FEMS Microbiology Letters 91 (19921 265-2711
© 1992 Federation of European Microbiological Societies 0378-11197/92/$05.110
Published by Elsevier
265
FEMSLE 04812
Enrichment of purple photosynthetic bacteria from earthworms
Howard Gest and Jeffrey L. Favinger
Photo~Tnthetic Bacteria Group. Department of Bioloh% Imliana Unicersity Bloomington IN, U.S.A.
Received 20 December 1991
Revision received and accepted 9 January 1992
Key words: Photosynthetic bacteria; Purple bacteria; Earthworms; Rhodomicrobium cannielii;
Rhodopseudomonas palustris
1. SUMMARY
2. INTRODUCTION
Numerous bacteria present in soil during its
passage through the alimentary canal of earthworms resist digestive action and the antimicrobial defenses of the worms. Thus, several kinds of
non-sulfur purple photosynthetic bacteria (Rhodospirillaceae) can be enriched from 'purees' of
washed earthworms (or from intestinal contents
of the worms) using a procedure that exploits the
capacity to fix N 2 during anaerobic phototrophic
growth with organic acid carbon sources. It appears that earthworm enrichments can be designed to provide highly selective methods for
isolation of Rhodomicrobium ,'annielii and
Rhodopseudomonas palustris, and perhaps of other
purple bacteria. The extensive burrowing activities of earthworms undoubtedly contributes to
the widespread dissemination of Rhodospiriliaceae in soils.
In 1944, van Niel [1] noted that "it is difficult
to collect samples of mud or surface water in
which, by proper enrichment methods, one cannot demonstrate the presence of the brown or
red non-sulfur purple bacteria." This statement
was later extrapolated by a number of investigators to mean that the non-sulfur purple bacteria
(Rhodospirillaceae) occur only or primarily in
"aquatic environments," Thus, decades later,
Biebi and Pfennig [2] stated that soil is a "very
poor' inoculum for isolation of non-sulfur purple
bacteria and that such organisms can be isolated
from terrestrial samples only "now and then."
Studies [3,4] in this laboratory, however, using
appropriate enrichment procedures have demonstrated that non-sulfur purple bacteria are essentially ubiquitous in soils. Since earthworms swallow soil extensively, soil is always present in their
alimentary canals, and we now report ti~e enrichment of Rhodospirillaceae from earthworms collected randomly. The enrichment procedure is
based on the capacity of virtually all non-sulfur
purple bacteria to grow photoheterotrophically
on organic acid carbon sources under strictly
Correspondence to: H. Gest, Photosynthetic Bacteria Group,
Department of Biology, Indiana University, Bloomington, IN
47405, U.S.A.
266
anaerobic conditions with N 2 as the source of
nitrogen.
3. MATERIALS AND METHODS
3.1. Media
Variations of enrichment medium GEM [3]
were used. GEM contains a mixture of organic
acids, namely, acetate, lactate, malate, succinate,
and citrate. In some experiments, lactate or
malate were successfully employed as sole carbon
sources. A low concentration of NH4CI (1.8 mM)
was added in a number of trials to provide a
rapidly assimilable nitrogen source, but this addition was not obligatory. Note that the original
recipe for medium GEM calls for addition of a
chemical reducing agent, but this was omitted in
the present experiments.
3.2. Inocula from earthworms
Earthworms were collected from various sites
in and near Bloomington, Indiana between early
June and late November 1991. For most of the
experiments, freshly collected worms were washed
repeatedly with sterile distilled water, then suspended in sterile distilled water (approx. 8
worms/25 ml) and comminuted in a small Waring Blendor (Eberbach, Ann Arbor, MI; capacity
50 ml) for several minutes. Ordinarily, a series of
increasing volumes (0.1-1.0 ml) of the 'puree'
thus obtained was used to inoculate 30-ml volumes of media contained in Hypovials (Pierce
Chemical, Rockford, IL) of 50 mi nominal capacity. In some instances, the inoculum consisted of
material extruded by manual pressure from the
intestinal contents of living worms. To ensure
that positive enrichments resulted from growth of
bacteria present within the worms, controls were
run with washed worms pretreated with 0.2 M
HgCI 2. The Hypoviais were gassed with N 2 and
sealed as described previously [3]; alternatively,
an atmosphere of 'anaerobic hood gas' consisting
of 85% N 2 + 10% H 2 + 5% CO 2 was also found
to be satisfactory.
3.3. Oxygen-free N 2
Commercially obtained N 2 was passed through
a scrubbing furnace containing reduced Cu filings
to remove all traces of 02.
3.4. h~cubation conditions
Cultures were incubated in a temperature-controlled cabinet at approx. 32°C with 400 footcandles (4300 Ix) of incandescent illumination provided by banks of 60 Watt Lumiline lamps.
3.5. hi vivo spectra
In vivo spectra of photosynthetic pigments were
determined using suspensions of cells in 30%
bovine serum albumin as described by Sojka et al.
[5].
4. RESULTS AND DISCUSSION
4.1. Hans Molisch's experiments with earthworms
The notion of testing earthworms as possible
sources of purple photosynthetic bacteria was
suggested by a hasty reading, and misinterpretation, of remarks made by Hans Molisch in his
autobiography [6]. He was a visiting professor at
the Bose Institute in Calcutta during 1928/1929,
and noted the following experience: "From time
to time during the preparation of the lectures,
something unexpected would take place. One day
I explained to my assistant, a very devout Hindu
who belonged to a particular sect whose followers
were not allowed to kill animals, how one could
obtain purple bacteria in the laboratory with relative certainty. For this purpose it was only necessary to fill a long glass tube with tap water (our
italics), put in pieces of a chopped up earthworm,
cover the water with a layer of oil and then let
the whole thing stand in the sun, whereupon the
purple bacteria appear in 1-2 weeks and color
the water red. When l checked up after a fe~
days on whether the assistant had initiated the
experiment correctly, I saw that everything was in
order, except that the earthworm was not cut up,
but moving around in a lively fashion at the base
of the tube. When I drew the assistant's attention
to this, he looked at me almost in fear, with wide
eyes, and asked quite remorsefully for forgiveness
for not having been able to bring it upon himself
to kill the earthworm. Attention may not be drawn
to such events in India when one considers that
here widespread sects of the Jains kill no animals
at all, not even fleas, lice and mosquitos; they
267
even have the evening meal before sunset, because if they would eat after sunset, many insects
would then fly into their lamps and be killed."
Assuming that Molisch knew that earthworms
harbored purple bacteria, and believing that this
was a reasonable proposition, we set up enrichments using earthworm puree as inoculum for
media containing organic acid carbon sources as
described in MATERIALSAND METHODS.Then, we
reread Molisch's 1907 paper [7] to see if earthworm enrichments were described in his classic
monograph. It became clear that minced earthworms were used by Molisch in 1907 not as
sources of photosynthetic bacteria, but rather to
provide organic carbon substrates for growth of
the purple bacteria present in Prague tap water!
Molisch states that the tap water in Prague at
that time was undrinkable (he and his family
drank only boiled water imported from elsewhere), but was a veritable [El] "Dorado" of
interesting microorganisms of many kinds. The
tap water in Calcutta in 1928/1929 must also
have been a "microbiological zoo". It should be
noted that aside from many other important discoveries made by Molisch [8], he was the first to
describe the photoheterotrophic growth mode of
purple bacteria [7].
Although we had set up our enrichments on a
mistaken premise, we allowed them to incubate
as described, and were pleasantly surprised to
observe that a number of the Hyl~ovials developed luxuriant enrichments of Rhodospirillaceae.
4.2. Prominent organisms in earthworm enrichment
cultures
Depending on the particular batch of earthworms used, red or brownish bacterial growth
characteristic of Rhodospirillaceae was evident to
the naked eye after 2-4 days, 6-8 days, or only
after several weeks of incubation. The duration of
the 'lag' obviously must be related to the concentration of viable purple bacteria in the inoculum
and the inoculum size, but other factors also
appear to have an influence. This was indicated
by the fact that the development of positive enrichments in a particular series of vials was frequently not a direct function of puree inoculum
size. It should be noted that there are some 1800
,¢
m
Wavelength (am)
Fig. I. In vivo absorption spectrum of cells from a primary
enrichment culture inoculated with a "puree" of earthworms.
The spectrum, characteristic of Rhodomicrobium t'annidii,
was obtained from a scan of cell masses (see text) dispersed in
30% bovine serum albumin.
different species of earthworms [9], and that the
worm batches used in our trials were not necessarily of a single species.
Thus far, the organisms most commonly enriched were Rhodomicrobium cannielii and
Rhodopseudomonas palustris. These bacteria are
readily identified by their distinctive morphological characteristics [10] and in vivo spectra of
photosynthetic pigments. A remarkable feature of
the earthworm enrichments is that often only one
species of purple bacteria is microscopically observed. In several experiments, Rhodomicrobium
developed in the form of small pigmented balls
(approx. 0.5 mm in diameter) that settled on the
bottom of the culture vials. The balls consisted of
masses of Rhodomicrobium cells, which showed
the characteristic in vivo absorption spectrum illustrated in Fig. 1.
There is little doubt that highly specific earthworm enrichments for Rhodomicrobium and R.
palustris could be designed by modifying the
growth medium. Thus, for Rhodomicrobium the
vitamin supplement can be omitted, and the initial pH adjusted to approx. 5.5 [2]. For R. palustris, use of an aromatic compound such as cinnamic acid as sole carbon source would provide
very selective growth conditions [11]. In one of
the current experiments, material from the
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hindgut of a worm inoculated into G E M medium
yielded a luxuriant culture of R. pah~stris.
In addition to Rhodomicrobium and R. palustris, other non-sulfur purple bacteria have been
occasionally observed in earthworm enrichments,
for example, Rhodobacter capsulatus. We anticipate that a wider range of Rhodospirillaceae can
be enriched from earthworms of different kinds
using appropriate modifications to optimize
growth of particular species.
4.3. Earthworm microbiology
Since the alimentary canal of earthworms ordinarily contains soil, it is evident that numerous
species of bacteria and other microorganisms will
be found in purees of washed earthworms. There
are a number of papers in the literature which
summarize studies on the normal microbial flora
of the coelomic fluid and gut of earthworms,
responses of the worms to bacteria pathogenic for
humans, effects of the worms on soil microflora
etc. [12-15]. Of special interest in connection
with the present results is a paper by Khambata
and Bhat [16] on the intestinal microflora of
Indian earthworms. Their report is particularly
noteworthy for a review of older literature and
experimental data on bacteria that survive digestive processes of earthworms and can be readily
enriched, for example, oxalate and cellulose decomposers. Among the oxalate decomposers were
several strains that resembled Bacillus extorquens,
a red-pigmented bacterium originally isolated
from earthworm excrement by Bassalik in 1914
[17]. Apparently similar, and probably closely related, strains have been described under a variety
of designations such as 'Vibrio extorquens', 'Pseudomonas extorquens ', and ' Protomonas extorquens' [18]. These aerobic organisms have been
reported to contain carotenoids and bacteriochlorophyll, but attempts to grow them with light as
the sole energy source have given negative resuits, i.e. they are not true phototrophs. The
possibility that production of photopigments in
aerobic bacteria of the 'extorquens' type is coded
by genes derived from recognized Rhodospirillaceae via 'lateral gene transfer' remains to be
explored.
5. C O N C L U D I N G R E M A R K S
Further detailed studies are required to define
the factors which contribute to persistence of
viability of particular purple bacteria in earthworms. Occurrence of cell forms relatively resistant to adverse environmental conditions, such as
the exospoles of Rhodomicrobium [19], no doubt
are important in this connection. Various vegetative cell properties (for example, capsules, as in
R. capsulatus) that may confer resistance to
phagocytosis also may be of significance. It appears likely that the extent of dissemination of
microorganisms through soil processing by earthworms has been underestimated in ecological
analyses. Renewed study of earthworms as 'microbial transfer agents' seems desirable.
ACKNOWLEDGEMENT
This research was supported by grant DCB8915037 from the U.S. National Science Foundation.
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