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Limnology Oceanography - January 1975 - Gunnison
Limnology Oceanography - January 1975 - Gunnison
Limnology Oceanography - January 1975 - Gunnison
Abstract
The susceptibility to microbial decomposition of species of 14 algae was assessed in
pond water and with inocula from several environments. Some of the algae were destroyed
in short periods, but others withstood microbial digestion for more than 4 weeks. The
production of toxins did not account for the resistance of those algae not readily destroyed
microbiologically. The suitability of the cell walls as a substrate for microorganisms was
correlated with the longevity of three susceptible ( ChZamydomonas reinhardtii, CyZindro-
spermum sp., and Ulothrix fimbrata) and three resistant (Fischerella muscicola, Pediastrum
duplex, and Staurastrum sp.) algae. Microorganisms able to digest walls of the susceptible
but not of the resistant algae wcrc also isolated. The differing susceptibilities to decom-
position may be related to the relative biodegradabilities of specific components of the
algal walls. -
Although algal growth can be controlled rpm under 5,400 lux constant illumination
or modified by chemical means and by for 18 to 23 days and used to inoculate an
reducing nutrient inputs into waters, little 18-liter carboy containing 16 liters of BBM.
is known of the biological control mecha- The culture was incubated at 22°C with
nisms that limit algal growth under normal illumination at 10,800 lux provided by two
conditions or cause the sudden termination fluorescent lamps mounted 5 cm from the
of aquatic blooms. This study was under- sides of the bottle. Filter-sterilized air was
taken to gain an understanding of some of passed through the medium at a rate of 10
the biochemical factors that may be impor- liters min-l. The cultures were harvested
tant in programs designed to effect a bio- after 19 to 23 days by centrifugation, and
logical control of algae and, specifically, to the cells were washed three times with cold
determine the basis for the resistance and 0.01 M phosphate buffer, pH 7.0, and then
susceptibility of algae to microbial attack. suspended in 100 ml of the buffer. The
We thank I. Feigenoff for her assistance. final suspension was termed the “washed
Support for the work was provided by a algal concentrate” and was either used
Ford Foundation training grant in the ecol- immediately or lyophilized and stored at
ogy of pest management. -17°C.
To test the susceptibility of algae to
Methods and materials decomposition, we used a method modi-
The algae, all in axenic culture, were fied from that of Gromov and Mamkaeva
selected to represent a range of planktonic (1969). Washed algal concentrate (2.5 ml),
and attached species, but the choice was tither living or steamed for 20 min, was
also governed by the ability of the organ- spread evenly on the surface of BBM solidi-
isms to grow in the fermentors used and fied with 2.0% agar in a petri dish to form
their suitability for harvesting by continu- a “lawn.” The preparation was then per-
ous flow centrifugation, Algae were grown mittcd to dry for 4-5 days at 20°C under
in 250 ml of a modified Bristol’s medium 10,800 lux illumination. Samples of soil and
(BBM) (Bold 1942) in 500-ml Erlenmeyer sediment (0.1 g) or natural waters (0.1 ml)
flasks at 22°C on a rotary shaker at 150 were added to the surface of the algal
1awns, with sterilized samples serving as
l Present address: Waterways Experiment Sta-
controls. The soil was either Williamson
tion, U.S. Army Corps of Engineers, Vicksburg,
Mississippi 39180. silt loam from a cultivated field or a garden
LIMNOLOGY AND OCEANOGRAPHY 64 JANUARY 1975, V. 20( 1)
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Microbial decomposition of algae 65
soil. Sediment samples came from a fresh- A sample block of 28 pipets was removed
water swamp or a water-filled ditch. Water from the raft every 7 days for 12 weeks,
was collected from Cayuga Lake, Jenning’s and the algal layer was examined micro-
Pond, or the primary settling tank of a scopically. At the same time about 0.5 ml
sewage treatment plant. The plates were of the control suspension was removed from
incubated in a moist atmosphere at 30°C each ampoule aseptically for examination,
in the dark for 30 days and examined daily but the control ampoules were then re-
for evidence of discoloration, visual disap- turned to the raft.
pearance of algal biomass, loss of algal To test the ability of culture filtrates to
walls, and release of algal cell contents. inhibit microbial growth, we sterilized the
To dcterminc the validity of the labo- cell-free liquid from a 21-day algal culture
ratory findings, WC conducted a study in by filtration and introduced portions of this
Cornell University experimental pond 201. liquid or sterile 13BM into stainless steel
This pond has a surface area of about 0.07 antibiotic assay cylinders supported on an
ha and a mean depth of 1.5 m (Hall et al. agar medium previously seeded with a test
1970). The pond is mesotrophic, lacks any heterotroph. The plates were incubated
emergent vegetation except Ear a stand of for 2 to 5 days at 30°C and examined for
Typha sp. in one corner, and has Chara sp. the appearance of clear zones around the
as the dominant bottom plant. cylinders.
A covered raft was placed in the middle The existence of intracellular compounds
of the pond which held 18 removable sam- with antimicrobial activity was also investi-
ple blocks submerged beneath the surface. gated. The washed algal concentrate was
Affixed vertically to each side of 15 of the suspended in an equal volume of distilled
blocks were 14 Pastcur pipets, with the water and disrupted by treatment for 15
wide end downward and the tips removed min with a sonic oscillator at a temperature
to provide openings of 2-3 mm. The wide not exceeding 20°C. The resulting suspen-
ends were packed with glass wool to a sions or those remaining after centrifuga-
tightness sufficient to prevent the passage tion at 3,000 x g were placed in antibiotic
of algae while permitting the entry of wa- cylinders and assayed as above.
ter and small microorganisms. Into each The method of preparing the cell walls
pipet was introduced 1.0 ml of washed al- of Fischerella muscicola, Pediastrum du-
gal concentrate; the glass wool plugs re- plex, Staurastrum sp., and [Jlothrix fim-
tained the algae while the buffer drained hrata, cell envelopes of Cylindrospermum
out. All 14 algae were on each side of the SP*, and wall fragments of Chlamydomonas
block, and each alga was thus represented reinhardtii will be described elsewhere
twice on one block. The pipcts were kept ( Gunnison and Alexander in prep. ) . The
in darkness about 10 cm beneath the pond procedure involved repeated rupture of
surface. The aquatic microbiota had access the cells with a sonic oscillator (or a French
to the algae through the unobstructed top pressure cell for C. reinhardtii) followed by
of the pipct, and the glass wool plug at the extensive washing of the material collected
bottom was sufficiently loose to permit the by centrifugation; the wall preparations
exchange of nutrients and dissolved gases were then suspended in sodium dodecyl
from the water. Controls were provided sulfate, treated again by sonic oscillation,
by lo-ml ampoules containing 2.0 ml of and finally suspcndcd in distilled water.
washed algal concentrate and 6.0 ml of For the isolation of microorganisms able
filter-sterilized pond water. The ampoulc to degrade cell walls, the salts solution of
tops were sealed with sterile parafilm, and Potgieter and Alexander (1966) was sup-
the ampoules were fastened to the rcmain- plemented with 0.001% yeast extract and
ing 3 sample blocks and held beneath the 0.1% algal ccl1 walls, 0.2% lyophilized algal
water surface at the same depth as the cells, or 0.2% of either xylan or MN cellu-
pipcts. lose 300 (Brinkmann Instruments). The
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66 Gunnison and Alexander
solution was inoculated with a sample of cells of some being rapidly destroyed while
soil or aquatic sediment and incubated at others are quite refractory to attack. Dif-
30°C. Serial transfers were made to fresh ferences are evident also in the rates of di-
medium when much of the wall material gcstion of a single alga by organisms from
had disappeared or after 40 days, which- the different habitats, but no consistent
ever came first; after several such transfers, trend is evident.
the enrichment was streaked onto the salts Anabaena flos-aquae and Anacystis ni-
medium solidified with 2.0% agar and over- dulans were quite susceptible to microbial
lain with a suspension of 0.1% walls or decomposition, the average time for their
0.2% algal cells in 1.5% agar. The plates digestion, considering samples from all
were incubated for l-5 days, and colonies the environments, being less than 5 days.
surrounded by clear zones were picked and Ankistrocbsmus falcatus, C. reinhardtii,
identified by the methods described by Cylindrospermum sp., Eugleena gracilis,
Skerman ( 1967). Gloeocystis &as, Ourococcus multisporus,
and U. fimbrata were somewhat more re-
Results sistant; the average time for the initiation
Decomposition in algal lawns of their decomposition ranged from 6-10
and natural water days. Stazwastrum sp. was less liable to
degradation with generally more than 20
Viable algae present on an agar surface days before decomposition was observed.
differed markedly in their susceptibility to Calothrix anomala and Chlorella sp. were
degradation by microorganisms present in
likewise moderately resistant, and attack
soils, sediments, and bodies of water (Ta-
was occasionally not noted at all in the test
ble 1). The values given represent the
period. On the other hand, I;. muscicola
times elapsed before decomposition was
evident microscopically; each is the aver- and P. duplex were never attacked appre-
age of three replicates. Observations ended ciably during the study period.
after 30 days. The data show appreciable The patterns of resistance and suscepti-
differences among the algae in their resis- bility were quite similar when dead algae
tance to digestion by microorganisms, the were used (Table 2). The values again
19395590, 1975, 1, Downloaded from https://aslopubs.onlinelibrary.wiley.com/doi/10.4319/lo.1975.20.1.0064 by Nigeria Hinari NPL, Wiley Online Library on [02/09/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
Microbial decomposition of algae 67
4
Anabaena
ms
flos-aquae
Ankistrodesmus
nidulans
falcatus
3
5
:6 : 2
8
3
3
7
Calothrix anomala 7 :: 1; 15 i
Chlamydomomnhardt -ii 8 7 8 6 4
Chlorella
Cylindrospermum
sp. -
sp.
197
6
17 13
5
16 24 28
8
4
Euglena gracilis
Fischerella muscicola
11
r30 730
z . 19
730
95
>30
1;
730
11
r30
:
23
Gloeocystis CJ&& 12 11 13 10 8 9 4
Ourococcus multisporus 5 5 5 5 8 8 4
Pediastrum duplex >30 >30 >30 r30 >30 >30 p30
Staurastrum sp. 230 >30 >30 22 26 >30 15
--Ulothrix fimbrata 9 6 9 13 8 7 3
represent the averages of three replicates. prescntcd in Table 3. The data for the two
Again taking into account the times for di- means of testing for degradability are in
gestion for samples from all environments, general agreement, but a few disparities
A. flos-aquae and A. nidulans are most exist. For example, C. anomala was not
prone to digestion. As with the viable al- subject to discernible attack for the first 18
gac, the average times for evidence of an days on lawns but was readily decomposed
attack 011A. falcatus, C. reinhardtii, Cylin- in the natural pond. Decomposition of P.
drospermum sp., E. gracilis, G. gigas, 0. duplex was never found, either in the 30-
multisporus, and U. fimbrata were 6-10 day laboratory trial or in the 12-week field
d ays. Nonviable cells of Chlorella sp. were evaluation.
still less susceptible, but the resistance of
C. anomala was markedly reduced when Possible rcsis tancc mechanisms
the cells were steamed. Fischerella musci-
cola, P. duplex, and Staurastrum sp. were As an explanation for the resistance of
resistant whether viable or heated; digcs- some of the algae, it sccmcd plausible that
tion of P. dupZex was never observed. The some spccics might excrete extracellular
cells of algae such as A. falcntus and Chlo- toxins that prevent the growth of lytic, par-
rella sp. also generally lost some of their asitic, or predatory organisms. I-Iowcver,
resistance as a result of steaming, whereas culture filtrates had no discernible effect
the resistance of G. gigas and Staurastrum on the development of Aspergillus niger,
sp. usually increased as a conscqucncc of Penicillium brefeldianum, or Saccharomy-
this treatment. ces cerevisiae on potato dextrose agar, Ba-
Marked differences in resistance to de- cillus subtilis, Micrococcus lysodeikticus,
composition were evident in the pond wa- Nocardia sp., or Streptomyces sp. on nutri-
ter (Table 3); many spccics were dcstroycd ent agar, or Rhixobium sp. on mannitol-
with comparative ease, while others per- yeast extract agar. These heterotrophs were
sisted for 2-3 weeks. Staurastrum sp, and selected to reprcscnt a diverse group of
F. muscicola showed no signs of signifi- organisms so that antimicrobial activities,
cant attack until the fourth week, and P. if prcscnt, might bc observed.
duplex endured for 12 weeks, when the Diffcrcnccs in susceptibility to attack
study ended. might also result from the presence of in-
For comparative purposes, the ranges of tracellular algal constituents inhibitory to
values for the lawns of viable algae arc also potential lytic, parasitic, or predatory or-
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68 Gunnison and Alexander
Table 4. Digestion of algal walls by individual microbial rcsidcnts of pond water. Fisch-
microorganisms. ereZZamuscicola was the only resistant blue-
green of the five examined, but still it was
C. rein-
Digestion of walls
Cylindro-
of
i. fim-
attacked to some extent after 4 weeks in
Isolate rardtii spermum sp. brata natural waters. The unsuitability of the
blue-green algae as food for higher organ-
Microbacterium sp.
+;" -I- isms is often cited, in particular for those
+;':
Brevibacterium
Corynebacterium
G2
sp.
+"' planktonic crustaceans grazing on algae
Streptomyces D8
+A
+ + (Hutchinson 1973). The grazing pressure
+': -E
Streptomyces G6
+
+:"
-4
cxcrtcd by zooplankton on blue-greens
Streptomyces F7
Brevibacterium G5 -I- + +':' ranged from variable to ineffective (Porter
+ + +;;
Streptomyces 64 1973 ) ) although a few blue-greens are
by themsclvcs able to meet the nutritional
;:Walls serving as carbon source for original isola- requirements of Daphnia pulex (Arnold
tion. 1971). I3y contrast, blue-green algae are
known to bc consumed, lysed, or parasit-
degradation and thinning of the surface ized by a wide range of microorganisms.
components were always evident. We For example, Ho and Alexander (1974)
foulld two microorganisms which caused reported the utilization of blue-greens as
this kind of digestion of 77. fimbrata walls. food sources by several spccics of amcbac.
We found no isolates that could grow on Viruses acting on blue-greens have been
or dissolve the walls of any of the three foulid ( Shilo 1971)) and bacteria lysing
resistant algae. blue-green algae have been isolated fre-
The bacteria and actinomycetcs wcrc quently (Daft and Stcwart 1971; Granhall
tested for their ability to digest algal walls and 13crg 1972; Gromov et al. 1972; Shilo
other than those present in the mediuln for 1970). TIcnce it is not mlexpectcd that
their original istilation. The isolates were most of the blue-greens we tested are sus-
streaked on a plate containing an inorganic ccp tiblc to decomposition.
salts-agar medium on top of which was Among the Chlorophyceac examined,
placed water agar supplemented with the only C. reinharcltii and 0. multisporus wcrc
cell walls. Walls of none of the resistant particularly susceptible to decomposition,
algae were solubilized or digested to an cx- and the green algae we studied wcrc gen-
tent evident as zones of clearing around the erally modcratcly or highly resistant. The
streaks. 13~ contrast, four of the eight iso- resistance of these Chlorophyceae might
lates degraded walls of all the susceptible seem to contradict the fact that many of
algae (Table 4), although the walls used these algae serve as food for grazing or-
for the initial isolation were always the ganisms. IIowever, those algae that fall in
more cxtcnsivcly destroyed. Two digested the susccptiblc to susceptible-intermediate
only the walls on which they were initially category in our studies arc often subject to
isolated. heavy grazing pressure. Chlamydomonas is
digested by zooplankton (Porter 1973); I-IO
Discussion and Alexander ( 1974) found that C. rein-
The rcsistancc of algae to microbial de- hardtii, 0. multisporus, and A. falcatus
composition may well be diffcrcnt from were consumed by various amebae, a1-
their suitability as food sources to higher though Chlorella sp. and Staurastrum sp.
organisms. For example, our results show were complctcly resistant to the protozoa.
that three of the genera of blue-green algae Arnold ( 1971) found that both A. falcatus
arc particularly susceptible to microbial at- and Chlorella vulgaris were capable of sup-
tack under all conditions tried. Even C. porting D. pulex; however, larger green
anomala, although somcwhat slowly uti- algae and desmids (like Staurastrum) arc
lized on lawns, was readily decomposed by ullaffccted by grazing pressure, lullike the
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70 Gunnison and Alexander
smaller nongelatinous green algae, which BOLD, H. G. 1942. The cultivation of algae.
decline under such pressure (Porter 1973). Bot. Rev. 8: 69-138.
DAFT, M. J., AND W. D. P. STEWART. 1971.
Algae produce many antimicrobial agents, Bacterial pathogens of freshwater blue-green
and it is quite possible that algal resistance a1gac. New Phytol. 70: 819-829.
is associated with toxic metabolites. The GLOMHTZA, K.-W, 1970. Antimikrobiclle In-
toxins can affect other algae ( Lefevrc et haltsstoffc in Algen. 2. Das Vorkommcn van
al. 1952) or nearby heterotrophs (Pratt Acryls#ure in verschicdenen Mcercsalgcn.
Planta Med. 18: 210-221.
et al. 1944). The toxicity may sometimes GI~ANIIALL, U., AND B. BERG. 1972. Antimicro-
even be attributable to the high oxidation- bial effects of CeZZ&brio on blue-green algae.
reduction potentials resulting from algal Arch. Mikrobiol, 84<: 234-242.
growth ( Glombitza 1970; Maksimova and GROMOV, 13 V., 0. G. IVANOV, K. A. MAMKAEVA,
Fcdenko 1965). The results of our study AND I. A. AVILOV. 1972. Flexibacterium lys-
ing blue-green algae. Mikrobiologiya 41:
suggest that toxic products-ex tracellular, 1074-1079.
intracellular, or localized at the cell sur- - AND K. A. MAMKAEVA. 1969. Sensitivity
face-are not important in regulating the of ‘diffcrcnt Scenedesmus strains to the endo-
growth of potential microbial agents of dc- parasitic microorganism Amoebo~$eZicZiz~m.
composition, at least for the algae we inves- Phycologia 7 : 19-23.
tigated. In nature, nevertheless, toxins may IIALL, D. J,, W. E. COOPER, AND E. E. WERNER.
1970. An experimental approach to the pro-
be of significance in dense blooms of duction dynamics and structure of frcshwatcr
aquatic algae, affecting either microbial dc- animal communities. Limnol. Oceanogr. 15 :
composition or the grazing activities of the 839-928.
zooplankton. Ho, T. S., AND M. ALEXANDER. 1974. The fccd-
ing of amebae on algae in culture. J. Phycol.
Our results do suggest, however, that the
10 : 95-100.
cell wall is a major determinant of algal I~UTCIIINSON, G. E. 1973. Eutrophication. Am.
resistance. Although walls of the readily Sci. 61: 269-279.
decomposable species were subject to deg- JA%W, A. 1964. The bacteriology of trickling
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refractory species were obtained. If cell MAKSLMOVA, I. V., AND E. P. FEDENKO. 1965.
walls are, in fact, major determinants of The effect of the oxidation-reduction potential
resistance, then they must possess chemical on the development of bacteria in algal cul-
constituents subject to rapid or slow de- tures. Mikrobiologiya 34 : 344-349.
struction, which determine the suitability POINTER, K. G. 1973. Selective grazing and dif-
fercntial digestion of algae by zooplankton.
of the walls or of the algae themselves to Nature ( Lond. ) 244 : 179-180.
microbial digestion. The identities of these POTGIETE~, H. J., AND M. ALEXANDER. 1966.
constituents are not known, but prelimi- Susceptibility and resistance of several fungi
nary studies suggest that polyaromatic to microbial lysis. J. Bactcriol. 91: 1526-
1532.
compounds may be important in determin-
PHATT, R., AND OTHERS. 1944. Chlorellin, an
ing the resistance of walls of some species antibacterial substance from Chlorella. Sci-
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data), Such information could be of con- SIITLO, M. 1970. Lysis of blue-green algae by a
sidcrable value in providing a biochemical myxobacter. J. Bacterial. 104: 453-461.
basis for aspects of the ecology of aquatic -. 1971. Biological agents which cause
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SKERMAN, V. B. D. 1967. A guide to the iden-
ZCeferences tification of the genera of bacteria. Williams
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seven species of blue-green algae. Limnol.
Submitted: 11 March 1974
Oceanogr. 16 : 906-920. Accepted: 22 August 1974