(DFMS) after acid hydrolysis of particle-free water and naturally occurring DFMS may
decline from 10 to approx. 1 within a 14 day incubation period. Data from lakes (Hama
and Handa, 1980) suggest that only 20 % of total DOC is of polymeric nature (carbohydrates, proteins).
The question is how these materials, a priori unsuitable for direct use by heterotrophic
bacteria, are introduced to the aquatic food chain and/or the pool of dissolved organic
monomers. Hollibaugh and Azam (1983) and Hoppe (1983) demonstrated through
different methodological approaches that products resulting from enzymatic splitting of
polymers are incorporated by bacteria. However, a varying fraction also contributed to
the DOC pool of small molecules.
In light of these general findings we constructed a few experiments on bacterial extracellular enzymatic activity concerning the following aspects: i) Origin of extracellular enzymatic activity investigated via fractionated filtration. This involved the abundance of free
dissolved enzymes. ii) Extracellular enzymatic properties of bacteria pure cultures and
axenic algal cultures were assessed in order to verify field observations where normally
little extracellular enzymatic activity was found in the algal fraction. iii) Since extracellular enzymatic activity seems to be closely related to the bacterial fraction of aquatic
organisms, the relationship between enzymatic and heterotrophic activity was investigated during an annual survey in two different biotopes of an eutrophic brackish water
fjord. Special emphasizes was placed on the relationship with phytoplankton development, a possible key factor in the supply of polymeric organic substances in the investigated area.
A strong correlation between protease activity and heterotrophic leucine uptake has
already been computed by Somville and Billen (1983) from data of different aquatic
biotopes. However, it is an open question as to whether an uncoupling of these processes
may occur seasonally and with changing environmental conditions.
The application of fluorigenic methylumbelliferyl-substrates (Hoppe, 1983) has proven to
be an useful tool in the study of extracellular enzymatic activities of bacteria in the field as
well as in experimental situations. The sensitivity of methods employing these substrates
seems to be on the same order of that using radioactively labelled polymers (Hollibaugh
and Azam, 1983). Results from both approaches, however, were not always comparable.
This may be a consequence of the great variety of combined amino acids ranging from
simple dimers to polymers of very different structure.
METHODS
Sampling
Water samples were taken from the pier of the Institut für Meereskunde (Kiel, FRG) and
from the adjacent brackish water Kiel Fjord. Stations were located in the polluted
harbour area (station Hauptpost) and in a more offshore position in front of the fjord’s
mouth (station Feuerschiff). Sterile 2 1-bottles in a surface sampler were used for water
collection.
Measurement of extracellular enzymatic activity:
For the determination of enzymatic activities fluorigenic methylumbelliferyl (MUF)
-linked substrates were used (MUF-α-D-glucopyranoside, MUF-N-acetyl-glucosamide,
MUF-phosphate and L-leucyl-methylcoumarin-HCl = MUF-leucine, provided by
Sigma or Fluca). Substrates of this nature were used for enzymatic measurements in
ecological studies by Petterson and Jansson (1978), Hoppe (1983) and Somville (1984).
120
decline from 10 to approx. 1 within a 14 day incubation period. Data from lakes (Hama
and Handa, 1980) suggest that only 20 % of total DOC is of polymeric nature (carbohydrates, proteins).
The question is how these materials, a priori unsuitable for direct use by heterotrophic
bacteria, are introduced to the aquatic food chain and/or the pool of dissolved organic
monomers. Hollibaugh and Azam (1983) and Hoppe (1983) demonstrated through
different methodological approaches that products resulting from enzymatic splitting of
polymers are incorporated by bacteria. However, a varying fraction also contributed to
the DOC pool of small molecules.
In light of these general findings we constructed a few experiments on bacterial extracellular enzymatic activity concerning the following aspects: i) Origin of extracellular enzymatic activity investigated via fractionated filtration. This involved the abundance of free
dissolved enzymes. ii) Extracellular enzymatic properties of bacteria pure cultures and
axenic algal cultures were assessed in order to verify field observations where normally
little extracellular enzymatic activity was found in the algal fraction. iii) Since extracellular enzymatic activity seems to be closely related to the bacterial fraction of aquatic
organisms, the relationship between enzymatic and heterotrophic activity was investigated during an annual survey in two different biotopes of an eutrophic brackish water
fjord. Special emphasizes was placed on the relationship with phytoplankton development, a possible key factor in the supply of polymeric organic substances in the investigated area.
A strong correlation between protease activity and heterotrophic leucine uptake has
already been computed by Somville and Billen (1983) from data of different aquatic
biotopes. However, it is an open question as to whether an uncoupling of these processes
may occur seasonally and with changing environmental conditions.
The application of fluorigenic methylumbelliferyl-substrates (Hoppe, 1983) has proven to
be an useful tool in the study of extracellular enzymatic activities of bacteria in the field as
well as in experimental situations. The sensitivity of methods employing these substrates
seems to be on the same order of that using radioactively labelled polymers (Hollibaugh
and Azam, 1983). Results from both approaches, however, were not always comparable.
This may be a consequence of the great variety of combined amino acids ranging from
simple dimers to polymers of very different structure.
METHODS
Sampling
Water samples were taken from the pier of the Institut für Meereskunde (Kiel, FRG) and
from the adjacent brackish water Kiel Fjord. Stations were located in the polluted
harbour area (station Hauptpost) and in a more offshore position in front of the fjord’s
mouth (station Feuerschiff). Sterile 2 1-bottles in a surface sampler were used for water
collection.
Measurement of extracellular enzymatic activity:
For the determination of enzymatic activities fluorigenic methylumbelliferyl (MUF)
-linked substrates were used (MUF-α-D-glucopyranoside, MUF-N-acetyl-glucosamide,
MUF-phosphate and L-leucyl-methylcoumarin-HCl = MUF-leucine, provided by
Sigma or Fluca). Substrates of this nature were used for enzymatic measurements in
ecological studies by Petterson and Jansson (1978), Hoppe (1983) and Somville (1984).
120