A Look through the Wormhole: Identification of Bacterial Isolates from Vermicompost
Rebekah Church, Sara Church
Microbiology
Dr. Stephen Baron
October 20, 2015
Purpose: To inoculate, incubate, isolate, inspect, and identify a Gram positive and negative
bacterium from the vermicomposting facility at Bridgewater College.
Introduction:
Vermicomposting is a very adept method of transforming solid organic waste into useful
and valuable resources (16). The conversion process involves a series of intricate interactions
that occur between certain species of earthworms and microorganisms in efforts to bio-oxidize
and stabilize organic matter (11). Presently, there has been considerable research conducted on
Eisenia fetida, a common composting worm, in regards to their interdependent relationship with
the various microbial communities found in vermicomposting facilities (1). These earthworms
play a critical role in the process of vermicomposting; however, it is the resident microbial
communities that serve as the active component involved in the biodegradation and conversion
processes during vermicomposting (1). Specifically, it is with the help of the aerobic and
anaerobic microbes in the gut of the earthworm that organic wastes are grinded up and digested
in order to be converted into valuable vermicompost (16). The vastitude of microbial species
associated with soil found in vermicomposting sites assured that the vermicomposting facility at
Bridgewater College would be a reliable source for cultivating isolates. The goal of this research
was to isolate and identify two bacterial species obtained from the vermicompost. Researchers
were able to successfully isolate and identify one Gram positive and one Gram negative specie of
bacteria. Researchers were able to cultivate, isolate, and identify the bacterial species Bacillus
cereus and Citrobacter freundii.
Materials and Methods:
The differential staining technique of Gram staining was administered to both the
bacterial isolate from sample D on the 1 cm EMB plate and sample B on the 1 cm PEA plate. In
addition, an endospore stain was administered to bacterial isolate from sample B on the 1 cm
PEA plate. The Gram staining technique is utilized to distinguish between Gram positive and
negative bacterium. The endospore stain distinguishes between an endospore containing microbe
and a microbe that does not produce an endospore. The following staining techniques used in the
identification of the unknown were adapted from the second edition copy of Microbiology
Laboratory Theory & Application by Leboffe and Pierce (13). Immediately after each staining
procedure was completed, the bacterial stain was viewed and inspected under oil immersion with
1000x magnification using bright field microscopy. The endospore stain procedures were
repeated three times due to the dye’s lack of adherence resulting from human error.
After the completion of the differential staining procedures and the determination of the
Gram reaction of the two bacterial isolates, the sequencing of the bacteria’s 16S rDNA was
conducted using the following procedures (5). For this procedure, the genomic DNA from each
bacterium was extracted from an isolated TSA slant culture through the utilization of a DNA
extraction kit (Quik-gDNA MiniPrep from Zymo Research). The extracted DNA served as the
template in the amplification of the DNA sequence along with universal primers in the
Polymerase Chain Reaction (PCR). The PCR procedure allowed for the amplification of a 1
kilobase pair (kb) sequence confined within the 16S rRNA genes present in the genomes of the
unknown bacterial isolates. The products obtained from the PCR amplification were purified
through the utilization of the nucleic acid based method of gel electrophoresis containing 1.0%
agarose. After the gel was ran at 125-150 volts for approximately 30 minutes, the 1 kb sequence
was excised from the gel using a gel extraction kit (Genelute Gel Extraction Kit, Sigma-Aldrich).
The DNA extracted from each of the bacterial samples was sent to a sequencing facility
immediately following the placement of the DNA sample in a vacuum (Speed-Vac). The Speed-
Vac was utilized to ensure the complete evaporation of water, thus bringing the DNA to a state
of dryness. The DNA sample from the first unknown isolate was sequenced using a universal
forward PCR primer (U341F) and the DNA sample from the second unknown isolate was
sequenced using a universal reverse PCR primer (UA1406R). Once the completed sequences
were retrieved from the sequencing facility, the sequences were exposed to the BLAST database
search against a collection of bacterial nucleotides to determine any similarities between the
unknown sequence and the identified bacteria (15).
The Gram positive and Gram negative bacterial isolates were subjected to several
biochemical tests using various types of alternative substrates. The tests were conducted in
accordance to the dichotomous key for selected Gram negative bacteria and the dichotomous key
for selected Gram positive/acid-fast bacteria published in Laboratory Exercises in Microbiology
(8). In order to confirm the identification of the isolate, additional biochemical testing was
conducted. The reactions of both bacterial isolates were meticulously observed and recorded.
The biochemical tests conducted on the Gram negative isolate include: Fermentation of
carbohydrates (glucose, lactose, mannitol), Sulfur Indole Motility (SIM), citrate test,
oxidation/fermentation, Vogues-Prokauer (VP), nitrate reduction, milk agar, starch agar, and
nutrient gelatin tests (4,6). The only biochemical tests conducted on the Gram positive isolate
included the fermentation of the carbohydrate mannitol and starch agar. For each of the
mentioned tests involving the use of alternate substrates, the specialized media was inoculated
with a bacterial isolate and incubated for a 24-48 hour period at 37°C. A phenol red broth test for
mannitol and glucose fermentation was performed, and the results of the initial test were
confirmed through a second test. For the mannitol fermentation test, the phenol red broth was
inoculated with the Gram positive isolate and incubated for a 24 hour period at 37oC. For the
glucose and lactose fermentation, two phenol red broths were inoculated with the Gram negative
isolate and incubated for a 24 hour period at 37oC. In order to determine if the isolated bacteria
produced the enzyme amylase, a starch agar plate was used to test for starch hydrolysis (6). Both
the Gram negative and the Gram positive isolates were inoculated onto a starch agar plate and
incubated for 48 hours at 37°C. After the 48 hour incubation period, both plates were flooded
with Gram’s iodine and observed for a clearing zone to determine whether the starch hydrolysis
had occurred.
Both the Gram positive and Gram negative microbial isolates were then subjected to a
catalase test and an oxidase test (4). The catalase test was conducted by placing a small amount
of the bacterial isolate onto a clean slide containing 3% H2O2 and observing and recording the
catabolic reaction resulting in the expulsion of O2 gas. This expulsion of O2 is characterized by
the appearance or truancy of fizz (4). The oxidase test was conducted by using a sterile cotton
swab to obtain microbial growth from the Gram negative isolate and then dousing it with ~ 100
microliters of oxidase reagent. An alteration in the color of the colony on the swab to the color
black was used to discern whether the Gram negative isolate contained cytochrome oxidase, Cyt
a/a3 (4). The oxidase test procedure was repeated twice to ensure the reliability of the results.
Results and Discussion:
Bacterial isolates from sample D on the 1 cm EMB plate developed in a circular colony
formation with a smooth texture. The isolated colonies were uniformly arranged with a shiny
appearance, a low convex elevation, and an entire margin. The isolated colonies presented a foul,
bitter smell. The results from the Gram staining procedure on the isolates from sample D on the 1
cm EMB plate showed a Gram negative bacterium with a rod-like formation. The cells were
present both singly and in a paired arrangement (Fig. 1). This bacterial isolate was successful in
the fermentation of glucose which was made evident by the color change of the phenol red broth
to yellow, indicating the production of organic acids as the byproduct. The isolate was able to
degrade citrate in order to be utilized as the sole carbon source for the construction of cellular
material. The conversion of citric acid into an alkaline product was indicated by the
bromothymol blue dye which turned blue when the pH of the isolate reached approximately 7.6.
When inoculated and incubated in the SIM medium, the bacterial isolate was determined to be
motile indicated by the presence of a cloudy growth throughout the entire medium. The isolate
was positive for the production of H2S through the degradation of sulfur containing amino acids
by the enzyme desulfurase. This result was indicated by the color black when the FeSO4 in the
medium reacted with the H2S product resulting in a FeS precipitate. The isolate did not produce
indole by the means of tryptophan metabolism. This was indicated by the medium turning a
yellowish color after the addition of the Kovac’s reagent rather than red which would have
indicated a positive test. The bacterial isolate did not contain the enzyme amylase used to
hydrolyze the starch polymer into maltose. This result was concluded by the absence of a
clearing zone when flooded with Gram’s iodine. The enteric isolate was negative for the enzyme
casein protease used in the degradation of casein. This was made evident by the lack of a
clearing zone surrounding the bacterial colony. The bacterial isolate does not contain the
exoenzyme, gelatinase, needed for the hydrolysis of the nutrient gelatin. The lack of gelatinase,
was made evident by the solidification of the gelatin after incubation for approximately 48 hours.
The bacterial isolate was capable of fermenting both mannitol and lactose. This conclusion was
reasoned by the change in color of both phenol red broths to yellow. The change in color
signified the acidity of the broth; thus, indicating the production of organic acids. The microbial
isolate was determined to be a facultative anaerobe through the observation of the isolate growth
throughout the entire tube. In both the nutrient oil sealed tube and the unsealed tube, the growth
was indicated by the color yellow which was present throughout the entire length of the tube.
The bacterial isolate was able to perform anaerobic respiration via the reduction of nitrate to
nitrite through the process of dissimilatory nitrate reduction. This result was indicated by the
change in color of the trypticase nitrate broth from a clearish color to cherry red, provided that
the reaction did not require the utilization of zinc. The results of the VP test indicated that the
isolate was unable to form acetoin via the butylene glycol pathway. These results were
concluded from the lack of color change present proceeding the addition of reagents ⍺-naphthol
and potassium hydroxide. The results from the oxidase test indicated that the Gram negative
isolate did not contain cytochrome oxidase as a major electron transport protein. The absence of
cytochrome oxidase was made apparent by the lack of color change following the addition of the
oxidase reagent. The isolate contained the enzyme catalase used to breakdown the toxic
byproduct, H2O2, formed during aerobic respiration. This was concluded by the fizzing that
resulted from the inoculation of the isolate into 3% H2O2.
The colony morphology and results of the biochemical tests served as the basis for the
decision that the unknown isolate from sample D on the 1 cm EMB plate was best categorized
under the phylum Proteobacteria, genus Citrobacter, and specie freundii. The properties
displayed by the bacterial isolate led to the determination that the phylum, genus, and specie
were consistent based upon the homologous characterization of the unknown and the identified
bacteria. Proteobacteria is the largest and most phenotypically diverse phylum, consisting of five
classes: 𝞪,,𝞬,𝞭,𝞮 (23). Collectively, these microbes exhibit a great deal of metabolic diversity.
They comprise the majority of the Gram negative bacteria known to have considerable
significance in medicine, agriculture, and industrial complexes (21). This phylum of bacteria
may have flagella, gliding filamentous stalks or other appendages, while others may have
vesicles filled with gas. Some species of this bacteria are capable of carrying out photosynthesis,
whereas others deposit sulphur within or outside the cells (21). The different subgroups of
proteobacteria are defined based on their rRNA sequences (21).Gammaproteobacteria is one
subgroup of proteobacteria that is comprised of both facultatively anaerobic and fermentative
Gram negative bacteria (17).
The genus Citrobacter resides under the class 𝞬 proteobacteria and consists of Gram
negative bacilli from the Enterobacteriaceae family (12). These microbes are most commonly
found in soil, water, sewage, food, and the intestinal tracts of animals, such as earthworms and
humans. The facultative anaerobic bacteria may appear as rods approximately 0.3-1 µm in
diameter and 0.6-5 µm long. Citrobacter bacteria are oxidase-negative bacilli that utilize citrate
as a sole carbon source. In addition, these bacteria are motile by means of peritrichous flagella.
Bacteria under the genus Citrobacter also ferment mannitol accompanied by the production of
gaseous H2S (12). The previously mentioned biochemical characteristics were revealed by the
isolate as a result of the specific reactions that occurred throughout a multitude of tests.
Citrobacter freundii is classified as an opportunistic pathogen responsible for a number
of significant infections seen in humans (2). It is known to be the cause of a variety of
nosocomial infections within the respiratory tract, urinary tract, and blood of patients seeking
treatment in medical facilities (2). In the environment, however, this infectious microbe plays a
much more beneficial role. C. freundii is classified as an enteric coliform used in the indication
of water contamination (18). Thus, ecologists and environmentalists test for this bacteria to
assess the health and quality of various bodies of water. Furthermore, as a facultative anaerobe,
Citrobacter freundii is capable of reducing nitrate to nitrite in the environment (18). This crucial
conversion is an important feature of the nitrogen cycle, which is essential for the maintenance of
Earth’s atmospheric conditions.
Microbial isolates from sample B on the 1 cm PEA plate were presented as off-white or
dull gray colored colonies with a rough, flat, and matted surface. The colonies were opaque with
irregularly shaped undulated margins. The isolates from sample B on the 1 cm PEA plate were
determined to be Gram positive due to the retention of the crystal violet dye when Gram stained
(Fig. 2). When Gram stained the bacterial isolate showed a rod like formation of cells in an
arrangement of tangled chains. The structural endospore stain of the Gram positive isolate
showed the inclusion of an endospore within the microbial specie. The presence of an endospore
was determined by the retention of the malachite green dye in the thick cell wall of the
endospore. The spores stained by the malachite green were observed to be endospores lysed from
the vegetative cells of the isolate (Fig. 3). The isolate contained the enzyme catalase used in
breakdown of the toxic byproduct, H2O2, formed during aerobic respiration. This was
determined by the fizzing that resulted from the inoculation of the isolate into 3% H2O2. The
oxidase test result indicated that the Gram positive isolate did possess cytochrome oxidase as a
major electron transport protein. The presence of cytochrome oxidase was made apparent by the
color change to black following the addition of the oxidase reagent. The bacterial isolate was
unable to ferment mannitol. This was concluded by the absence of a color change in the phenol
broth indicating that an alkaline product was formed.
The decision to designate the unknown isolate from sample B the 1 cm PEA plate as a
bacterium belonging to the phylum Firmicutes, genus Bacillus, specie cereus was based on the
observation of colony morphology and the results of the biochemical tests. This interpretation of
the unknown isolate was based on characteristics of the phylum and genus that were similarly
displayed by the isolate. The phylum Firmicutes constitute as one of the largest groups of phyla
within the domain Bacteria (9). The bacteria of the phylum are all Gram positive with a low
percentage of Guanine and Cytosine DNA content (9). This lineage of phylum is highly diverse
in regards to their oxygen preference, morphology, variety of habitats, and reproductive lifestyle
(10). Some Firmicutes form an endospore, which is a differentiated cell that is resistant to
specific conditions present in nature (10). This specific property of the members of the phylum
Firmicutes is found in four representative classes of the phylum: Clostridia, Erysipelotrichi,
Negativicutes, and Bacilli (10).
Bacilli are characterized as Gram positive rods that are able to produce endospores
resistant to unfavourable external conditions (14). Bacilli can be differentiated from alternative
spore-formers due to their oxygenic preference, rod-shaped cells, and ability to synthesize
catalase (20). This is noticeable when looking at Bacillus cereus, a specie of bacteria
characterized by their rod shaped cells. This specie of Bacillus is distinguished by a thick layer
of peptidoglycan that allows it to retain the crystal violet dye; thus, Bacillus cereus is identifiable
as a Gram positive bacteria. This specie is a ubiquitous facultative anaerobe that forms
endospores and is commonly found in soil. A sizable population of Bacillus cereus present
throughout the vermicomposting process is used as an indicator of the quality and maturity in
that particular compost (3). In addition to the indicative qualities, Bacillus cereus also produces
an abundance of chitinolytic enzymes which play a major antagonistic role in the biocontrol of
microbial pathogens such as Rhizoctonia solani (3). Bacillus cereus is also known to
manufacture antibiotics that are utilized in the nullification of fungal diseases that occur in the
rhizosphere (22).
Conclusion
The gut of Eisenia fetida contains a variety of microflora that interact with the common
composting worm in a symbiotic relationship. Throughout the process of vermicomposting, the
dead, organic matter enters the intestinal tract of the worm and is chemically and physically
altered. This alteration occurs through the joint effort of Eisenia fetida and the microbial
community present in the gut. The microbial community, namely Citrobacter freundii and
Bacillus cereus, plays an important role by acting as catalyst in the catabolism of organic matter.
These microbes also serve as factors in the stimulation of other free microbial flora to further
degrade the organic matter into vermicast. Consequently, the production of vermicast leads to an
increase in the population of microbial species, enzymatic activity, bacterial respiration,
colonization of nitrogen fixing and nitrifying bacteria (19).
Description
Max
Score
Total
Score
Query
Cover
E
Value
Ident
Pseudomonas aeruginosa gene for 16S ribosomal RNA,
partial sequence, strain: MF106
1792
1792
97%
0.0
99%
Uncultured bacterium partial 16S rRNA gene, clone
16sps23-2f08.w2k
1788
1788
96%
0.0
99%
Pseudomonas aeruginosa strain NRRL B-14935 16S
ribosomal RNA gene, partial sequence
1788
1788
97%
0.0
99%
Pseudomonas sp. CIFE_HT1 16S ribosomal RNA
gene, partial sequence
1786
1786
97%
0.0
99%
Pseudomonas sp. LB-5 16S ribosomal RNA gene,
partial sequence
1784
1784
96%
0.0
99%
Table 1: Results from the BLAST database search of adjacent 16S rDNA sequence from the
unknown isolate from sample D on the 1 cm EMB plate (15).
Figure 1. Photomicrograph for the Gram stain of isolate from sample D on the 1 cm EMB plate
under oil immersion at 1000x magnification using Bright field microscopy
Figure 2. Photomicrograph for the Gram stain of isolate from sample B on the 1 cm PEA plate
under oil immersion at 1000x magnification using Bright field microscopy
Figure 3. Photomicrograph for the endospore stain of the isolate from sample B on the 1 cm
PEA plate under oil immersion at 1000x magnification using Bright field microscopy.
Comparison of the unknown sample
and Identified bacterium
Citrobacter Freundii
Unknown sample D on
1 cm EMB plate
Gram Reaction
Negative
Negative
Catalase Test
Positive
Positive
O/F Test
O/F, facultative
anaerobe
O/F, facultative
anaerobe
VP Test
Negative
Negative
Milk Agar
Negative
Negative
Starch Agar
Negative
Negative
Nutrient Gelatin
Negative
Negative
Nitrate Reduction
Positive
Positive
Oxidase
Negative
Negative
Citrate
Positive
Positive
Glucose Fermentation
Positive
Positive
Lactose Fermentation
Positive
Positive
H2S Production
Positive
Positive
Indole Production
Positive or Negative
Negative
Motility
motile
motile
Table 2: Biochemical Test results for Citrobacter freundii, derived from Bergey’s Manual of
Determinative Bacteriology (7).
Comparisons of the unknown
sample and the identified
bacterium
Bacillus cereus
Unknown sample B on 1 cm
PEA plate
Gram reaction
Positive
Positive
Endospore stain
Positive
Positive
Mannitol Fermentation
Negative
Negative
Oxidase
Positive
Positive
Catalase
Positive
Positive
Table 3: The biochemical test results Bacillus cereus and the order by which the Gram positive
tests were conducted was derived from Laboratory Exercises in Microbiology (8).
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