more frequently at the
pulp mill while others such as Zalerion maritimum (Linder)
Anastasiou, were isolated less frequently.
Oceanographic measurements were carried out in an
attempt to explain the distributional effects. The pH,
salinity, and temperature values differed little when
12 m depths at the pulp mill and a control station were
compared. Dissolved oxygen at the 12 m pulp mill sta-
tion was lower than that at the control station. At a
depth of 30 cm dissolved oxygen, salinity, and pH values
iii
were both lower and more variable at the pulp mill than
at the control station. Temperature readings at a depth
of 30 cm were similar at the pulp mill and control station.
Dry weight and oxygen uptake studies showed that
Z . maritimum was tolerant to a wide range of salinity and -
pH conditions. Dry weight was higher in caustic Kraft
pulp mill effluent plus nutrients than in seawater plus
nutrients. Dry weight was lower in acidic bleach plant
effluent and alkalized bleach plant effluent plus nutri-
ents than in seawater plus nutrients.
Oxygen uptake was higher in caustic effluent plus
nutrients than in seawater plus nutrients. Oxygen uptake
was lower in acidic bleach plant effluent with and without
nutrients than in seawater with and without nutrients. Al-
kalized bleach plant effluent did not significantly affect
oxygen uptake values.
In Phialophora melinii caustic effluent and acidic
effluent without nutrients did not significantly affect
oxygen uptake. Oxygen uptake was lower in acidic bleach
plant effluent with nutrients than in seawater with nutri-
Fig. 22 The effect of Kraft pulp mill acidic bleach plant
effluent plus basal nutrients on oxygen uptake in
Phialophora melinii. A-A= seawater plus nutri-
ents, e--...e = 50% effluent plus nutrients, m--- = 10O0A
effluent plus nutrients. Vertical lines show the
means 3 one standard error. Means followed by the
same letter do not, at 120 minutes, differ signi-
ficantly at the 95% probability level.
timum, both 50 and 100% acidic bleach plant effluent plus
nutrients significantly reduced respiration rates when
cbmpared with seawater plus nutrients.
To summarize, - 2. maritimum was tolerant to a wide
range of salinity and pH conditions, as shown in studies
of oxygen uptake and growth measured as dry weight. Caustic
effluent increased oxygen uptake and dry weight relative to
those observed in seawater, and acidic bleach plant effluent
decreased dry weight and oxygen uptake relative to seawater.
When the bleach plant effluent was alkalized, a toxic effect
was demonstrated in dry weight studies but not in oxygen up-
take experiments. In g. melinii, neither caustic effluent
with or without nutrients nor acidic bleach plant effluent
without nutrients significantly affected oxygen uptake.
Oxygen uptake in 100% acidic bleach plant effluent with
added nutrients was approximately half that in seawater
and 50% solutions.
cent to a pulp mill and in "control" areas to determine the
effects, if any, of Kraft pulp mill effluents on the species
I composition of filamentous marine fungi. The principal "con-
I
troll8 area chosen, Horseshoe Bay, was a site distant from both
Howe Sound pulp mills. Panels were frequently lost from other
areas which were tested as control sites. The Keats Island and
Gambier Island stations were both considerably closer to the
pulp mill. However, oceanographic and species composition data
from these sites and Horseshoe Bay were very similar.
The field survey indicated some differences in species
composition between the pulp mill and control stations. There
were a small number of species in common at the pulp mill sta-
tion and control stations, a fact supported by Sorensents Sim-
ilarity Index. Comparison of the species composition at the
three control stations indicated a high degree of similarity.
However, the small number of isolations of certain species
limits the statistical validity of Sorensents Similarity Index.
All groups of fungi. were poorly represented on the
bottom panels at Port Mellon, This is difficult to explain
because the pH, salinity, and temperature at this depth
-75-
differed little with that at the same depth at Horseshoe
Bay. There was some variation in dissolved oxygen levels
at the Port Mellon bottom station; however, marine fungi
are tolerant of low oxygen pressures (52). Moreover, the
concentration of effluent reaching this depth is low (37).
The best explanation for the paucity of marine fungi at this
depth is that the panels which rest on the bottom are buried
in the fibre bed and fungal spores are physically prevented
from landing and germinating on the wood substrate. When
the fibres themselves were examined, the only fungal struc-
tures observed were biflagellate zoospores.
Phycomycetes, Ascomycetes, and ~ungi Imperfecti were
all isolated from the pulp mill surface station and control
areas during the course of the study. The Phycomycetes,
which were isolated from leaves only, occurred at the pulp
mill as frequently as at Horseshoe Bay. This may reflect
the fact that the marine Phycomycetes can usually grow
under low salinity conditions (2, 52). The data would also
suggest that these fungi may tolerate high concentrations of
acidic and caustic effluent as well as low pH conditions.
Labyrinthula - was found on leaves from both levels
at Port Mellon and Horseshoe Bay. This is not unexpected,
as Labyrinthula has been shown to be tolerant to a wide range
of salinity, temperature, and pH conditions (52).
The results from the species composition data indi-
cate an almost complete absence of marine Ascomycetes at the
pulp mill station. Other authors (5, 38, 48) have studied
the two Ascomycetes most commonly isolated from the control
stations, Ceriosporopsis halima and ~ulworthia floridana,
and related their growth and distribution to certain en-
vironmental parameters. Although Barghoorn and Linder (5)
found that Ceriosporopsis halima grew more readily on fresh-
water agar than on seawater agar, Hughes (48) isolated this
species from medium and high salinity areas only. Labora-
tory studies by Barghoorn and Linder (5) showed that Cer-
iosporopsis halima grew best over a pH range of 5.2 - 9.2
(the highest pH tested) and a temperature range of 15 - 25'~.
Lulworthia floridana, the only representative of
the marine Ascomycetes isolated from Port Mellon, was iso-
lated from the bottom sampling depth where conditions of
temperature, salinity, and pH were closest to those at the
control stations. The temperature and salinity tolerances
of Lulworthia floridana were studied by Gold (38) who
found that this fungus did not occur in water of salinities
under 2 0 o/oo . The absence of Ascomycetes at the pulp mill surface
station in the summer months may be explained by low salinity
values. This explanation would not apply from October through
March as during this time salinity generally ranged from 15 -
30 o/oo. Nor can the lack of Ascomycetes be explained in
terms of competition from other species, as there was no
heavy fungal growth on panels removed from the mill.
A point to note in regard to Ascomycetes and other
marine fungi relates to marine borers such as Teredo and
~imnoria. ~arine borer cavities were conspicuously absent
on panels submerged at t k pulp mill. Although panels sub-
merged 6 months at Horseshoe Bay or other control stations
were thoroughly penetrated and close to disintegration
from borer attack, panels at Port ello on were still intact
after a submergence period of 1 year. There is some evi-
dence that attack by Limnoria may be facilitated by the -
presence of marine fungi, upon which they may feed (52).
The corollory, that fungi infect cavities formed by marine
borers, is also true although the cavities are not a nec-
essary prerequisite to fungal attack. Perhaps the inabil-
ity of borers, bacteria, and other marine organisms to de-
compose the panels at the pulp mill could make infection
and breakdown of wood materials more difficult for the marine
fungi .
The Fungi Imperfecti isolated were a heterogeneous
I group and showed different degrees of tolerance to the con-
ditions existing at the pulp mill surface station. Mono-
dictys pelaqica, a very common marine Imperfect species,
appeared to grow more readily at Port Mellon than at Horse-
shoe Bay. This phenomenon could have resulted from an abil-
ity to utilize some component of Kraft mill effluent. On the
other hand, Monodictys pelaqica could have become established
as a result of the limited competition from other marine
fungi such as Z. maritimum, a pioneer species which heavily
infects submerged panels.
Z. maritimum, perhaps the most common wood-inhabit- -
ing species in the Pacific Northwest (49, 6 3 ) , was isolated
less frequently from Port Mellon than from Horseshoe Bay.
Although physiological studies by Barghoorn and Linder ( S ) ,
Gustafsson and Fries (41) and the present study show that
Zalerion is capable of growing in media made with distilled
water, H6hnk (46) found Z. maritimum to be absent in waters
below 7 o/oo in salinity. Hughes (50) found that - Z. mari- -
timum occurred as frequently in areas of low salinity as ---
high salinity. Ritchie (71) showed that the higher the
temperature, the higher the salinity optimum for this fungus.
The results from the salinity measurements in the
field suggest that in the months May through September low
salinity conditions could have prevented the occurrence of
this species at the pulp mill station. However, salinity
conditions did not explain the lower frequency of occur-
rence of this fungus in the winter months. Temperature
did not appear to be a contributing factor, as temperature
conditions at the mill were not seriously affected by the
effluent. Moreover, Barghoorn and Linder (5) demonstrated
that - Z . maritimum could grow over a wide range of temperature
levels. They also investigated pH tolerances, and found
that - Z. maritimum could grow over the whole range tested,
4.4 - 9.2. It would appear that none of the oceanographic
parameters monitored could explain the decreased frequency
of occurrence of - Z. maritimum at the pulp mill station. For
this reason the physiological experiments, which will be dis-
cussed in a later section, were carried out.
The Imperfect fungus which occurred exclusively at
the pulp mill was Phialophora melinii, a species not comrnon-
ly isolated from marine habitats. studies have been carried
out on fungi which may occur in slime accumulations within
pulp mills (9-13, 34-36, 69, 72, 94), and several members
of the genus ~hialophora, - P. fastiqiata (Lagerb. & Melin)
Conant, g. richardsiae (Nannf.) Conant, - P. lignicola (Nannf.)
Goidanich, and P. alba van Beyma, have been isolated from
this source. Phialophora spp. have also been recorded as
occurring on stored wood, wood pulp, and wood chips (4, 57,
63, 72). The species isolated from Port Mellon was very
closely related to g. fastiqiata, differing only in the
size of the conidia (87).
My attempts to determine the source of Phialophora
melinii at the pulp mill station were inconclusive. The
results suggest that the spDres of this fungus were deposit-
ed on the panels in the field, but did not develop in the
sea. ~xperiments indicated that the source of the spores
was not the panels themselves, or either of the two effluents,
although it is possible that the spores were present in the
effluent in such low concentrations that they were not found
in the samples tested. The temperature of the effluent ranged
from 5 to 10 C in the winter and 20 to 25 in the summer (60),
neither low nor high enough to kill the spores of this fungus.
Aerially born spores are another p~ssible means of infection.
Some species of ~hialophora grow in wood chip piles ( 5 8 ) ,
and spores from such a source could have been carried by
wind and deposited in the sea. Whatever the source of
inoculum, the spores of this fungus were abundant in waters
in the vicinity of the pulp mill.
The oceanographic data was collected in an attempt
to explain the species composition differences. The measure-
ments in this survey were taken in the zone of highest ef-
fluent concentration, at a point approximately 100 yards
from each effluent pipe. Farther from the effluent pipes
salinity, temperature, and dissolved oxygen quickly approach-
ed values common in local coastal waters (37).
The B.C. Research Council (37) surveyed at different
depths as well as distances from the mill and found that the
effluent had little effect on salinity, temperature, dis-
solved oxygen, and pH at a depth of 30 feet.
The measurement of effluent concentrations under-
taken by the B.C. Research Council (37) indicated that
dependent upon tides, currents, and wind conditions .05%
to 100% effluent (only caustic effluent was monitored in
this study) was found at the closest station to the point
where the panels were submerged. Only low concentrations,
.5% or less, could be traced at a distance of 1 mile from
t h e e f f l u e n t p ipe . The e f f l u e n t tended t o move i n a north-
e r l y o r sou the r ly d i r e c t i o n depending on t i d a l cond i t ions ;
s i t e s t e s t e d very c l o s e l y t o t h e m i l l could show no t r a c e
of e f f l u e n t . The a r e a of 5 t o 100% e f f l u e n t concent ra t ion
extended l e s s than 2000 f e e t from t h e o u t f a l l , whereupon
t h e e f f l u e n t was r a p i d l y d i l u t e d a s t h e d i s t a n c e beyond
t h e m i l l i nc reased . Ef f luen t concent ra t ion decreased rap-
i d l y wi th inc reas ing depth , p a r t i c u l a r l y beyond t h e immediate
a r e a around t h e o u t f a l l s . Concentrat ions measured a t t h e
30 f o o t depth a t t h e s t a t i o n n e a r e s t where t h e panels were
loca ted were 6%, 5.4% and 0.2%.
The v a r i a b i l i t y which can be seen i n e f f l u e n t con-
c e n t r a t i o n s was a l s o c h a r a c t e r i s t i c of t h e oceanographic
parameters which were measured a t t h e s u r f a c e pulp m i l l
s t a t i o n . S a l i n i t y , d i s so lved oxygen and pH were not only
v a r i a b l e from day t o day, but a l s o could change cons iderably
over a 24 hour pe r iod . Hardon (43) conducted a survey a t a
s i t e very c l o s e t o where t h e panels were submerged and found
t h a t temperature, s a l i n i t y , d isso lved oxygen, and p H va r i ed
from 9 t o 1 2 C , 15 t o 2 1 o/oo, 3 mg/l t o 7 rng/l, and 6.0 t o
8.5 r e s p e c t i v e l y i n 24 hours . Low readings u s u a l l y co r res -
ponded t o low t i d e l e v e l s , when e f f l u e n t d i l u t i o n c l o s e t o
-83-
the outfalls was at a minimum.
When the results from the oceanographic measurements
at Port Mellon are interpreted, it should be kept in mind
that certain parameters such as salinity were considerably
affected by the nearby Rainy River. Thus, the low salinity
values in the summer months should be to a large degree
attributed to runoff conditions. The low temperature of
the freshwater may also have compensated for the higher temp-
eratures of the effluent stream. However, pH and dissolved
oxygen effects were mainly due to the presence of the efflu-
ents. Perhaps the most acute effect of the pulp mill efflu-
ent was to consistently lower pH values.
It was difficult to correlate the species composition
data with salinity, pH, dissclved oxygen, temperature, or
effluent conditions as these factors varied daily at the
mill. In an effort to understand the distribution of Z.
maritimum and g. melinii, physiological experiments on growth
and oxygen uptake were carried out.
Although there are some unique problems associated
with manometric techniques as applied to the filamentous
fungi (1, 16), I felt that the results from such studies
would be useful when considered in conjunction with the re-
sults from dry weight experiments. One problem with res-
piration studies in the fungi has been the high rates of
endogenous respiration, caused by the accumulation of re-
serve materials from the overly rich media usually employed
in studies of fungal physiology (1, 16). The media used in
this study therefore employed the relatively low concentra-
tion of lg/l glucose. Some factors which should be con-
sidered in respect to inoculation techniques are that mac-
eration may partially destroy the cells (16, 29), that the
dry weight of mycelium may change during the course of the
experiment (29), and that oxygen uptake should not be
correlated to dry weight unless the ratio of respiring to
non-respiring protoplasm is constant (29).
Preliminary studies were carried out with Z. mari-
timum to determine what inoculation technique would yield
the smallest variation in oxygen uptake values between
replicate flasks. These experiments showed that oxygen
uptake rates were most uniform when pellets or mycelial
fragments of the same diameter were used. Dry weight of
the inoculum among replicate flasks was also fairly uniform,
averaging 2.1 k 0.3 mg. However, those flasks with the
highest oxygen uptake values did not necessarily correspond
to those flasks with the highest dry weights at the con-
clusion of the experiment. Therefore, the direct oxygen
uptake method in which total oxygen uptake is plotted
against time but not related to dry weight (85) was used
to present the respiration data.
All dry weight and respiration studies on Zalerion
maritimum were carried out at 2 4 ' ~ , found by Barghoorn and
Linder (5) and G.C. Hughes (51) to be the optimal temperature
for growth. Although salinity and pH optima (5) have been
derived for this fungus by measuring radial growth on agar,
I felt that these experiments should be repeated using
aerated liquid media. The dry weight and respiration ex-
periments show that Z. maritimum has a broad tolerance to
varying salinity conditions; however, this principle does
not necessarily apply under field conditions, where the
concentration of essential nutrients may be low. The results
from the salinity experiments agree with those of Barghoorn
and Linder, who found that ~elicoma maritimum could grow
on freshwater medium, although not as well as on seawater
medium. The dry weight and respiration studies indicate
that Z. maritimum is tolerant to a wide range of pH levels
under laboratory conditions, as was also shown in the work
of Barghoorn and Linder (5). unlike many fungi, the optimum
pH for growth of Z. maritimum is in the alkaline range,
suggesting an adaption to growth in the marine environment.
However, when supplied with basal nutrients, this fungus
can grow to a limited extent at pH 3. The pH and salinity
studies in the laboratory do not explain the limited occur-
rence of Z. maritimum in the pulp mill area.
When growth and oxygen uptake of Z. maritimum were
measured in caustic effluent, caustic effluent with added
basal nutrients stimulated rather than inhibited fungal
metabolism. Perhaps growth and oxygen uptake increased
owing to utilization by the fungus of the sugars and/or
lignin content of the effluent. Caustic effluent alone
did not stimulate dry weight or oxygen uptake. This con-
firms the findings of ~nkvist (33) and others (61, 84)
who found that certain basal nutrients were required for
growth of fungi in pulp mill effluents. Perhaps one of
the most interesting aspects of the present study was the
discovery that one of the organisms natural to the marine
environment could utilize some component in caustic pulp
mill effluent. Suggested areas of future research are:
to determine what nutrients should be added to the effluent
for optimal growth; to determine whether lignin and lig-
nosulphonic acids are utilized; and to determine
whether this organism could be used in a microbial waste
treatment process.
other authors have investigated the growth of fungi
in pulp mill effluents. Some fungal species may grow in
sulphite and sulphate liquors if certain nutrients are
supplied (33, 61, 84). The fungi may use the sugars in
the effluent, reducing the biochemical oxygen demand (33)
and in some cases producing as end products economically
useful compounds such as fumaric acid (74). The use of
pulp mill wastes as a source of nutrition for commercial
yeast has also been investigated (74). Certain fungi
have the ability to degrade lignin (21, 42) and it has
been shown that some species may break down the lignin in
pulp mill liquors (7, 33, 54, 61, 84).
A series of experiments was carried out to deter-
mine whether the acidic bleach plant effluent could cause
the lower frequency of occurrence of 3. maritimum at Port
Mellon. The low pH (2.0 - 2.4) of the bleach plant efflu-
ent was probably responsible for the toxic effect of un-
alkalized media. However, bleach plant effluent adjusted
to the pH of seawater still retained some toxicity for the
fungus. In the unbuffered medium, the drop in pH which
-88-
occurred as growth proceeded could have been partially respon-
sible for the toxic effects. Perhaps the toxic component
had a more pronounced effect on the fungus under conditions
of low pH. When tris buffer was added to the alkalized bleach
plant effluent, the toxic effect shown in the unbuffered
medium was partially, but not completely overcome. The
toxic component of the alkalized bleach plant effluent did
not affect normal oxygen uptake by the fungus. When the
experiment testing the effects of alkalized bleach plant
effluent plus nutrients on oxygen uptake was repeated, dif-
ferent results were obtained. Two different samples of ef-
fluent do not necessarily have the same chemical composition,
and perhaps the effluent used in the second experiment con-
tained a higher percentage of carbohydrate and lignin, and
a lower percentage of the toxic component.
The results from the physiological studies suggest
that the combination of high concentrations of bleach plant
effluent and low pH conditions may be at least partially
responsible for the lower frequency of occurrence of 2.
maritimum at the pulp mill site during the winter months.
Oxygen uptake studies were carried out with - P.
melinii to determine whether this fungus showed a greater
tolerance to pulp mill effluents than did Z. maritimum.
Unlike Z. maritimum, oxygen uptake in g. melinii was not
stimulated by some component of the caustic effluent. The
results from the acidic bleach plant effluent studies indi-
cated that high concentrations of unneutralized bleach plant
effluent had no significant effect on oxygen uptake when
compared to seawater. With the addition of basal nutrients,
oxygen uptake was decreased only in lo@/, unneutralized bleach
plant effluent. The frequent isolation of - P. melinii from
Port ello on may be due to the tolerance of this fungus to
low pH conditions and to high concentrations of bleach
plant effluent.
V SUMMARY
I effluents cause certain changes in the species composition
I of marine fungi in an area very close to a pulp mill efflu-
ent discharge. Certain fungi such as Zalerion maritimum - were isolated less frequently at the pulp mill station than
at other stations. However, certain fungi such as Phialo-
phora melinii appeared to develop more readily on panels
submerged at the pulp mill station.
Physiological studies with g. maritimum indicated
that this fungus utilized some component or components of
the caustic effluent, resulting in greater growth and oxygen
uptake than found in control flasks. Although other fungi !
have been demonstrated to have this ability, this is the
first report of a marine fungus able to utilize pulp mill
I effluent . other work done on Z. maritimum indicated that the
salinity values prevailing at the pulp mill might inhibit
the growth of this fungus during the summer months. However,
it was difficult to correlate the absence of Zalerion during
the winter months with any of the measured physical parameters.
~ h y s i o l o g i c a l experiments ind ica ted t h a t t h e b leach p l a n t
e f f l u e n t might con ta in a substance t o x i c t o ~ a l e r i o n , par-
t i c u l a r l y a t low pH va lues . I t i s t h e r e f o r e suggested t h a t
t h e b leach p l a n t e f f l u e n t combined wi th low pH values may
be re spons ib le f o r t h e lower frequency of t h i s fungus a t t h e
pulp m i l l .
S tud ies wi th Phialophora - d i d not i n d i c a t e t h e source
of t h e spores of t h i s fungus. ~ h y s i o l o g i c a l experiments
showed t h a t c a u s t i c e f f l u e n t d i d no t a f f e c t oxygen uptake,
bu t t h a t t h i s s p e c i e s was p a r t i c u l a r l y t o l e r a n t t o high
concent ra t ions of a c i d i c bleach p l a n t e f f l u e n t . I t i s
suggested t h a t t h i s spec ies developed we l l on pane l s sub-
merged a t 30 cm a t t h e m i l l because of i t s t o l e r a n c e t o low
pH l e v e l s and high concen t ra t ions of b leach p l a n t e f f l u e n t .
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APPENDIX I
Table A-1 . Analysis of effluent and water samples for