Marine fungi

The metabolism of filamentous marine fungi has received little attention
either from the specific or comparative standpoints. As part of
an overall investigation of the carbon metabolism of this heterogeneous
group of Ascomycetes and Deuteromycetes, a rapid, semi-quantitative
method has been adapted to the screening of their NAD- and NADP-linked
dehydrogenases. The Ascomycete, Halosphaeria mediosetigera (700), and
Deteromycetes, Culcitalna achraspora (230) and Humicola alopallonella
(710), were grown in a semi-synthetic, artificial sea water broth in submerged
culture, from quantitatively standardized inocula, and harvested
in the linear growth phase when respirometric data indicated maximum
endogenous oxygen uptakes. Based upon dry weight, culture 700 gave
QO2 maxima of 9.3-14.6 at 72 hr growth, while culture 710 values were
12.4-16.2 for the same time; culture 230 gave values of 13.8-18.4 at
96 hr growth. Harvested mycelia were washed, quantitated and subjected
to sonic disintegration at 21 kHz for 20 minutes at 5-10 C in pH 7.0
phosphate buffer containing cysteine. Debris was removed by centrifuging
at 12,000 X g at 4 C for 30 minutes and the resulting turbid, yellow
supernatant fraction clarified by two successive 2 hr centrifugings at
4 C and 41,000 X g. Whatman #1 filter paper sheets, divided into 1 cm. squares, were pretreated with pH 8.0 Tris buffer containing MgCl2, and
air-dried. The papers were spotted at the grid intersections with appropriate substrates. Cell extracts were mixed with NAD or NADP in Tris and
the mixture superimposed upon the substrates. Within 2 hr the papers
were viewed under ultraviolet light at 3600 A for spot fluorescence from
the reduced coenzymes. In all extracts (5-10 mg protein per ml),
dehydrogenases were indicated for glucose, glucose-6-phosphate, gluconate,
6-phosphogluconate, isocitrate and malate. Less definite were indications
of oxidoreductases for galactose, lactate and glycerol. It is therefore indicated that these filamentous marine fungi utilize glucose carbon via
the hexose monophosphate pathway and the tricarboxylic acid cycle.

Selected species of marine filamentous fungi, Halosphaeria
mediosetigera, Humicola alopallonella, Culcitalna achraspora and
Orbimyces spectabilis, grown in a semi-synthetic medium containing
glucose 0.5, ammonium nitrate 0.24, tris(hydroxyrnethyl)aminomethane
0.12 and yeast extract 0.1% (Medium A) and modifications thereof
have been studied with regard to cellular composition, carbon fate
and growth kinetics. Cultures were aerated with sterile, carbon
dioxide-free air and washed mycelia were analyzed for macroconstituent
elementary composition, total carbon, protein, chitin and
lipids. Culture filtrates were tested for total carbon, .extracellular
carbohydrates and organic acids. Physiological profiles
were obtained to ascertain and correlate the ·extent and rate of
growth; and changes in pH, concentration of glucose . and anthronepositive
materials. · Elementary analyses of washed fungal mycelia
showed 41.0 to 49.0% carbon, 4.7 to 6.0% nitrogen and 5.4 to 6.5% ·
hydrogen. Optimal growth was accompanied by a concomitant fall in
pH followed by a shift back to the alkaline side. Following autolysis
at 8 days, almost one-third of the glucose substrate reappeared in
the medium accompanied by smaller amounts of mannose with Halosphaeria.
With Culcitalna, following exhaustion of glucose in the medium,
autolysis was accompanied by the reappearance of glucose. Pentoses
and a hexose other than glucose also were indicated by relative
mobilities on thin layer chromatograms. The most profound pH changes
were obtained with Humicola reaching a low of 2.4 in 15 days, but
the pH at 45 days had stabilized around 5.3. Increasing the glucose concentration to 5.0% (Medium B) did not increase the mycelial
yields to any substantial degree except for Halosphaeria. In
all cases pH fluctuated around 4 between 10 and 25 days, hut the final
pH was about 6. Production of mycelia was always accompanied by
substantial utilization of the glucose in the medium. When the
glucose concentration was raised to 5%, to force the buildup of
metabolic products, and yeast extract omitted (Medium C), substantial
amounts of glucose were utilized, but this was not manifested by
parallel increases in growth. Carbon balance studies confirmed that
pH changes were not due to the production of organic acids, but
probably to preferential utilization of ailllllonium cations. The initial
substrate carbon was found almost exclusively in the fungal mycelia
and evolved carbon dioxide. Carbon recoveries obtained under standard
conditions were 97.9, 97.4, 98.2 and 94.0% respectively for
Halosphaeria mediosetigera, Humicola alopallonella, Culcitalna
achraspora and Orbimyces spectabilis in the standard medium containing
0.5% glucose. In 5% glucose (Medium B) the recoveries were 93.0,
90.6 and 84.7% for the first three organisms mentioned. Time did
not allow a complete investigation of the causes. of decreased carbon
recoveries, but greater production of incompletely oxidized metabolites
appeared probable. Economic coefficients were determined for three
species in three media while determinations on the fourth species are
in progress. Culcitalna gave values of 71 in Medium A, 40 in Medium B
and 3 in Medium C; Halosphaeria gave 78 in A, 45 in B and 15 in C;
Orbimyces gave 75 in A and 7 in C . The effect of omission of yeast extract and of elevated glucose concentration on the economic
coeffic ients was pronounced . The only products detected under exaggerated
carbon conditions were acetic acid in the culture broth of Culcitalna
and a greenish pigment in the culture filtrate of Halospaeria. The
analogously-suggested accumulation of 2-ketogluconic acid in the latter
case could be indicative of the functioning of the hexose monophosphate
shunt or another primary aerobic pathway.

Cellulolytic enzymes were induced in the marine fungal isolates,
Culcitalna achraspora, Halosphaeria mediosetigera, and Zalerion
xylestrix by native cotton and related substrates. Marine
environmental conditions were maintained in an artificial
seawater medium. Induced c1 and ex enzymes and cellobiase were
determined colorimetrically. Induction also occurred on
cellobiose, but not on glucose and lactose. Permenter studies
with glucose and with toilet tissue carbon sources gave close
correlations between mycelial dry weight, enzyme component
activity, and cell nitrogen. In cotton substrate media,
Tween 80 increased C1 activity by 148%, and Cx activity by
160%, on the average. Millipore ultrafiltration was the best
technique for concentrating cellulase for Sephadex G-75 column
chromatography, while conventional techniques were unsuccessful.
Fractionation diagrams for all cultures were similar with
2 distinct peaks. The low molecular weight fraction contained
mainly Cx enzyme activity, while the high molecular weight
fraction contained C1 and Cx enzymes and cellobiase. These
fungi possess a cellulase similar, but not identical, to that
found in terrestrial species.

Cellulolytic marine isolates, Culcitalna achraspora and
Halosphaeria mediosetigera, were shaker-grown at 25 C in
artificial seawater containing glucose, NH4NO3 , tris(hydroxymethyl)
aminomethane and yeast extract, pH 7.5. Cultures were
harvested in the late linear phase where whole cell endogenous
respiration was maximal. Spectrophotometric determinations on
acetone powder extracts revealed the presence of aconitase,
NAD-linked isocitrate dehydrogenase, succinate dehydrogenase,
fumarase, malate dehydrogenase and isocitratase. Culcitalna
also showed NADP-linked isocitrate dehydrogenase activity.
Manometric examinations of acetone powder and dialyzed sonic
extracts were largely confirmatory. Oxidation of citric acid
cycle intermediates by extracts were observed with Culcitalna
for all but succinate with sonic extracts and for all but
succinate and 2-oxoglutarate in acetone powder extracts. With
Halosphaeria, neither extract could oxidize citrate or succinate,
while acetone powder extracts were unable to degrade cis-aconitate
and 2-oxoglutarate. Native mycelia were active only against
malate. Attempts to permeate cell walls with ethyl esters of
intermediates failed.

Halosphaeria mediosetigera and Culcitalna achraspora have been studied
to elucidate the mechanism by which they degrade cellulosic materials in
the sea. H. mediosetigera (700 LC 1), a C1-less variant, was also employed
in this work. Standardized shake-cultures were grown on cellulose and
cellulose derivatives supplemented with NH4N03, tris(hydroxymethyl)aminomethane
and yeast extract in artificial sea water (Lyman and Fleming), pH 7.5, at
25 C. Induced C1 and Cx enzyme and cellobiase activities were determined
calorimetrically by correlation with cotton fiber weight loss, by the
formation of reducing sugars from carboxymethylcellulose and by Glucostat,
respectively. Optimum pH values for both Cx enzyme and cellobiase of
C. achraspora were 6.0 at 37 C while those for H. mediosetigera (700 LC 1)
were 5.0 and 6.0, respectively, at 37 C. In both cultures, Cx enzymes
had a variable optimum temperature ( 37-50 C) at pH 6.0, depending on the
length of the incubation period, while cellobiases had the same optimum
temperature (50 C) at pH 6.0. Thermostability observations on Cx enzyme
and cellobiase of C. achraspora showed almost complete deactivation at
100 C in 15 min for the former and 50 C in 15 min for the latter. Thermostability observations on Cx enzyme and cellobiase of H. mediosetigera
(700 LC 1) showed almost complete deactivation at 45 C in 15 min for the
former and at 50 C in 15 min for the latter. Data indicate that at least
three enzymatic functions are involved in cellulose breakdown by these
cultures.