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Biotechnological potential of bacteria isolated from Greenlandic marine sediments
Biotechnological potential of bacteria
isolated from Greenlandic marine
sediments
SANDRA WINGAARD THRANE (S082403)
STUD. M.SC.ENG. BIOTECHNOLOGY
SUPERVISOR: PROFESSOR LONE GRAM
DTU FOOD, GROUP OF BACTERIAL ECOPHYSIOLOGY AND INDUSTRIAL BIOTECHNOLOGY
AND ARTEK CENTER FOR ARCTIC TECHNOLOGY
SPECIAL PROJECT: 15 ECTS
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Biotechnological potential of bacteria isolated from Greenlandic marine sediments
PREFACE
The present project is the final report for a 15 ECTS points special course performed as part of the
Arctic Technology field course offered by ARTEK Center for Arctic Technology. The project
consisted of the planning and execution of a 3 week field project in Sisimiut, Greenland, in august
2013 as well as the analysis of the gathered samples after returning to Denmark. The entire
project was supervised by Professor Lone Gram and the group of bacterial ecophysiology and
industrial biotechnology at DTU Food.
I would like to thank ARTEK Center for Arctic Technology for making a field project like this
possible. The chance to do field work and the planning of such an extensive project, gives a rare
insight into project management. I think it has been a great experience to be able to
independently, as a student, take a project from idea to finished report. Also I would like to thank
the group for bacterial ecophysiology and industrial biotechnology for help, guidance and good
company in the lab. Lastly I would like to extend a big thank you to Professor Lone Gram, for
helping me and giving invaluable guidance all the way to the finished report.
I hope you enjoy reading my project.
________________________________________________________________________________
Sandra Wingaard Thrane, 31/1-2013
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Biotechnological potential of bacteria isolated from Greenlandic marine sediments
INDEX
ABSTRACT
4
THE SEARCH FOR BIOACTIVE MARINE BACTERIA
5
THE DIVERSITY AND BIOTECHNOLOGICAL POTENTIAL OF ACTINOBACTERIA
6
Selecting for bioactive marine Actinobacteria
11
FUTURE POTENTIAL
12
EXPORT- AND RESEARCH LICENSE
12
MATERIALS AND METHOD
13
Sampling for gram positive marine bacteria (adapted from Jensen et al 2005)
Agar media used for plating samples
Gram reaction of isolated strains
Bioactivity of isolated strains
RESULTS
Characterization of isolated strains
Biotechnological potential of isolated strains
13
14
15
16
19
19
22
DISCUSSION AND FUTURE PERSPECTIVE
26
REFERENCES
30
APPENDIX 1: EXPORT PERMISSION
34
APPENDIX 2: SURVEY LICENSE
35
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Biotechnological potential of bacteria isolated from Greenlandic marine sediments
ABSTRACT
The need to find novel substances with potential to treat cancer, multiresistant microbial
infections and inflammatory diseases is more urgent than ever, why the focus on isolating
Actinobacteria, known for their ability to produce bioactive compounds, is increasing. Previously it
was generally accepted, that marine Actinobacteria did not exist, yet in recent years
Actinobacteria have been discovered with confirmed marine adaptation. The marine
Actinobacteria have shown to comprise a diverse group of slow growing, Gram positive bacteria,
with many different morphologies, making it difficult to select for only them during culture and
isolation. In the present study isolation methods for recovering marine Actinobacteria were tested
as part of a field project in Sisimiut, Greenland. Marine sediment from Ulkebugten was sampled
and heat-treated prior to inoculation and incubation. A total of 26 isolates were recovered, from 9
samples taken from 3 different locations in the bay. All isolates were analyzed for phenotypic
traits (Gram-test, microscopy) as well as production of bioactive compounds (degradation of chitin
and carrageenan, inhibition of Vibrio anguillarum and Staphylococcus aureus). Several isolates are
proposed to be Actinobacteria and several strains showed chitinolytic-, proteolytic- and/or
antibacterial bioactivity, implying that the tested isolation methods favored growth of marine
Actinobacteria and other bioactive bacteria.
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Biotechnological potential of bacteria isolated from Greenlandic marine sediments
THE SEARCH FOR BIOACTIVE MARINE BACTERIA
Defining the biotechnological potential of a niche, a bacterial species, a geographical area or
simply a project is impossible, since it is never possible to know what you are going to find. In
order to estimate biotechnological potential, we look at previous studies and experiences, that can
give indications of where to look, how to look or what to look for. In previous years, the
biotechnological potential of the marine environment has been the target of several studies, and
with the first products derived from marine microbes emerging, marine organisms are being
regarded as a potential source of many novel substances (Imhoff et al. 2011). Marine
macroorganisms were for many years considered to be the source of the isolated novel bioactive
metabolites, yet studies showed that the compounds in many cases came from the
microorganisms colonizing the macroorganisms, rather than the macroorganisms themselves
(Imhoff et al. 2011). Several studies have attempted to evaluate what the remaining potential of
discovering new marine microbes is, and it is estimated that we today know less than 0.1-0.01% of
the microorganisms in the ocean (Simon and Daniel 2009b).
Marine microorganisms are a relatively unknown area, which means that traditional culturing
techniques must be revised, in order to ensure that the marine bacteria can be recovered and
investigated in the laboratory. The knowledge of culturing techniques and media formulations for
isolation of marine bacteria is limited, and this is one of the reasons for the rumor of “marine
bacteria being difficult to cultivate” (Bhatnagar and Kim 2010). Several studies have shown that
marine bacteria can be cultivated successfully when the proper cultivation methods are applied
(Davidson 1995; Fenical 1993; Kobayashi and Ishibashi 1993; Okami 1993; Jensen et al. 2005).
Studies of the microbial biodiversity of the ocean, can indirectly favor growth of fast growing
bacteria, by use of non-selective media and standard incubation time. They therefore describe
Gram negative bacteria such as Vibrio, Pseudoalteromonas, Ruegeria and others, which are
abundant in the ocean and grow fast (Gram et al. 2010). On land, the Gram positive bacteria have
shown to be some of the most potent producers of novel bioactive compounds. These include soil
bacteria such as the Streptomyces, found in a variety of niches on land (Bérdy 2005). Therefore
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Biotechnological potential of bacteria isolated from Greenlandic marine sediments
looking for these slow growing Gram positive bacteria, and similar Actinobacteria, in the marine
environment, may uncover new strains and a whole new research area with biotechnological
potential.
THE DIVERSITY AND BIOTECHNOLOGICAL POTENTIAL OF ACTINOBACTERIA
Actinobacteria is the common name for the bacteria of the order Actinomycetales, known to be
potent producers of secondary metabolites with industrial applications (Bérdy 2005). They
comprise a diverse group of soil bacteria, forming filamentous colonies and showing mycelial
growth and dormant spore-formation, allowing spread of the bacterium to new locations
(McCormick and Flärdh 2012). Actinobacteria show a high degree of diversity shown by the wide
range of morphologically and developmentally different strains such as the simple coccoid cells of
the Micrococcus genus, the industrially important rod-shaped and pleiomorphic Corynebacterium,
pathogens such as the Mycobacterium and the characteristic mycelial growth of the filamentous
Streptomyces (McCormick and Flärdh 2012).
Actinobacterial strains isolated from the ocean have throughout time been thought to come from
dormant spores that had been washed into the sea from land, rather than from marine
Actinobacterial variants (Goodfellow and Haynes 1984). Yet studies have begun to investigate
whether, marine Actinobacteria occur naturally in the ocean, presenting a new niche of bacteria
with biotechnological potential (Jensen et al. 2005; Hames-Kocabas and Uzel 2012). The traditional
way of selecting Actinobacteria, do not favor growth of marine variants, and a new way of
selecting and thinking, is suggested to be needed to discover whether these bacteria can also
thrive in the marine environment, and produce unknown novel secondary metabolites, to be
useful in the pharmaceutical industry.
In 1988 two-thirds of all known naturally derived antibiotics were discovered from cultured
Actinobacteria (Okami and Hotta 1988) and Actinobacteria, and soil bacteria in general, have since
the discovery of penicillin in the 1920’s, been thought to be the largest source of new novel drugs
(Bhatnagar and Kim 2010). Bioactive compounds are secondary metabolites of microorganisms
that have an effect that we can utilize to our advantage. Actinobacteria account for around 45% of
the known microbial bioactive secondary metabolites, and their metabolites have many different
applications (Bérdy 2005). These applications range from different types of medicin, that can cure
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Biotechnological potential of bacteria isolated from Greenlandic marine sediments
certain physiological states (neuropsychiatric conditions, carcinomas, coronary heart disease) or
aid us when foreign matters or organisms are attacking our bodies (infections with one or more
bacteria, immunosuppressant’s for transplant patients), to simple compounds that can be used in
the industry to help us in our everyday lives (enzymes, food additives, nutraceuticals) (Bhatnagar
and Kim 2010). This has made bioactive compounds one of the main focus areas of the
pharmaceutical industry (Bhatnagar and Kim 2010).
A large group of the bioactive compounds isolated from Actinobacteria, comprise a range of
different antibiotics (figure 1). 28% of these antibiotics have been found to also have other
biologically useful activities, and around 14% of the isolated compounds have a biological activity
other than antibiotic (figure 1). In total 10.100 bioactive compounds had been isolated from
Actinobacteria in 2005 (figure 1) and it is estimated that the remaining potential within these
microorganisms is many times the size of this (Kurtböke 2012).
10000
8000
6000
4000
2000
0
Figure 1. Illustration of the known number of bioactive
compounds isolated from Actinobacteria and whether
they have antibiotic activity. Data from Bérdy 2005.
Many of the bioactive compounds isolated from Actinobacteria originate from strains of the
Streptomyces genus. The Streptomycetales are a group of Gram positive and GC-rich bacteria
belonging to the phylum of Actinobacteria, producing a wide range of industrially applicable
compounds. They have a very large genome which is meant to be part of the explanation for their
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Biotechnological potential of bacteria isolated from Greenlandic marine sediments
large secondary metabolite profile (Goodfellow et al. 1986). 80% of the known bioactive
compounds from Actinobacteria have been isolated from Streptomycete species, making up 7.600
different compounds (Goodfellow 2010). The Streptomycete species are also known for their
complex life cycle, involving multicellular behavior and intercellular communication which is neatly
coordinated with various physiological states (McCormick and Flärdh 2012). Streptomycete species
develop on laboratory agar plates over the course of a few days, where they form mycelium, to
explore the surrounding environment for nutrients, and excrete hydrolytic enzymes to degrade
and release the nutrients from the surrounding environment. Quickly the Streptomycete species
form multicellular communities, with many different cell-types, why they can behave very
differently depending on the conditions they are grown under (Chater 1998).
The Actinobacteria is a diverse group of bacteria, and models have shown that there may still be a
potential for discovering new bioactive compounds within the group. By 1994, 8000 antibiotics
had been identified, originating from Actinobacteria – 80% of these from Streptomyces species,
yet other genuses such as the Micromonospora are also being considered to have high potential as
novel producers of bioactive compounds. Watve et al. 2001 suggests that at least 150.000
bioactive compounds are still to be discovered from the Streptomyces genus alone. They think the
decline in new compounds that have been discovered is due to a lack in proper screening
methods, both when isolating bacteria and when purifying and characterizing new compounds,
rather than a lack of new compounds to be discovered (Baltz 2005). This knowledge in relation to
the fact that it is estimated, on the basis of 16S rRNA sequence data, that 99% of the
microorganisms in the environment are unculturable or simply uncultured, show the potential of
this research area (Bull et al. 2000; Bull 2004). The same was concluded by Hames-Kocabas and
Uzel 2012 who after studying isolation strategies for marine-derived Actinobacteria, conclude that
you must supply nutrients that mimic the natural environment, meaning that they are present in
low concentrations and can sustain growth. They also conclude that long incubation times are
crucial, to allow slow-growing strains to grow as well as the aspect of sampling geographically
diverse and special areas, in order to isolate niche-strains, with yet undiscovered abilities. Only by
taking these principles into consideration can new Actinobacteria strains be isolated. And as it is
concluded by Zengler et al. 2002: “There is an urgent need for the development and application of
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Biotechnological potential of bacteria isolated from Greenlandic marine sediments
new strategies for the detection, isolation, dereplication and subsequent description of novel
organisms, including actinomycetes, from natural habitats”.
Even though focus on the area is increasing, marine microorganisms and their bioactive products
have not been given very much attention through the years, and to date the literature on the
subject is limited (Bhatnagar and Kim 2010). Especially marine Actinobacteria is a relatively
unknown area, due to the fact, that they for many years were considered to come from spores
from land that were led into the ocean by outside factors, such as wind and machines, rather than
be exclusively marine derived strains (Goodfellow and Haynes 1984). Later it has been shown that
strains of marine Actinobacteria exist, by isolation of the bacteria from the ocean, and recultivation of the bacteria with and without seawater, to show their need for seawater to grow
and thereby their marine adaptation (Jensen et al. 2005). Jensen et al. 2005 found, that by
sampling seawater and sediment, they could isolate five new marine phylotypes, ranging from
Salinispora to Streptomyces, showing existence of diverse populations of pylogenetically distinct
Actinobacteria of marine origin, all with marine adaptation. It can be hypothesized that the great
variation within marine Actinobacteria strains, is an indication of the diversity in the marine
environment, and may represent many unknown niches with bioactive potential. To uncover
these, more knowledge of the mechanism of action of the bioactive compounds, understanding of
the microbial interactions in the marine environment and appropriate assays and selection
methods are needed (Bhatnagar and Kim 2010).
From the known isolated marine Actinobacteria, bioactive compounds are already being isolated.
Examples of some of these, as well as their structure and diverse biological activities are shown in
table 1.
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Biotechnological potential of bacteria isolated from Greenlandic marine sediments
Table 1. Selected marine Actinobacteria and their bioactive secondary metabolites, with terms to name, structure,
bioactivity and reference. The table is adapted from Bhatnagar and Kim 2010.
Bacterium
Compound
Streptomyces
Resistoflavine
Structure
Biological activity
Litterature
Anticancerous and
Gorajana et al. 2007
antibacterial
Marinispora
Marinomycin A
Antitumor and
Kwon et al. 2006
antibiotic
Streptomyces
Daryamide C
Antitumor
Asolkar et al. 2006
Chromobacterium
Violacein
Antiprotozoal
Matz et al. 2008
Bioactive compounds from marine Actinobacteria range from antibiotics, to antitumor and
antiprotozoal drugs (table 1). They also vary greatly in structure, from small aromatic compounds,
to larger more complex molecules (table 1). Several Actinobacteria strains have been found to
produce these compounds, and it is believed to be a trait in common amongst Actinobacteria
(Baltz 2005).
An example is the marine derived actinomycete Nocardiopsis lucentensis (strain CNR.712). Cho et
al. 2007 performed a study, where they looked for antitumor compounds from marine
actinomycetes. They successfully isolated four new peptides, Lucentamycins A-D (figure 2), and
when they were tested in vitro for cytotoxicity against HCT-116 human colon carcinoma cells, two
of the four were positive. In a similar study performed by Hawas et al. 2009 a total of 13
compounds were isolated including isoquinoline quinines and Mansouramycin A-D (figure 2), all by
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Biotechnological potential of bacteria isolated from Greenlandic marine sediments
making a ethyl acetate extract of a marine variant of the Streptomyces sp. (Strain Mei37). All the
isolated compounds were tested against a range of tumor cells, and all showed cytotoxicity against
these. These studies demonstrate the abundance of bioactive compounds coming from
Actinobacteria, and show that when the search for these strains and their metabolites is done in a
structured manner, the success rate is high.
Figure 2. The chemical structure of two marine derived bioactive compounds (Bhatnagar and Kim 2010)
SELECTING FOR BIOACT IVE MARINE ACTINOBACTERIA
The increasing attention on marine Actinobacteria is resulting in a better understanding of these
organisms, and their characteristics and what distinguishes them from the land-based
Actinobacteria. They are found in many niches such as soil, sludge and water. Especially
Streptomyces sp. is known for their ability to degrade a range of types of organic matter, and to be
abundant in many locations (Blunt et al. 2004; Burja et al. 2001; Fuesetani 2000). Several of the
marine strains isolated, have belonged to already known genuses such as the Streptomyces genus,
yet previously unknown genuses have also been isolated from the marine environment. These
include Salinispora, Salinibacterium, Sciscionella, Serinicoccus and Marinactinospora (Fenical and
Jensen 2006; Han et al. 2003; Maldonado et al. 2005; Tian et al. 2009a; Yi et al. 2004; Tian et al.
2009b). The marine Actinobacteria species have in common with known Actinobacteria from land,
that they are potent producers of bioactive compounds, including a range of antibiotics
(Bhatnagar and Kim 2010).
In the present study the waters surrounding the Greenlandic city of Sisimiut was sampled, in order
to examine sediment that had not otherwise been investigated for marine Actinobacteria. Hereby
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Biotechnological potential of bacteria isolated from Greenlandic marine sediments
it was tested whether marine Actinobacteria can also be found in arctic waters, and if they are
similar to the strains found on other geographical locations, or if they present a completely new
niche of Actinobacteria.
FUTURE POTENTIAL
This study presents information about how a field project can be planned, executed and
presented, and serves as a proof of concept, for a new way of selecting for specific bacteria. The
results serve as means to evaluate the tested methods and can be of importance to the
microbiological knowledge of arctic regions and their diversity, meaning that otherwise
undiscovered areas can help us learn more about the microbial diversity in different regions of the
world. Also as described, Actinobacteria are a diverse group of bacteria that produce a wide range
of bioactive compounds. The discovery of new compounds, can be of great importance to the
medical and pharmaceutical industries, and may help cure certain diseases in the future. If such
new compounds are identified it will be of importance to the Greenlandic home rule, not only
because of a financial outcome, but also to highlight the diversity and unknown territory found in
and around Greenland. This may lead to additional projects in the area, and focus from the
microbiological society on the arctic area in general.
EXPORT- AND RESEARCH LICENSE
Prior to the field work, export- and research licenses were acquired from correspondence with the
Greenlandic home rule. This was done in order to get permission for sampling in Sisimiut,
Greenland, as well as to clarify for how long we could work with the samples and what any
potential new discoveries would mean, with respect to profits. Per Aksel Petersen
([email protected]) from the Ministry of Domestic Affairs, Nature and Environment was the contact
person, and he supplied an “Application form for survey licence for collection and/ or acquisition of
biological resources for research purpose”. This granted permission to take 20 samples of 50 mL
volume, as well as to work with them, after the return to Denmark, until May 30 th 2013. At the
same time an export-permission was acquired, for transporting the samples back to Denmark.
The export permission and research permission can be seen in Appendix 1 and 2.
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Biotechnological potential of bacteria isolated from Greenlandic marine sediments
MATERIALS AND METHOD
During this project, samples of sediment from the coast around the Greenlandic city, Sisimiut,
were taken by boat. The sediment was plated on agar plates in the lab in Greenland, and shipped
to Denmark, where strains were isolated and further characterized and tested. The isolation
methods used in this project, were tested prior to departure to Greenland on sediment collected
by the Danish coast around Bellevue in Charlottenlund and on plain soil samples collected on DTU
campus as a positive control.
SAMPLING FOR GRAM POSITIVE MARINE BACTERIA (ADAPTED FROM JENSEN ET AL 2005)
Samples of water and sediment from the seafloor off the coast of Sisimiut were taken from a boat
from three different locations:
The sediment was collected in clean buckets with lids and marked with location and sample name.
Samples of sediment from the seafloor were collected and marked as being sediment with mostly
sand-sediment (G) or mostly mud-sediment (D).
20 g of the wet sediment was weighed in a 50 mL falcon tube and diluted/dissolved in 20 mL of
sterile 0,9% Sigma sea salts (S9883, Lot: 120M0011V) solution. The samples were then further
processed by two different methods and characterized by a range of tests and analysis.
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Biotechnological potential of bacteria isolated from Greenlandic marine sediments
Using method 1, samples were serially diluted in 0,9% Sigma sea salt solution to 10-7. The diluted
samples were vortexed and allowed to settle for a few minutes in order for the sediment to form a
pellet, and the spores and bacteria to still remain in the water phase. 100 µL of each of the
dilutions (10-1, 10-2, 10-3, 10-4, 10-5, 10-6 and 10-7) were surface spread on agar plates (see next
section). The plates were incubated 1 months at room temperature and periodically examined for
Actinobacteria colonies (especially looking for mycelium growth and small rubbery colonies).
Using method 2, the samples were treated as above and after dilution the samples were heated to
55oC for 6 min. by placing them in an incubator in a water bath (to kill most heterotrophic bacteria
and leave Actinobacteria spores). The diluted samples were vortexed and allowed to settle for a
few minutes, to let the sediment form a pellet, and the spores and bacteria still remain in the
water phase. 100 µL of each of the resulting dilutions (10-1, 10-2, 10-3, 10-4, 10-5, 10-6 and 10-7) was
surface spread on agar plates (see next section). The plates were incubated 1 month at room
temperature and periodically examined for Actinobacteria growth (especially looking for mycelium
growth and small rubbery colonies).
AGAR MEDIA USED FOR PLATING SAMPLES
Samples were plated on two different agar-based media, both originally designed for the selection
of Actinobacteria (Jensen et al. 2005). One was a rich medium (AMM) and the other a minimal
medium (SRC), in order to isolate and select for a wider range of different Actinobacteria. Growth
was observed on the rich medium (AMM) after only a couple of days growth at room temperature.
The minimal plates (SRC) were shipped home in a container, and examined after return to
Denmark (approximately one month after the plates were inoculated) and only very little growth
was observed.
The AMM plates were made by mixing 1,8% agar (AppliChem, A7254.1000), 1% starch (from
potato, Sigma S-2004, Lot: 108711313), 0,4% BactoTM Yeast Extract (REF 212750), 0,2% BactoTM
Peptone (REF 211677) and 0,9% Sigma sea salts. All ingredients were dissolved in dH2O. If needed
the solution was heated for all additives to dissolve before autoclaving at 121oC for 15 min.
The agar was prepared in Denmark, shipped to Greenland in the bottles, and in Greenland the
agar was boiled until it melted. After melting the agar was cooled to approx. 46oC and 100 µg/mL
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Biotechnological potential of bacteria isolated from Greenlandic marine sediments
cycloheximide (diluted in ethanol) was added to inhibit growth of fungi. The agar was then plated:
approx. 20 mL pr. petridish and stored in the refrigerator (5oC) until use.
The SRC plates were made by mixing 1% agar and 0,9% Sigma sea salts that were dissolved in
dH2O. If needed the solution was heated for all additives to dissolve. The solution was transferred
to blue cap bottles and autoclaved.
The agar was shipped to Greenland in the bottles, and when in Greenland the agar was boiled
until it melted. After melting the agar was cooled to approx. 46oC and 100 µg/mL cycloheximide
(diluted in ethanol) and 5 µg/mL rifampicin (diluted in DMSO) was added to inhibit growth of fungi
and heterotrophic bacteria. The agar was then plated: approx. 20 mL pr. petridish and stored in
the refrigerator (5oC) until use.
GRAM REACTION OF ISOLATED STRAINS
all strains were streaked onto AMM agar and incubated at 25oC for 3 days. A single colony was
picked and dissolved in one drop of 3% KOH solution on a glass slide. The loop was used to mix the
colony thoroughly with the KOH, and it was then lifted gently from the glass surface. When strings
were observed from the liquid to the loop the isolate in question was Gram negative. If no strings
were observed the isolate was Gram-positive. Vibrio anguillarum strain 90-11-287 (Skov et al,
1995) was used a positive control (Gram-negative), sterile dH2O as a negative control.
It is noted that several isolates formed very dense rubbery colonies, which would not dissolve in
the KOH, and it is hypothesized that this could impact the result of the gram test.
The Gram reaction was also tested using the Microbiology Bactident® Aminopeptidase (MBA)
(MERCK 1.13301.0001) sticks.
All strains were incubated 2 days in liquid AMM (as AMM plates, but without addition of agar)
while shaken at 200 rpm at 20oC. 1 mL culture was transferred to a sterile 1,5 mL eppendorf tube
and spun 1 min. at 10000 g. The supernatant was removed and the bacterial cell pellet redissolved
in sterilized dH2O. 20 uL of the resulting bacterial suspension was further diluted in 180 uL
sterilized dH2O in clean durrham glass tubes (small). A MBA stick was placed in each tube, the pad
exactly covered by the suspension. The sticks were then incubated for 10 min. at 37 oC followed by
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Biotechnological potential of bacteria isolated from Greenlandic marine sediments
placement at room temperature for 30 minutes to rest. The result was then recorded. If yellow
coloration of pad and liquid was observed to any extend, the tested strain was Gram negative.
Only if no coloration was observed was the sample Gram positive.
Vibrio anguillarum strain 90-11-287 was used as positive (Gram-negative) control, sterilized dH2O
as a negative control.
BIOACTIVITY OF ISOLATED STRAINS
All strains were examined for their “biotechnological potential”, by testing for production of a
range of different bioactive compounds in agar-based assays. Strains were both tested for
degradation of known compounds, and the hereby resulting enzymes, as well as tested for
inhibition of other strains, to test for production of bacteriostatic or bacteriocidal compounds.
Chitinolytic activity: Chitin is one of the major components of the shell of shrimp and other
crustaceans (Brück et al. 2011) and bacteria or bacterially derived enzymes, that are able to
degrade chitin, can play an important role in the exploitation of waste from the seafood industry.
All strains were tested for degradation of chitin, by use of chitin plates. Chitin was prepared from
crab shells (practical grade, Sigma C7170) by the method previously described by Wietz, (2011).
Ten g of chitin was hydrolyzed in ice-cold 37% HCL, in a glas beaker, until it started to solubilize.
Then it was diluted with 5 L of dH2O and placed at 4oC overnight for the chitin to settle at the
bottom of the beeker. The supernatant, or water phase, was then removed, and the chitin was
redissolved in 5 L of dH2O. This was done 4-5 times, to lower pH of the solution. Finally the water
phase was removed, leaving a final volume of approximately 900 mL. The pH was adjusted to
approximately 7 using KOH pellets and 2M KOH to fine adjust. The chitin solution was then
transferred to 200 mL Schott-bottles and autoclaved. The chitin concentration, in the solution, was
determined by drying 10 mL of the chitin solution overnight at 70oC. The chitin concentration was
1,4% in the chitin solution used in this study.
The chitin plates were prepared by mixing 0,05% BactoTM Peptone, 0,01% BactoTM Yeast Extract,
0,001% ironphosphtaetetrahydrate, 1,5% agar and 4% Sigma sea salt in dH2O. The solution was
autoclaved, and after cooling to 45oC, 150 mL/L 1,4% chitin solution was added. The agar was
poured, and the plates dried before use.
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Biotechnological potential of bacteria isolated from Greenlandic marine sediments
All strains were grown on AMM plates and re-streaked onto the chitin plate (in lines). The plates
were incubated at 20oC for 7 days, and inspected for clearing zones, where the chitin in the plate
was degraded. Vibrio anguillarum strain 90-11-287 was used as positive control.
Carrageenolytic activity: Carrageenan is a component of red seaweed, and is a compound of
biotechnological relevance as the possibility of utilizing seaweed as biomass for biofuel
production, has become an area of research. Therefore bacteria able to degrade carrageenan, or
enzymes derived from these, are important for future progress in the field.
Carrageenan plates were used for screening for strains for the ability to degrade carrageenan. The
plates were prepared by dissolving 37.4 g/L Difco Marine Broth 2216 in dH2O. The media was
boiled for 2 minutes. 20 g of carrageenan (Fluka 22048) was kept in a bottle, and stirred with a
magnet. The medium was poured into the carrageenan and the medium was autoclaved. The
medium was cooled to 70oC and the plates were poured. The plates were set to dry for one hour,
before being used .
All strains were grown on AMM plates and re-streaked onto the carrageenan plate (in spots). The
plates were incubated at 20oC for 7 days, and inspected for strains degrading the carrageenan by
looking for formation of shallow holes in the plate. Pseudoalteromonas carrageenovora strain
DSM 6820 (Akagawa-Matsushita et al, 1992) was used as a positive control, Vibrio anguillarum
strain 90-11-287 was used as negative control.
Proteolytic activity: Proteases are enzymes that are already used commercially in detergents and
food processing. Therefore proteolytic bacteria may present a source of new proteases applicable
in biotechnological industry.
All strains were tested for proteolytic activity, by use of skim milk plates. The plates were made by
mixing 10% DifcoTM Skim Milk (REF 232100), 2% Sigma sea salt, 0,33% BactoTM Casaminoacids (REF
223050) and 1% agar. All components were dissolved in dH2O and autoclaved. The agar was then
cooled to 45oC and poured into plates. All strains were grown on AMM plates and re-streaked
onto the skim milk plates (in lines).
The plates were incubated at 25oC for 7 days, and inspected for clearing zones, where the protein
particles in the plates were degraded.
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Biotechnological potential of bacteria isolated from Greenlandic marine sediments
Antibacterial activity: To test the isolated strains for production of either bacteriostatic or
bacteriocidal compounds, two different agar-based well diffusion assays were performed. All
isolated strains were tested for inhibition of Vibrio anguillarum strain 90-11-287 and
Staphylococcus aureus strain NCTC 8325 (Novick, 1967).
3% Instant Ocean® salts, 0,3% w/V BactoTM Casaminoacids and 1% agar in dH2O were mixed and
autoclaved. The agar was cooled to 45oC and 2% of 20% w/V filter-sterilized glucose solution was
added. Following this 5 uL of culture of either Vibrio anguillarum (grown in MB for 2 days at 20oC)
strain 90-11-287 or Staphylococcus aureus (grown in BHI for 2 days at 20oC) strain NCTC 8325 was
added to the agar, and it was quickly poured into plates.
After the plates had set, 0,9 mm wells were made and a 4 day culture of the isolated strains grown
in liquid AMM was used, when adding 20 uL of each strain to the wells in the plate. The strain in
the plate itself, was added to a well as a negative control.
The plates were incubated at 20oC and observed for lack of growth of the pathogen in the plate,
seen as inhibition zones around the wells after 3 days and 7 days. The inhibition zones were
measured and recorded.
Page 18 of 38
Biotechnological potential of bacteria isolated from Greenlandic marine sediments
RESULTS
During this study three locations by the coast of Sisimiut were sampled in triplicate for sea
sediment of a muddy or sandy character, in order to isolate Gram positive spore forming bacteria.
From a total of 18 samples, 26 different isolates were cultivated in the lab to a pure culture, and
further analyzed for phenotypic traits and bioactivity profile. 22 of the isolates were recovered
from a rich medium AMM, and were isolated in Greenland, where samples had been heat-treated
prior to inoculation. Four isolates were recovered from a minimal medium, SRC, which had been
transported from Sisimiut to Denmark by container, and after one month isolated in a laboratory
in Denmark. Both media were designed to isolate preferably Gram positive spore forming bacteria,
and the results will work as a proof-of-concept of a low labour way of isolating selected types of
bacteria with biotechnological potential, from remote locations.
CHARACTERIZATION OF ISOLATED STRAINS
Colonies appeared on the AMM (rich) medium in Greenland after 2-3 days incubation at room
temperature. Sediment samples had prior to inoculation been heat-treated and serially diluted.
Colonies varied from small round and shiny colonies that were either white/grey/beige or
yellow/orange in color to rough rubbery colonies, with clear structures and folds in the surface
also white/grey/beige or yellow/orange in color. Other morphologies were observed, but these
were the predominant ones.
Through serial dilution it was attempted to determine the CFU of the sediment, yet due to
particles with higher CFU it was not possible to quantify the CFU in the sediment. It was observed
that the CFU varied a lot, and that there were no samples with CFU below 10 -3 CFU/mL.
All strains were re-streaked on AMM, and the plates were packed in parafilm and plastic bags, and
brought bag to Denmark as a part of my luggage. Here all strains were re-streaked again 2-3 times,
to make sure, that all strains were pure.
After approximately one month the SRC (minimal) plates arrived in Denmark, after having been
shipped in a container from Sisimiut. The plates were examined, and colonies were re-streaked
both onto AMM, SRC and MB. After approx. one week, colonies appeared on the AMM plates.
They were clearly filamentous in appearance and resembled fungi on the agar plate.
Page 19 of 38
Biotechnological potential of bacteria isolated from Greenlandic marine sediments
The 22 strains were tested for Gram-reaction and their morphology observed in the microscope
using 1000 x magnification. Based on the KOH-test, 21 of the 26 isolates were classified as Gram
positive, however using diaminopeptidase-strips, only 7 isolates were Gram-positive (Table 2).
Table 2. Characterization of isolated strains
Isolated Sampling
from
place
Sand
AMM
2
3
Sand
SRC
1
Mud
AMM
1
2
3
Mud
SRC
1
Name
KOH
MBA
stick
Morphology in microscope (x1000)
G2.1a
G+
G-
Rod shaped bacteria with visible spores
G2.3a
G+
G+
Tetracocci bacteria
G2.3b
G+
G-
Tetracocci bacteria and formation of clusters
G2.3c
G+
G-
Diplococcic bacteria
G2.3d
G+
ND
Tetracocci bacteria and formation of clusters
G2.3e
G+
G-
Tetracocci bacteria and formation of clusters
G3.3a
G+
G+
Coccobacilli bacteria with visible spores
G3.3b
G+
G-
Cocci (some diplococcic) bacteria
G3.3c
G+
G-
Tetracocci bacteria and formation of clusters
G1.1m
ND
ND
-
G1.3m
ND
ND
-
D1.1a
G+
G-
Rod shaped bacteria with visible spores
D1.1b
G+
G-
Rod shaped bacteria with visible spores
D1.1c
G+
G-
Rod shaped bacteria with visible spores
D1.2a
G+
G-
Tetracocci bacteria
D1.2b
G+
G+
Streptococci bacteria
D1.2c
G+
G+
Filamentous growth, with branching structures and mycelium
formation
D2.3a
G+
G+
-
D2.3b
G+
G-
Tetracocci bacteria
D2.3c
G+
G-
Rod shaped bacteria with some chain formation
D2.3d
G+
G+
-
D2.3e
G-
G-
Cocci bacteria
D3.1a
G+
G-
Tetracocci bacteria and formation of clusters
D3.1b
G+
G+
Tetracocci bacteria and formation of clusters
D1.2am
ND
ND
-
D1.2bm
ND
ND
-
All strains were examined at 1000x magnification under a microscope. The bacteria were taken
from a liquid culture onto a glasslide. Some strains were very similar when examined, yet a variety
of morphologies was observed (Table 2).
Strains G2.3b, G2.3d, G2.3e, G3.3.c, D1.2a, D2.3b, D3.1a and D3.1b all developed tetrahedral
structures comprised of cocci cells, and in several isolates also octahedral structures were
observed (figure 3). The structures formed clusters, and there were no planktonic cell growth in
Page 20 of 38
Biotechnological potential of bacteria isolated from Greenlandic marine sediments
the samples (figure 3). Cell size varied slightly, but all these isolates seemed to be related in
structure, and cell-arrangement.
Strain D1.2c as the only strain showed branching and mycelia growth (figure 3). The strain had
white filamentous morphology on an agar plate, and grew in cell pellets, when in a liquid medium.
Figure 3. Microscopy of strain D3.1a (left) and D1.2c (right) with 1000x magnification.
Other morphologies included streptococci, diplococcic and a variety of rod shaped bacteria and to
a large extend either planktonic og diploid growth was observed. Strain G2.3c, G3.3b and D2.3b all
showed cocci and diplocci growth (figure 4 (left)). The remaining strains all comprised a variety of
rod shaped bacteria, many with visible spore formation (figure 4 (right)).
Figure 4. Microscopy of strain G3.3b (left) and G3.3a (right) with 100x magnification.
For strains G1.1m, G1.3m, D2.3a, D2.3d, D1.2am and D1.2bm no one morphology was observed,
and nothing is therefore recorded in table 2. All the m-strains were isolated from the SCR agar,
Page 21 of 38
Biotechnological potential of bacteria isolated from Greenlandic marine sediments
had clear filamentous growth on agar plates, and formed pellets when grown in liquid medium.
These were hypothesized to be fungi, which could explain the difficulty of characterizing their cell
morphology. Strains D2.3a and D2.3d, had yellow rubbery colonies when grown on agar plates,
but were not filamentous. They can both be seen at 1000x magnification in figure 5, and both
samples could be hypothesized to show pieces of hyphae and conidiophores, yet this would not
correspond with their appearance when grown on a solid medium.
Figure 5. Examples of the strains, where it was not possible to get a proper sample, for examination under
microscope. Strain D2.3a (left) and D2.3d (right) with 1000x magnification.
After consulting with Professor Jens Christian Frishvad from DTU Systems Biologys Center for
Microbial Biotechnology, who is an expert in characterizing filamentous fungi, all isolates
recovered from the SRC (minimal) medium was identified as filamentous fungi. Therefore no
bacterial isolates were recovered using this isolation method.
BIOTECHNOLOGICAL POTENTIAL OF ISOLATED STRAINS
To investigate the biotechnological potential and frequency of bioactivity, all isolated strains were
tested for degradation of a range of compounds and inhibition of other bacterial strains. All these
tests were performed using agar-based assays and were only used to determine if they had
bioactivity, not the efficiency of the bioactivity (Table 3) .
Page 22 of 38
Biotechnological potential of bacteria isolated from Greenlandic marine sediments
Table 3. Bioactive profiles of isolated strains
Isolated
from
Sampling
place
SAND
AMM
2
3
SAND
SRC
1
Mud
AMM
1
2
3
Mud
SRC
1
Name
Vibrio**
Staph**
Chitin***
Protein***
Carrageenan*
+
-
-
-
-
-
-
+
-
-
-
-
-
+
-
-
-
-
-
-
-
-
-
-
-
-
-
-
G2.3e
-
-
-
-
-
+
-
3 days
5 days
3 days
5 days
G2.1a
-
-
+
G2.3a
-
-
G2.3b
-
G2.3c
-
G2.3d
G3.3a
-
-
-
-
-
-
-
G3.3b
-
-
-
-
-
-
-
G3.3c
-
-
-
-
-
+
-
G1.1m
-
-
-
-
-
-
-
G1.3m
-
-
-
-
-
-
-
D1.1a
(+)
+
-
-
-
-
-
D1.1b
+
+
-
-
-
-
-
D1.1c
-
-
-
-
-
-
-
D1.2a
-
-
-
-
-
+
-
D1.2b
-
-
-
-
-
-
-
D1.2c
-
-
-
-
+
(+)
-
D2.3a
-
-
-
-
-
-
-
D2.3b
-
-
-
-
-
+
-
D2.3c
-
-
-
-
-
-
-
D2.3d
-
-
-
-
-
-
-
D2.3e
-
-
-
-
-
(+)
-
-
-
D3.1a
-
-
-
-
-
D3.1b
-
-
-
-
-
D1.2am
-
-
-
-
-
+
-
D1.2bm
-
-
-
-
-
-
-
-
*Degradation of carrageenan was detected by looking for a shallow hole in the plate. +: hole observed, -: no hole
**Inhibition was measured as an inhibition zone. +: zone>1cm in diameter, (+): zone<1cm in diameter, -: no zone
***Degradation of chitin or protein was measured as the clearing zone around the bacteria on the plate.
+: visible clearing zone, (+):intermediate, -: no visible clearing zone
Strains D1.1a and D1.1b inhibited Vibrio anguillarum in a well diffusion assay (table 3 and figure 6
(left)) yet neither of these strains inhibited growth of Staphylococcus aureus when tested in an
assay of the same type. Only one strain, G2.1a, inhibited growth of Staphylococcus aureus (table 3
and figure 6 (right)). Increased growth of Staphylococcus aureus was observed around the wells in
the plate (figure 6 (right)), yet since this was also seen in the negative control containing only
sterile medium, it is hypothesized to be due to the medium rather than the bacteria.
Page 23 of 38
Biotechnological potential of bacteria isolated from Greenlandic marine sediments
Figure 6. Examples of the agar plates containing Vibrio aguillarum (left) or Staphylococcus aureus (right) in the
medium. Strains were added to the wells, and inhibition zones of the background was recorded, see table 3.
Degradation of carrageenan, chitin and protein were tested by use of carrageenan, chitin and skim
milk plates, respectively. No strains were found to be carrageenolytic (table 3). The ability of the
bacteria to degrade the chitin/protein was seen by a clearing zone around the well in the plate. In
table 3 the degradation is recorded either as negative (-) where no zone was observed,
intermediate ((+)) where the clearing zone was hard to distinguish or positive (+) with a visible
clearing zone (figure 7).
Figure 7. Examples of the skim milk (left) and chitin (right) plates. Strains to be tested were streaked onto the agar,
and the plate was observed for clearing zones. Results can be seen in table 3.
Of the 26 isolated strains seven (D1.2am, D2.3b, D1.2a, G3.3c, G2.3a, G2.3b and G2.3e) were
proteolytic, and able to degrade protein in an agar-based experiment, two additional strains
Page 24 of 38
Biotechnological potential of bacteria isolated from Greenlandic marine sediments
(D2.3e and D1.2c) were found to be intermediate proteolytic. Considering the number of isolated
strains, and the occurrence of proteolytic activity, it could be hypopethized that proteolytic
activity is a trait either selected for with the applied selection method or a trait selected for in the
environment, from where the samples were taken. Only a single strain, D1.2c, was found to have
chitinolytic activity (table 3, figure 7 (right)).
Out of the 26 isolated strains, a final of 22 were found to be bacterial strains, and therefore
relevant for the results in the present study. The SRC medium seemed to be unfit for isolation of
bacteria, even though it was successful when tested on sea-sediment from Denmark. This might
be due to the composition of the sediment in Greenland or the prevalence of the target bacteria
at the sample location. Of the 22 bacterial strains, 7 were Gram positive strains and these were
evenly obtained from all three locations. Equally, proteolytic activity was found from strains from
all three locations, whereas bacteriostatic/-cidal activity was only found at location 1 and 2, and
only single chitinolytic isolate was recovered from location 1.
Page 25 of 38
Biotechnological potential of bacteria isolated from Greenlandic marine sediments
DISCUSSION AND FUTURE PERSPECTIVE
In the present study cultivation methods to isolate marine Actinobacteria from sea sediments
were investigated, as a part of a field project in Greenland. A range of different bacteria (and
fungi) were successfully isolated and further studied in the laboratory. Several isolates were found
to be Gram positive, spore-forming and produced bioactive compounds; mainly hydrolytic
enzymes degrading proteins, but also single isolates with chitinolytic or bacteriocidal activity. With
the limited time frame, and relatively few samples, it is suggested that the media and cultivation
methods used favored growth of Actinobacteria (and close relatives) with biotechnological
potential.
Several different colony morphologies were observed after isolation and investigation of the
bacterial strains. It is proposed that isolate D1.2C, which showed filamentous growth, mycelium
formation and produced bioactive compounds, is a member of the Streptomycetaceae family.
Other marine Streptomyces strains have previously been isolated (Hawas et al. 2009), yet
investigation of the marine adaptation of the isolate is needed in order to confirm that it is not a
soil contaminant from land. Several isolates formed clusters of 4-8 coccoid cells, and considering
their appearance under a microscope and where they were isolated are potentially Actinobacteria
of the species Micrococcus luteus. For several isolates, microscopy was not possible, and these
strains should be further investigated by 16S rRNA sequencing.
In the present study the bacteria were identified by means of phenotypic tests (microscopy, Gramreaction, colony morphology) as is the case in other studies of the diversity of Actinobacteria in
sea sediments (Becerril-Espinosa et al. 2012, Jensen et al. 2005). In these studies, 16S rRNA gene
sequence analysis was used to confirm the phenotypic identification, and to allow species level
identification. This gives a higher degree of certainty, and allows for more extensive community
studies, and phylogenetical analysis. With prices of sequencing going down, it is possible that
whole genome sequencing will play a role in studies like this in the future. Alternatively other tests
have been developed for the characterization of Actinobacteria. These include analysis of fatty
acid methyl esters, fatty acid composition by gas chromatography, pyrolysis mass spectrometry of
cells and other sequence and PCR based methods (Zhao et al. 2004).
Page 26 of 38
Biotechnological potential of bacteria isolated from Greenlandic marine sediments
Maldonado et al. 2005 used 16S rRNA gene sequence analysis for community DNA analyses, prior
to medium- and pre-treatment selection. By sampling the marine sediment and using sequence
tools to analyze “who’s there”, narrow selective media were subsequently used together with
specialized pre-treatments in order to sustain growth of the Actinobacteria species found on the
given location. For Maldonado et al. 2005 the selection method was directed towards the genera
Amycolatopsis
and
Pseudonocardia
and
to
the
families
Streptomycetaceae
and
Thermomonosporaceae. They consequently isolated over 800 actinomycetes, using a total of 6
different media (Maldonado et al. 2005). Hereby few false positives were obtained, and the
success rate was higher. In the present project the time frame did not allow such a pre-study, yet
it should be considered for future studies.
The results of the Gram-reaction using KOH performed in this study did not show the same result
as the aminopeptidase sticks. It is assumed that the aminopeptidase sticks give a more accurate
result, due to industrial standardization and that their procedure was attempted adapted to the
rough morphology of the bacterial colonies on agar plates. Taking this into consideration it is
noted, that several Gram negative strains were isolated, showing that the selective medium could
not completely rule out growth of bacteria other than Actinobacteria. This should not simply be
regarded as negative, since Gram negative which survive on the selective media after the pretreatment, could also produce bioactive compounds and are therefore relevant to include in the
analysis of the biotechnological potential of the sampled location. With this mentioned, a possible
way of specializing the isolation method, and ruling out more of the heterotrophic bacteria, could
be to use a higher temperature in the pre-treatment of the sediment. In this study the sediment
was inactivated at 55oC, in order to kill heterotrophic bacteria, and leave bacterial spores, which
could then germinate on the agar plates. This temperature may have been too low, allowing for
survival of some heterotrophic bacteria. With this in mind, the temperature could be raised to
80oC, since this would kill off heterotrophic bacteria more efficiently and still leave the spores in
the sediment.
The minimal medium (SRC) used in this study, was derived from a medium first described by
Jensen et al. 2005, and consisted of agar, antibiotic and fungicide. In this study the very minimal
conditions and long incubation time, only resulted in isolation of fungi, and no Actinobacteria.
Page 27 of 38
Biotechnological potential of bacteria isolated from Greenlandic marine sediments
Considering this, the applied fungicide may need to be revised, in order for the medium to work
more efficiently. A future study may supplement the medium with several fungicides, in order to
better prevent growth of all types of filamentous fungi. Since Greenlandic sediments have not
previously been screened by this method, another possibility is that growth of Actinobacteria
found in this location, was not supported by the very minimal medium. The rich medium (AMM)
resulted in isolation of a range of different bacterial strains, some of which are proposed to be
Actinobacteria, which indicates that the SRC medium should be completely revised in the given
context.
Another aspect which could be looked into is incubation times. The minimal plates used in this
study were incubated approximately four weeks. This was chosen since similar studies had found
that most Actinobacteria colonies from sediment samples were visible after approximately one
month incubation (Becerril-Espinosa et al. 2012, Jensen et al. 2005). Yet the same studies report
that new colonies were observed up until 12 weeks incubation, and they chose to incubate all
medium-types for the full 12 weeks. This longer incubation may result in isolation of some of the
more rare/specialized isolates.
Other physio-chemical factors could also be taken into consideration when handling the recovered
samples. These include oxygen availability, temperature and pH (Bhatnagar and Kim 2010). When
choosing a growth medium, focus is on carbon utilization and abundance of nutrients, yet to
completely emulate the natural environment these other factors may play a role. Some sediment
layers are anaerobic, and depending on the local sea-environment, pH and temperature will vary.
This should be considered when determining culture conditions for production of secondary
metabolites, since it can be assumed, that these are expressed under certain conditions, and not
constitutively. This also emphasize that a better understanding of the genetic regulation of the
known bioactive compounds from Actinobacteria, may help in the discovery of new bioactive
compounds, where to look for them and under what conditions they are produced.
In the marine environment bacteria are found on surfaces and in the presence of other bacterial
species (Bowman 2007). This indicates that the interplay between different bacteria, as well as the
difference in conditions when under planktonic growth and when growing in biofilms, as a part of
bacterial communities, will affect the regulation of the bacterial metabolism, and thereby the
Page 28 of 38
Biotechnological potential of bacteria isolated from Greenlandic marine sediments
production of bioactive compounds (Bowman 2007). An example is the production of marine
microbial antifouling compounds, which seems to directly or indirectly involve signal transduction
pathways (Bhatnagar and Kim 2010). If pathways like these, can be understood and analyzed, it
will be clear whether other bacteria give the cue to produce these compounds by fore example
quorum sensing, if environmental factors are the cue or if it is a combination.
This study has evaluated the use of two different media for isolation of marine Actinobacteria
from sea sediments in the arctic region. It has demonstrated that it is possible with few growth
media and little equipment to select particular bacterial strains, which produce bioactive
compounds, from sea sediments. It is concluded that the rich medium (AMM) is suitable for
isolation of bioactive marine bacteria, yet that the procedures must be further revised in order to
isolate only Gram positive bacteria.
Page 29 of 38
Biotechnological potential of bacteria isolated from Greenlandic marine sediments
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APPENDIX 1: EXPORT PERMISSION
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Biotechnological potential of bacteria isolated from Greenlandic marine sediments
APPENDIX 2: SURVEY LICENSE
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Biotechnological potential of bacteria isolated from Greenlandic marine sediments
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