Accepted Manuscript

Title: Rare actinomycetes Nocardia caishijiensis and

Pseudonocardia carboxydivorans as endophytes, their
bioactivity and metabolites evaluation
Author: Rabia Tanvir Imran Sajid Shahida Hasnain Andreas
Kulik Stephanie Grond
PII:
DOI:
Reference:

S0944-5013(16)30003-9
http://dx.doi.org/doi:10.1016/j.micres.2016.01.003
MICRES 25855

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28-8-2015
23-11-2015
20-1-2016

Please cite this article as: Tanvir Rabia, Sajid Imran, Hasnain Shahida,
Kulik Andreas, Grond Stephanie.Rare actinomycetes Nocardia caishijiensis and
Pseudonocardia carboxydivorans as endophytes, their bioactivity and metabolites
evaluation.Microbiological Research http://dx.doi.org/10.1016/j.micres.2016.01.003
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Rare actinomycetes Nocardia caishijiensis and Pseudonocardia carboxydivorans as

endophytes, their bioactivity and metabolites evaluation
Dr. Rabia Tanvir1,4* rabiatanvir@hotmail.com rabiatanvir@yahoo.com, Imran Sajid1,
Shahida Hasnain1,2, Andreas Kulik3, Stephanie Grond4
1

Department of Microbiology and Molecular Genetics, University of the Punjab, Quaid-

e-Azam Campus, 54590, Lahore, Punjab Pakistan

2

Department of Microbiology and Molecular Genetics, The Women University, Multan,

Punjab Pakistan
3

Mikrobiologie/Biotechnologie, Interfakultres Institut fr Mikrobiologie und

Infektionsmedizin, Eberhard Karls Universitt Tbingen, Auf der Morgenstelle 28,

72076 Tbingen, Germany
4

Institut fur Organische Chemie, Eberhard Karls Universitt Tbingen, Auf der

Morgenstelle 18A, 72076 Tbingen, Germany

*

Corresponding author at: Department of Microbiology and Molecular Genetics,

University of the Punjab, Quaid-e-Azam Campus, 54590, Lahore, Punjab Pakistan. Tel.:
+92-322-4439472, Fax: +92-42-35952855.

Abstract
Two strains identified as Nocardia caishijiensis (SORS 64b) and Pseudonocardia
carboxydivorans (AGLS 2) were isolated as endophytes from Sonchus oleraceus and
Ageratum conyzoides respectively. The analysis of their extracts revealed them to be
strongly bioactive. The N. caishijiensis extract gave an LC50 of 570g/ml-1 in the brine
shrimp cytotoxicity assay and an EC50 of 0.552 g/ml-1 in the DPPH antioxidant assay.
Antimicrobial activity was observed against Methicillin resistant Staphlococcus aureus
(MRSA) and Escherichia coli ATCC 25922 (14mm), Klebsiella pneumoniae ATCC
706003 (13mm), Staphlococcus aureus ATCC 25923 (11mm) and Candida tropicalis
(20mm). For the extract of P. carboxydivorans the EC50 was 0.670 g/ml-1 and it was
observed to be more bioactive against Bacillus subtilis DSM 10 ATCC 6051 (21mm),
Candida tropicalis (20mm), Staphlococcus aureus ATCC 25923 (17mm), MRSA
(17mm), E. coli K12 (W1130) (16mm) and Chlorella vulgaris (10mm). The
genotoxicity testing revealed a 20mm zone of inhibition against the polA mutant strain
E. coli K-12 AB 3027 suggesting damage to the DNA and polA genes. The TLC and
bioautography screening revealed a diversity of active bands of medium polar and
nonpolar compounds. Metabolite analysis by HPLC-DAD via UV/Vis spectral
screening suggested the possibility of stenothricin and bagremycin A in the mycelium
extract of N. caishijiensis respectively. In the broth and mycelium extract of P.
carboxydivorans borrelidin was suggested along with -pyrone. The HPLC-MS
revealed bioactive long chained amide derivatives such as 7-Octadecenamide, 9, 12
octadecandienamide. This study reports the rare actinomycetes N. caishijiensis and P.
carboxydivorans as endophytes and evaluates their bioactive metabolites.
Keywords: Actinomycetes; Ageratum; Endophytes; Nocardia; Pseudonocardia;
Sonchus

1. Introduction
Actinobacteria are widely recognized as the most important sources of bioactive
secondary metabolites and also due to their major role in environmental processes. They
have been widely reported to inhabit different environments, especially soil. But in the
recent years, another ecological niche, the endophytic environment of plants has been
found to be a prosperous source of these biotechnologically productive bacteria
(Kaewkla and Franco, 2013). Endophytic actinomycetes are also known as the
microbial chemical factories present inside the plants (Gandotra et al. 2012). Since
endophytes have modified their ability to utilize available nutrients in plants such as
their sugar moieties, carbohydrate polymers, amino acids and peptides present in
varying concentrations. Therefore, larger genetic diversity of novel and rare species can
be obtained if the range of host plants is widened. Hence, it is crucial that targeted
selection of source plants should be done if programming the isolation of rare and novel
actinobacterial endophytes. Plants which are endemic and unique to specific areas are
more suspected to yield a high diversity of endophytes (Kaewkla and Franco, 2013).
Thus, unexplored habitats especially rare niches are a benefit to the science of drug
discovery (Vollmar et al. 2009).
Rare actinomycetes produce excellent antibacterial potency with the most diverse,
unique, unprecedented, and occasionally complicated compounds usually with low
toxicity. Several chemically simple compounds like terpenoids or benzenoids are almost
completely absent. However, compounds like vancomycin, ristocetin type that are
complicated glycopeptides are produced exclusively by various rare actinomycetes
species. At the present moment more than 50 rare actinomycete taxa are reported to be
producing 2,500 bioactive compounds (Kurtbke, 2012). For the purpose of finding
novel microbial resources for new bioactive compounds many rare actinomycetes are

now being isolated from plants. For example, in the culture broth of a novel endophytic
actinomycete Streptosporangium oxazolinicum sp. nov. strain, new antitrypanosomal
compounds, the spoxazomicins, have been found. This strain was isolated from the
roots of orchid plants. Other novel genera, Phytohabitans suffuscus and Actinophytocola
oryzae have also been discovered. Thus, plants have proven to be a prospective source
for newly discovered actinomycetes (Tiwari and Gupta, 2012). Recently the endophytic
actinomycetes that have been isolated recently from the tissues of the healthy plant
mostly belonged to the genera, Actinosynnema, Actinomadura, Microbispora,
Micromonospora, Streptomycetes and Nocardia (Nimnoi et al. 2010).
The systematics of actinomycetes remains in a state of flux as novel taxa are
continuously discovered. Nonetheless, by using a combination of chemical, molecular
and morphological criteria, unknown actinomycetes can readily be assigned to new or
existing described taxa. For example, members of the family Pseudonocardiaceae form
a distinct phylogenetic line, with a variable morphology. They contain
mesodiaminopimelic acid in their peptidoglycan and arabinose, galactose in whole
organism hydrolysates but lack mycolic acids. This family also contains the genera
Actinopoiyspora, Amycolatopsis, Kibdelosporangium, Pseudonocardia,
Saccharomonospora and Saccharopolyspora, some members of which are a source of
commercially significant bioactive compounds (Goodfellow et al. 1997).
The genus Nocardia belongs to the group of actinomycetes that contain mycolic acid,
i.e. the suborder Corynebacterineae which includes the genera Corynebacterium,
Dietzia, Gordonia, Mycobacterium, Nocardia, Rhodococcus, Skermania, Tsukamurella,
Williamsia and the genus Turicella deficient in mycolic acids. Much of the emphasis in
their systematics has been emphasized on the causal agents of actinomycetoma and
nocardiosis despite the fact that it is apparent that Nocardia are common in natural

habitats, especially in the soil. It is also becoming gradually clearer that nocardial
species diversity is underestimated therefore it is important to discover the species
richness of Nocardia. A study have shown the isolation of a novel species of Nocardia
by the name Nocardia caishijiensis spp. nov (Zhang et al. 2003).
The genus Pseudonocardia was first recognized by Henssen (1957). It at present
comprises 27 species and contains organisms with the following characteristics
depending on the species: vegetative and aerial mycelia with spore chains produced by
acropetal budding or fragmentation, meso-diaminopimelic acid, galactose and arabinose
in the peptidoglycan of the cell wall composition, MK-8(H4) as the major menaquinone,
iso-branched hexadecanoic acid as the predominant fatty acid, phospholipid type PII or
PIII pattern and a DNA content of G+C 6879 mol% (Park et al. 2008).
The present study was conducted to inspect the rare actinomycetes residing as
endophytes in the native Asteraceae plants. Since no earlier reports exist on the isolated
strains and their metabolites this study may lead to opening new horizons in these
specific species.

2. Materials and methods

2.1. Plant collection and strain isolation
Collection of the plants A. conyzoides L. and S. oleraceus L was carried after careful
scrutiny of the area around the department of Microbiology and Molecular Genetics
(MMG), University of the Punjab, Lahore. Intact plants were taken after cautious
digging of the roots and the plants were put in labelled bags for transportation to the lab.
Voucher specimen data of the plants is described in our previous study Tanvir et al.
(2014). The surface sterilization was done as described by Tanvir et al. (2013). Strain
AGLS 2 and SORS 64b were isolated on glycerol casein KNO3 agar (Kster and

Williams 1964) and actinomycetes isolation agar (Difco laboratories) plates that have
been incubated at 28C for three to four weeks following inoculation of cut plant parts.

2.2. Morphological, physiological and genetic characterization

The morphological properties of the isolates SORS 64b and AGLS 2 were documented
by sub culturing on GYM agar medium that have been incubated at 28C for up to 1821 days. For morphological observation, well grown 21 day incubated cultures were
observed under the microscope (Leica 4000 DM, Germany). The slide cultures were
prepared as described by Keiser et al. (2000) for the observation of spores in situ.
According to this method micro colonies of the strains were grown on a thin layer on a
glass slide. Observations were made by covering the slide with a cover slip and filling
the air space with water to obtain the required phase contrast conditions under the
microscope. Gram staining characteristics were also observed for the selected
actinomycetes strains.
The two strains were studied for an assortment of physiological properties by using
standard International Streptomyces Project (ISP) (Shirling and Gottlieb 1966). The
genetic confirmation by 16S rRNA amplification was carried out by Macrogen
sequencing facility (Macrogen Inc., Seoul, Korea). The obtained sequences were
analyzed by BLAST (www.ncbi.nlm.nih.gov/blast). The data was submitted to the
GenBank, and the accession numbers were obtained.
Phylogenetic trees of the two strains were made using the neighbour joining (NJ)
method (Saitou and Nei, 1987) in the software MEGA 5.2 (Tamura et al. 2011). An out
group using a sequence of closely related genus was also used for comparison.

2.3. Crude extract preparation

For pre-culture, 2 200ml GYM broth was prepared and pH was adjusted to 7.8.
Inoculation was done by adding agar blocks cut from well grown plates. Linear shaking
incubation was done at 180rpm for 3 days at 28 C. For crude extract preparation, 2
300 ml-1 of GYM broth for each strain was prepared and the pH was adjusted to 7.8
with 1N NaOH and 1N HCl. For each strain duplicate flasks were prepared and two
flasks were kept for negative control. The prepared media were autoclaved at 121 C for
20 minutes. Each flask was inoculated with the 10% pre-culture and incubated at
180rpm on linear shakers for 3 days at 28 C.
Extraction process was carried out for broth and mycelium separately. Before the
extraction process, the pH of the media was checked and celite (Diatomite product,
USA) was added. The whole mixture was separated using vacuum filtration. The
mycelial cake was dissolved in 2 volume of methanol and left for 30 minutes to let the
metabolites disperse. For the broth, a separating funnel was used and 3 volumes of
ethyl acetate added to it. Upper layer of ethyl acetate was separated and these processes
were repeated three times. In order to avoid the degradation of the compounds
evaporation was carried out immediately and the extracts dissolved in absolute
methanol and stored at -20 C.

2.4. Assays for bioactivity testing

2.4.1. Brine shrimp cytotoxicity assay
The cytotoxicity assay was performed according to the procedure described by Tanvir et
al. (2014). The extracts were tested at the concentrations of 10, 50, 100, 150, 500,
1,000g/ml and the mortality rate (M) was calculated by the formula:
M= (A
(G

Where,

N)
N)

100

M= % of the dead nauplii after 24 h

A= No. of dead nauplii after 24 h
B= Average no. of dead nauplii in control after 24 h
N= No. of dead nauplii before starting
G= Total nauplii

2.4.2. Antioxidant assay using diphenylpicrylhydrazyl (DPPH)

For the antioxidant assay the procedure of Tanvir et al. (2014) was followed. The
extract concentrations used for testing were 100, 500, 1,000, 5,000 and 10,000g/ml.
The antioxidant activity (EC50) was calculated by the formula:
[Abscontrol- Abssample] / Absample 100
Where,
Abscontrol = Absorbance of ascorbic acid standard at 517nm
Abssample = Absorbance of extracts at 517nm
EC50 = Dilution of the extract required to reduce initial DPPH chemilumininescence
intensity by 50%.

2.4.3. Antimicrobial assay

The antimicrobial activities of the crude extracts were determined using the agar well
diffusion method as described by Gebreyohannes et al. (2013) with slight modifications.
For the pathogenic bacteria, L-broth was used whereas for the clinical isolate of yeast
peptone dextrose (YPD) and for microalgae bold basal medium was used. The flasks
were inoculated with 80 l of cryo cultures and incubated on a shaker for 24 h for
bacterial and yeast strains. For the microalgae, incubation was carried out at room
temperature under a 12:12 h light-dark cycle with an illumination intensity of 3000 lux

for 96 h. After incubation, centrifugation was done for 20ml of broth at 8000 g for 10
mins. The resulting cell pellet suspended in 10 ml of normal saline and its O.D (optical
density) was adjusted to 1.3 at 546 nm. Then 2 ml of this cell suspension was added to
200 ml of the prepared agar medium (1:100) and poured in plates. Agar wells of about 5
mm in diameter were made with a sterile cork borer and 60 l of the crude extracts (5
mg/ml-1 in absolute methanol) were added to each well. The plates were left at room
temperature for 2 h in order to let the crude extract diffuse and afterwards incubated at
37C and 30C for 18-24 h. After incubation, zones of inhibition were measured in
mm(s).

2.4.4. DNA damage repair test (DDRT)

The test was performed as explained by Zhang et al. (2012). The strain E. coli K-12 AB
3027 was used for the test and the test was performed using disk diffusion method
(Shan et al. 2010). On each disk, 15l extract was added and the plates were incubated
at 37C for 24 hrs. The zones of inhibition were measured in mm(s).

2.5. Thin layer chromatography (TLC) and bioautography

For TLC the method of Kirchner (Kirchner, 1978) was followed. A NP silica gel plate
(TLC Silica gel 60, F254, Merck, Germany) was used for non-polar to medium polar
compounds whereas for very polar compounds a RP silica gel plate (TLC Silica gel 60
RP-18 F254, Merck, Germany) was used. The NP plate was developed with
CHCl3/MeOH (9:1) solvent system whereas for extremely polar compounds the RP
TLC plate was developed with MeOH/H2O (8:2) solvent system. Observation was
carried out at 365 nm and 254 nm and the compounds showing UV absorbance were
marked. Bioautography for the active metabolites of the two strains were performed as

described by Marston (2011) with a few modifications. Briefly, TLC of the selected
samples was performed and the visible bands were marked. The plate was cut into two
parts; one half was used for bioautography and the other half was sprayed using
anisaldehyde/ H2SO4 reagents and observed for different pattern of color development..
The test organism E. coli was grown on nutrient agar (Difco laboratories) slants; the
cells were scrapped off and added to 10ml of sterilized normal saline (0.85%). The cell
suspension was compared with 0.5MCF (Mc Farland) turbidity standard (BD
diagnostics, USA) to achieve the desired turbidity. This cell suspension was added to
15ml of 0.6% N agar in the ratio 1:100 and mixed. The developed TLC plate was
sterilized under UV lamp for 15 minutes then placed on the surface of a sterilized petri
plate. A thin layer of N agar was made on the surface of the TLC plate and it was
allowed to solidify by placing at 4C for 1-2 hours and then incubated at 37C for 24
hours. After incubation, 0.5mg/ml concentration of thiazolyl blue tetrazolium blue
(MTT) solution was sprayed on the plate surface. The plate was observed after 10
minutes for appearance of zones around the bands marked by yellow color whereas
inactive area was indicated with purple color.
2.6. XAD-16 column chromatography
Glass columns ranging from 20-75cm length with diameter ranging between 2-4cm
were filled with Amberlite XAD 16N (Dows chemical company, Dow Europe Gmbh).
The column was purified prior to elution with the following solutions: five bed volume
(BV) MeOH/ 1NHCl (9:1), five BV of acetone, five BV 1N NaOH and neutral wash
with 5 BV of deionised water. The last wash was done to neutralize the column and to
set the pH according to the pH of the sample. The crude extract dissolved in methanol
was evaporated and dissolved in 10ml of deionised water set at the specific pH of the
sample. The extracts was loaded onto the column and eluted by a stepwise gradient of 2

BV of MeOH/water as following:
Fraction 1: 100% water
Fraction 2: 80% water: 20% MeOH
Fraction 3: 60% water: 40% MeOH
Fraction 4: 40% water: 60% MeOH
Fraction 5: 20% water: 80% MeOH
Fraction 6: 100% MeOH
Fraction 7: 100% Acetone (wash)
The column system separated the fractions depending on their polarity. The fractions
were collected manually and the flow rate of the solvent was calculated according to the
length and diameter of the column used.

2.7. Preparative TLC (PTLC)

For this method, silica gel TLC glass plates (silica gel 60 F254 TLC plates 20 20 cm,
Merck Germany) were used. A line was drawn 2cm above the TLC plate surface and
2ml of the sample was loaded on it in a single straight line. The plate was developed in
CHCl3/MeOH (9:1) solvent system. After developing the plates, the bands of active
compounds were marked under the 254 and 365 nm UV wavelength and in cases of no
absorbance through the identification of reference band near the compound in question.
The silica gel with the bands was then carefully scrapped from the surface using a
scalpel and crushed to convert it into a fine powder. This powder was loaded on a frit
(Frits: Agilent DAS 35 Si small empty w/frits, NP, RP 20pk, Great Britain) and eluted
with CHCl3, MeOH and acetone. After separation, the solvent was evaporated on a
rotary evaporator to get the purified compound.

2.8. HPLC screening

2.8.1. HPLC-DAD UV Visible spectral analysis
The crude extract analysis of the strains was done using HPLC-DAD UV Vis database
(Fiedler 1993). The specifications of the system were as follows:
HPLC-DAD system: Agilent 1200 HPLC series equipped with a binary HPLC pump,
auto sampler and diode array detector, and an Agilent LC/MSD Ultra Trap System XCT
6330 (Agilent, Waldbronn, Germany); Column: Nucleosil 100 C18 column (100 2
mm, 3 m, equipped with a pre-column 10 2 mm, Maisch, Ammerbuch, Germany);
ESI MS: LC/MSD Ultra Trap System XCT 6330, Agilent Technology; Flow rate: 0.4
ml min1; 3 l injection volume; Program gradient: linear gradient from 10% to 100 %
of eluent B in 15 min (0.1% formic acid as eluent A, 0.06% formic acid in acetonitrile
as eluent B, Merck Germany); UV detector (UV/VIS) with wavelength monitoring at
230, 260, 280, 360 and 435 nm ESI: Ultra Scan mode, positive and negative; Capillary
voltage: 3.5 kV; Drying gas temperature; 350 C; Acquisition Program: Agilent 6300
series ion trap LC/MS software 6.1; Acquisition method: ESI MZ400.M; Databank:
visible spectral database (Fiedler, 1993, Biologisches Institut, LB
Mikrobiologie/Antibiotika, Universitt Tbingen, Germany).

2.8.2. Ultra High Resolution (UHR) HPLC-MS analysis

Further analysis of the crude extract was carried out using (UHR) HPLC-MS analysis.
The specifications of the system were as follows:
HPLC system: Dionex RSLC 3000 system, MS system: Bruker Daltonics maXis 4G
equipped with electrospray ion source; column: Nucleosil 100 C18 column (100 2
mm, 3 m, equipped with a pre-column 10 2 mm, Maisch, Ammerbuch, Germany);
flow rate: 0.3 ml min1; 3 l injection volume; program gradient: linear gradient from

0% to 100 % of eluent B in 20 min, 100% eluent B for 3 min (999.4% water, 0.6%
formic acid as eluent A, 999.9% methanol, 0.1% formic acid as eluent B (Merck
Germany); UV detector (UV/VIS) with wavelength monitoring at 210, 220, 254 and
272 nm; ESI: positive and negative mode; capillary voltage: 3.5 kV; drying gas
temperature; 350 C; acquisition program: Agilent 6300 series ion trap LC/MS software
6.1; scan began at 50 m/z and end at 1000 m/z; control software: Agilent 6300 series ion
trap LC/MS software 6.1; software for analysis: Brucker Daltonics DataAnalysis 4.1.

2.9. Statistical analysis

The experiments were carried out in triplicates and their overall results taken as mean
values. The LC50 was calculated through probit regression analysis and the EC50 was
taken out with the help of dose response curve. Analysis was done using statistical
package SPSS version 22.0 (SPSS, USA).

3. Results and discussion

3.1. Characterization of N. caishijiensis and P. carboxydivorans
Two strains SORS 64b and AGLS 2 were isolated from wild plants S. oleraceus and A.
conyzoides respectively. These plants were found to be growing in the vicinity of the
Department of Microbiology and Molecular Genetics (MMG), University of the Punjab,
Lahore. The isolate SORS 64b stood out from others since the color of its substrate
mycelium was light orange with light pink aerial mycelium and limited sporulation
(Supplementary Fig. 1a). The Gram staining revealed it to have a Gram positive
branched substrate mycelium (Supplementary Fig. 1b) similar to the results reported by
Zhang et al. (2003). The color of the colony also coincides with the color of the colony
of the genus Nocardia described in the same study as having an orange to brown

substrate mycelium with white to pinkish aerial hyphae which were either scarce or
plentiful.
The isolate AGLS 2 was different from the other strains despite being a hard and
embedded colony but the color of its aerial mycelium was sandy brown, the substrate
mycelium was light brown (Supplementary Fig. 1c). The Gram staining of the aerial
mycelium showed it to be in clusters of rod shaped spores and the substrate mycelium
as Gram positive filamentous chains (Supplementary Fig. 1d). The color of the substrate
mycelium can be correlated to the study by Park et al. (2008) which described a colony
of the genus Pseudonocardia to be producing brown substrate mycelium and white
aerial mycelium. The study also describes the aerial mycelium to be in the form of rod
shaped spores (Table 1).
The physiological characterization of these endophytic actinomycetes revealed a range
of versatile phenotypic properties. The strain SORS 64b gave negative melanin
formation test results but both strains gave positive test results for ten sugars used as a
carbon source. By comparing the physiological characteristics with those of known
actinomycetes as described in the Bergeys manual of determinative bacteriology (Holt,
1994) the results clearly indicate that the two strains belonged to rare genus in the group
actinomycetes. The two strains were also compared with similar physiological studies
done by Zhang et al. (2003), Park et al. (2008) and Huang et al. (2002) as described in
the table 1.
The analysis of the 16S rRNA gene sequences by BLAST showed that the strains
SORS64b belonged to the genus Nocardia with 99% homology for N. caishijiensis
(GenBank accession no. KC191699). In the neighbour joining (NJ) phylogenetic tree
analysis the strain was also found to be closely related to the same genus and gave a
99% homology to N. caishijiensis when Turicella otitidis was used as an out group (Fig.

1a). The BLAST analysis for the strain AGLS 2 belonged to the genus Pseudonocardia
with 99% homology for P. carboxydivorans (GenBank accession no. KC191696). In
the neighbour joining (NJ) phylogenetic tree analysis the strains AGLS 2 exhibited 99%
homology to P. carboxydivorans with being most close to Saccharomonospora viridis
as an out group (Fig. 1b).

3.3. Assays for bioactivity testing

The crude extract of the strain N. caishijiensis gave an LC50 of 570 g/ml and when
compared to the values provided by Geran et al. (1972) this value was within the toxic
range (Table 2). Prior studies have actively reported the cytotoxic potential of Nocardia
spp. with potent cytotoxic compounds such as Brasilibactin A from Nocardia
brasiliensis (Tsuda et al. 2005), Nocardicyclin A from Nocardia pseudobrasiliensis
(Tanaka et al. 1997), Nocardiones A from Nocardia spp. (Otani et al. 2000) and
chemomicin A from Nocardia mediterranei subspp. kanglensis (Sun et al. 2007).
Despite reports of many such compounds exist giving an interesting prospective on
cytotoxic ability of Nocardia spp. however either the producer species were isolated as
human pathogens or from soil environment. Only few studies have reported cytotoxic
activity of Nocardia spp. particularly endophytic in medicinal plants. One such study by
Huang et al. (2012) reported endophytic Nocadia niigatensis to possess bioactivity
against anti-tumor cells. Interestingly, no report on the cytotoxic study of N.
caishijiensis exist.
In the case of the antioxidant activity, the extract gave an impressive EC50 value of
0.552 g/ml which was closer to the control value of the ascorbate standard 0.338 g/ml
(Table 3). The reports on antioxidant activity of endophytic Nocardia spp. are scarce. A
recent study by Li et al. (2014) has explored the antioxidant potential of Nocardia spp.

YIM 64630 for antioxidant potential. However, no study to the best of our knowledge
exists on the anti-oxidant potential of N. caishijiensis. The crude extract of N.
caishijiensis was also found to be producing active antimicrobial compounds with the
most antifungal activity (20mm zone of inhibition) observed against clinical isolate
Candida tropicalis. The strain also displayed significant bioactivity against the
multidrug resistant MRSA and E.coli ATCC 25922 with 14mm zone of inhibition.
Against the ATCC standard strain K. pneumoniae ATCC 706003 and a nosocomial
pathogen Pseudomonas spp. a 13mm zone of inhibition were observed. It is interesting
however if our results are co related with prior studies such as by Qin et al. (2009)
which reported an endophyte Nocardia carnea isolated from the roots of a medicinal
plant with no antimicrobial activity. Another study by Verma et al. (2009) while
exploring the endophytes of Azadirachta indica A. Juss. discovered a Nocardia spp.
mostly active against B. subtilis (8-15mm zones) and Candida albicans (6-10mm zone).
The antimicrobial activity reported by the strain N. caishijiensis appears to be more as
compared to other endophytic Nocardia spp. isolated earlier. However, since no prior
study has been done on its antimicrobial potential therefore it is difficult to speculate.
The extract of N. caishijiensis however gave no genotoxic activity against E. coli K-12
AB 3027 in the DDRT test (Table 4). The DNA damage repair test (DDRT) is chiefly
used to distinguish active substances with DNA damaging ability and its application is
basically for antitumor pre-screening since many anticancer drugs act by damaging
DNA (Zhang et al. 2012). The E.coli K-12 AB 3027 is a strain defective in
endonuclease activity for apurinic sites in DNA, PolA genes (Ljungquist et al. 1976)
and is a SOS-defective recA, lexA mutant (Alam et al. 2009). Any damage to the DNA
and related genes would therefore be indicated by a strong inhibition of the AB3027
strain. This inhibition can therefore be related to anti-tumor activity through the

mechanism of DNA damage. A negative activity of the SORS 64b crude extract
indicated that there was no damage to the DNA and polA genes.
The crude extract of the strain AGLS 2 identified as P. carboxydivorans gave a
nontoxic value for LC50 i.e. 1100 g/ml. Nevertheless, as compared to the ascorbate
standard the EC50 value was quite significant (0.670 g/ml). Impressive antimicrobial
activity was also observed against B. subtilis ATCC 6051 with a maximum zone of
21mm. A very significant antifungal activity of 20mm zone was also observed against
clinical pathogenic fungal strain of Candida topicalis. A good activity with zones of
inhibition between 15-17mm was observed against S. aureus ATCC 25923, MRSA, E.
coli K12 (W1130) and nosocomial pathogens Enterobacter spp., and Pseudomonas spp.
Interestingly, the crude extract also displayed antialgal activity against Chlorella
vulgaris with 10mm zone of activity. The significant results of P. carboxydivorans
bioactivities are difficult to co-relate with other studies on endophytic Pseudonocardia
spp. as not much studies have been reported. A study by Qin et al. (2009) reported
endophytic Pseudonocardia alni to be moderately active and Pseudonocardia
zijingensis to be slightly active against S. aureus. Another study by Luo et al. (2012)
reported Pseudonocardia spp. from Stemona tuberosa Lour displaying activity against
pathogens such as Staphlococcus aureus and Pseudomonas aeruginosa. However, the
findings of our study remain unique as no prior study on Pseudonocardia strains has
reported such significant and wide bioactivity. Most important is the fact that no study
has been carried out on the strains reported in our study. The crude extract of P.
carboxydivorans also gave a very notable genotoxic activity with a prominent zone of
20mm in the DDRT test against E. coli K-12 AB 3027 (Fig. 2). It clearly showed that
the extract possessed anti-tumor properties along with the results of cytotoxicity and
antioxidant activities, further strengthening this idea.

3.4. TLC and bioautography screening

Thin layer chromatography (TLC) is used widely to separate or purify mixtures of
chemical and biological compounds. It is convenient due to its advantages of simplicity,
economy, easy operation, and the need for only small amounts of solvent. It is also
considered the most suitable separation methods for high throughput analysis because it
is performed under ambient conditions (Cheng et al. 2011). The TLC screening of the
crude extracts of the two actinomycetes revealed the production of both medium polar
and non-polar bands. On a normal phase silica plate the strain SORS 64b broth and
mycelium extract produced three medium polar bands at the center of the silica plate
whereas one nonpolar band was observed at the top of the TLC plate. The bands gave
near negligible absorbance at UV 254nm but more absorbance was seen at 365nm (Fig.
3a). Interestingly, in the mycelium extract the non-polar band was absent. In the case of
AGLS 2 extracts the bands observed were also either medium polar or non-polar in
nature. Only one polar band was observed in the mycelium extract. Poor absorbance
was observed at both 254 and 365nm wavelengths. No noticeable color was observed in
the anisaldehyde/H2SO4 reagent staining of the bands; only dull yellow color was
observed for the non-polar bands. Among the two strains the bioautography results for
AGLS 2 mycelium extract, showed the entire area containing the poor UV absorbing
medium polar and non-polar bands to be active with prominent yellow color against E.
coli (Fig. 3b). Since either low UV absorbing bands or no bands were observed here
therefore this gave an idea that there may be active compounds present in this area that
were non UV absorbing.

3.5. HPLC-DAD UV Visible spectral screening

Considering the previous results of TLC and bioautography the HPLC-DAD UV
analysis of the extracts of N. caishijiensis and P. carboxydivorans was done. It was
carried out for the UV absorbing compounds and compared with the actinomycetes
metabolites from the institute database of approximately 1000 known actinomycetes
compounds (Fiedler, 1993 Universitt Tbingen). A similarity in peaks 995 match
factor (UV spectral match) was considered to be precise. The strain SORS 64b
mycelium extract gave interesting matches however with minor precision with the
compounds in the database. The most similarity was found to the antibiotic stenothricin
with 995.189 match factor and a 994.685 match factor to bioactive compound trytophol
in the database (Fig. 4a). Stenothricin is reported to be a peptide antibiotic capable of
inhibiting the cell wall of bacteria (Hasenbhler et al. 1973). It has only been recently
reported from an actinomycetes Streptomyces roseosporus (Liu et al. 2014). Trytophol
had been previously reported from soil Streptomyces spp. (Amar et al. 2012). Other
match factors were found to be less accurate such as 992.814 match factor to fatty acid
2,4,7-decatrienic acid, 992.923 to HAG 010767-A2 a compound recently reported from
Streptomyces spp. (Dalmas et al. 2013). A match factor of 992.069 was observed with
the cyclic decapeptide antibiotic tyrocidin NK which has been reported to be produced
only by Bacillus brevis (Mootz and Marahiel 1997), and 990.147 to another cyclic
decapeptide antibiotic streptocidin D reported only from Streptomyces spp. T 6071
(Hltzel et al. 2001). A 992.397 match factor with antibiotic bagremycin A was also
observed in the mycelium extract. Bagremycin A has been previously described as
precursors of para-coumaric acid and 3- amino-4-hydroxybenzoic acid produced by
Streptomyces spp. It was observed to be active against Gram-positive bacteria such as
Bacillus subtilis, Arthrobacter aurescens whereas weakly activity against

Saccharomyces cerevisiae and Candida albicans. No antitumor activity was observed

for this antibiotic in the study (Bertasso et al. 2004). In our study, the crude extract
displayed the LC50 value in the toxic range exhibiting antitumor potential. Overall no
other study has reported these bioactive metabolite findings from N. caishijiensis as
endophyte or otherwise.
In the broth extract of AGLS2 identified as P. carboxydivorans an antibacterial agent pyrone was an interesting find however with a less significant 993.214 match factor
(Fig. 4b). Its derivatives have been reported from Streptomyces violascens (Zhang et al.
2013), marine derived Nocardiopsis spp. (Kim et al. 2013), Nocardiopsis dassonvillei
(Fu et al. 2011) and from Streptomyces spp. (Shin et al. 2014). Only one report exists of
its derivative from endophytic actinomycetes, Streptomyces sundarbansensis (Djinni et
al. 2013). In the mycelium extract a match factor 994.892 was found with the macrolide
antibiotic borrelidin initially isolated from Streptomyces rochei and then from
Streptomyces griseus, Streptomyces parvulus and Streptomyces californicus. Borrelidin
was reported to be widely bioactive with activities such as antibacterial, antiviral,
antitumor, antimalarial, insecticidal as well as herbicidal activity (Bhikshapathi et al.
2010). A match factor of 993.461 was found with -pyrone (Fig. 4c), a compound also
observed in the broth extract. Despite less precise matches, the metabolite analysis
reveals interesting results since P. carboxydivorans is only known for the production of
carbon monoxide (Park et al. 2008).

3.6 Ultra High Resolution (UHR) HPLC-MS analysis

Further metabolites analysis of the two rare actinomycetes species were carried out
using (UHR) HPLC-MS. It was considered important because prior HPLC-DAD UV

Vis spectral analysis revealed match factor values that were not very substantial. Also
because the bioautography of P. carboxydivorans (AGLS 2) mycelium extract had
revealed non or poor UV absorbing compounds that gave prominent activity against E.
coli. For this purpose, the extract was absorbed onto the XAD-16 column (pH 6.1) and
eluted out with the seven gradient fractions. The compounds in question were
considered to be non-polar and non UV absorbing. The TLC monitoring of the fractions
and bioautography was done along with the antimicrobial activity to determine the
bioactivity of the target compounds (Fig. 5a, b). Fractions 3 and 4 were observed to
contain bioactive bands with prominent zone of inhibition of 21mm against B. subtilis
ATCC 6051 and minor activity against E. coli (Fig. 5c, d). Further purification was
carried out using PTLC and final analysis by HPLC-MS resulted in the purification of
three active compounds. The entire work up scheme is shown in Supplementary Fig. 2.
The obtained molecular formula and molecular weight was compared with the reference
data in various databases like SciFinder, dictionary of natural products (DNP) and
chemical abstracts available.
3.6.1. Compound 1 (Molecular formula C18 H33 NO2)
The ion chromatogram at ESI +ve ionization showed the compound 1 (white powdered,
48.2 mg) was present at the retention time of 17.7-17.9 minutes. At m/z 296.2 a [M+H]+
signal was present, a [M+Na]+ signal was present at m/z 318.2 (Fig. 6a) fixing the
molecular weight as 295.2 daltons and a molecular formula C18 H33 NO2. It was
identified to be 7-Octadecenamide.

3.6.2 Compound 2 (Molecular formula C18 H33 NO)

Compound 2 was a white powdered non-polar compound (42.7 mg) with negligible UV
absorbance at 254nm. After staining with the anisaldehyde/H2SO4 staining reagent

violet color was observed. The MS analysis revealed the compound on the ion
chromatogram at ESI +ve ionization at a retention time of 18.2-18.7 minutes. At m/z
280.2 a [M+H]+ signal was present, a [M+Na]+ signal was present at m/z 302.2 (Fig. 6b)
and a [2M+H]+ signal was detected at m/z 560.4 calculating the molecular weight to be
279.2 daltons and molecular formula C18 H33 NO. It was identified to be 9, 12Octadecadienamide (Linoleamide).

3.6.3. Compound 3 (Molecular formula C16 H31 NO)

Compound 3 was a white solid of 14 mg weight. It was a non-polar compound with
weak UV absorbance at 254nm which gave violet colour upon staining. The MS
analysis on the ion chromatogram at ESI +ve ionization showed the compound present
at the retention time of 19.1-19.4 minutes. At m/z 254.2 a [M+H]+ signal was present, a
[M+Na]+ signal was present at m/z 276.2 (Fig. 6c) and a [2M+H]+ signal was detected at
m/z 508.2 deducing the molecular weight to be 253.2 daltons and a molecular formula
C16 H31 NO2.
The active compound identified as 7, octadecenamide (C18 H33 NO2) has been
previously reported in plants (Henry et al. 1987; Shibahara et al. 1993; Butovich and
Lukyanova, 2008; Abirami and Rajendran, 2012; Li et al. 2012; Rameshwar et al. 2012;
Sagwan et al. 2012; Igwe and Okwu, 2013), algae (Vick and Zimmerman, 1989;
Hamberg et al. 1992; Zimmermann et al. 2012; Zhang et al. 2013), actinomycetes
(Schumann et al. 1997; Shaaban et al. 2013) such as Streptomyces species (Rezanka et
al. 1984). Whereas, the compound identified as 9, 12 octadecandienamide (C18 H33 NO)
was found to be reported from algae (Bertin et al. 2012), plants (Morikawa et al. 2003;
Xu et al. 2004; Piazza and Foglia, 2005; Hanh et al. 2011; Rios 2012) and
actinomycetes (Jain et al. 1992).

It has been observed that such long chained amide derivatives of fatty acids are selfdefensive agents and possess various biological activities (Hanh et al. 2011). They have
established bioactivities with some of them being the most prominent like oleic acid
which was found to have a potential role in diminishing brain-related disorders such as
dementia and Alzheimers disease along with having the highest in vitro inhibition of
prolyl endopeptidase (PEP) enzyme that is believed to have a role in amyloid formation
in the brain. They have also been observed in the therapeutic management of colorectal
cancer and against cardiovascular and inflammatory diseases (Aluko 2012). According
to reports, derivatives of fatty acids exhibit greater antimicrobial activity than the
unchanged molecules and their amidation convey a broad range of activity against
bacteria, and fungi. Owing to this improved functionality and momentous properties of
bioactivity in secondary fatty amides, there is a growing curiosity in their manufacture
and characterization (Khare et al. 2009).

4. Conclusion
The study explores two rare actinomycetes N. caishijiensis and P. carboxydivorans
isolated as endophytes from the plants Sonchus oleraceus and Ageratum conyzoides
respectively. No prior study has reported them as endophytes and explored their
bioactive metabolite analysis so this study reports new finding. The strain N.
caishijiensis were found to be displaying cytotoxic and significant anti-oxidant values
along with being broadly antimicrobial. The strain P. carboxydivorans displayed
significant antimicrobial activity and genotoxicity against E.coli K-12 AB 3027. The
metabolite analysis by HPLC-DAD-UV Vis spectral screening in an in-house database
revealed minor indications of stenothricin, trytophol and borrelidin. Whereas, the

HPLC-MS results confirmed some long chained amide derivatives that have not been
previously reported from these actinomycetes species and are described to be widely
bioactive. Our findings encourage the exploration of these rare genus to further our
understanding as well as advance analysis of this particular niche may lead to finding
new drug targets.

Acknowledgements
Dr. Yi Zhang at the Guangdong Ocean University China is acknowledged for providing
the E.coli K-12 AB 3027 strain. A grant 1-/HEC/HRD/2012/2301 by Higher Education
Commission (HEC) Pakistan by the IRSIP program is greatly acknowledged.

Conflict of interest
The authors declare they have no conflict of interest.

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Figure Captions

Fig. 1a Neighbour Joining phylogenetic tree of 16S rRNA gene sequence of

actinomycetes strain SORS 64b and its twenty nearest homologues. SORS 64b was
clustered together with other Nocardia spp. gene sequences. For the measure of
reliability of the constructed trees, 100 replicates of boot-strap test (Efron and
Tibshirani, 1993) were used. A single phylogenetic tree was formed to compare all
the sequences making the group. The gene sequence of Turicella otitidis was used as
an out group

Fig. 1b Neighbour Joining phylogenetic tree of 16S rRNA gene sequence of

actinomycetes strain AGLS 2 and its twenty nearest homologues. AGLS 2 was
clustered together with other Pseudonocardia spp. gene sequences. For the measure
of reliability of the constructed trees, 100 replicates of boot-strap test (Efron and
Tibshirani, 1993) were used. A single phylogenetic tree was formed to compare all
the sequences making the group. The gene sequence of Saccharomonospora viridis
was used as an out group

Fig. 2 Antimicrobial activity of the crude extracts of rare endophytic actinomycetes. a)

Activity of N. caishijiensis against MRSA b) Activity of N. caishijiensis against k.
pnemoniae ATCC 706003 c) Activity of P. carboxydivorans against B. subtilis ATCC
6051 d) Activity of P. carboxydivorans against S. aureus ATCC 25923 e) Activity of P.
carboxydivorans against Pseudomonas spp. f) Activity of P. carboxydivorans against
MRSA g) Activity of P. carboxydivorans against Enterobacter spp. h) Activity of P.
carboxydivorans against E. coli K-12 AB 3027

Fig. 3a Chemical screening of strain SORS 64b by thin layer chromatography. Band
observation was carried out for both broth and mycelium extracts under UV 254 (shown
by ( ) symbol) and 365nm (shown by [ ] symbol) wavelength. The spots were
superimposed with 0.5ml of each extract. The TLC plates were developed with freshly
prepared MeOH/H2O (9:1) mobile phase. As a negative control the GYM media extract
was also run
Fig. 3b Chemical screening of strain AGLS 2 by thin layer chromatography.
Observation of bands was carried out for broth and mycelium extracts under UV 254
(shown by ( ) symbol) and 365 nm (shown by [ ] symbol) wavelength. The spots were
superimposed with 0.5ml of each extract. The TLC plates were developed with freshly
prepared MeOH/H2O (9:1) mobile phase. The staining of the active bands was done
with anisaldehyde/H2SO4 and bioactivity was observed through bioautography (Arrows
indicate the inhibition area with some non UV absorbing compounds)

Fig. 4a Total UV absorption spectra of N. caishijiensis mycelium extract at 230 (blue),

260 (red), 280 (light green), 360 (pink), 435 (green) nm wavelength and UV spectra
match with tryptophol and stenothricin (extract = blue, database= red)

Fig. 4b Total UV absorption spectra of P. carboxydivorans broth extract at (blue), 260

(red), 280 (light green), 360 (pink), 435 (green) nm wavelength and UV spectra match
with -pyrone (extract = blue, database= red)

Fig. 4c Total UV absorption spectra of P. carboxydivorans mycelium extract at (blue),

260 (red), 280 (light green), 360 (pink), 435 (green) nm wavelength and UV spectra
match with borrelidin and -pyrone (extract = blue, database= red)

Fig. 5a Observation of active bands under UV 254 in P. carboxydivorans (AGLS 2)

mycelium extract fractions 3 and 4 after XAD-16 column chromatography. Minor
absorbance is observed in bands marked from a-f
Fig. 5b Observation of active bands under UV 365 in P. carboxydivorans (AGLS 2)
mycelium extract fractions 3 and 4 after XAD-16 column chromatography. Minor
absorbance is observed in bands marked 4, c, e, i
Fig. 5c Antimicrobial activity monitoring of the active bands in P. carboxydivorans
(AGLS 2) mycelium extract fractions 3 and 4 after XAD-16 column chromatography.
Slight indication of activity is hinted in bands a and c against E. coli
Fig. 5d Antimicrobial activity monitoring of the active bands in P. carboxydivorans
(AGLS 2) mycelium extract fractions 3 and 4 after XAD-16 column chromatography.
The fraction 3 and 4 are both significantly active against B. subtilis ATCC 6051

Fig. 6a The +ve ionization mass spectra for compound 1. The signal for [M+H]+ is
visible at m/z 296.2 and a [M+Na]+ at m/z 318.2

Fig. 6b The +ve ionization mass spectra for compound 2. The signal for [M+H]+ is
visible at m/z 280.2 and a [M+Na]+ signal at m/z 302.2

Fig. 6c The +ve ionization mass spectra for compound 3. The signal for [M+H]+ is
visible at m/z 254.2 and a [M+Na]+ at m/z 276.2

Tables
Table 1 Morphological, physiological and genetic characterization of N. caishijiensis and P.

carboxydivorans

Morphological characterization

SORS 64b

AGLS 2

Growth Pattern

Well grown/ Partitioned

Well grown/
Partitioned

Substrate mycelium

Light Orange

Light Brown

Aerial mycelium

Light pink

Reddish brown

Soluble Pigments

No

No

Hard/
Embedded
Pin
Point

Hard/
Embedded
Pin
Point

Shape

Circular

Circular

Margin

Filamentous

Filamentous

Texture

Rough

Rough

Sporulation

Excellent

Good

7d

+*

+*

14d

+*

+*

7d

+*

+**

14d

+*

+**

7d

+**

+*

14d

+**

+*

7d

+**

14d

+**

7d

+*

14d

+*

7d

+*

+**

14d

+*

+**

7d

+**

+*

14d

+**

+*

Consistency
Size (mm)

Glu

Gal

Ara

Physiological characterization
using sugar utilization as carbon
source and melanin

Man

Sor

Suc

Manni

Raf

Fru

Lac

Mel

UA

UO

FO

HEA

Physiological characterization
using nine biochemical tests
HUA

LV

HT

LY (4d)

7d

+*

+**

14d

+*

+**

7d

+**

14d

+*

+**

7d

+**

+*

14d

+**

+*

8d

+*

14d

+*

6d

9d

++

++

16d

++

++

7d

11d

17d

5d

9d

5d

++*

++

10d

++*

++

5d

_*

_*

9d

_*

_*

3d

_*

5d

8d

2d

5d

0.1ml

+*

+**

0.5ml

+*

+**

1ml

+*

+**

NaCl (4d)

16S rRNA sequencing

GenBank
accession
no.

0g

+*

+*

2g

+*

+*

3.5g

_**

+*

5g

_**

+*

6.5g

KC191699

KC191696

Utilization of carbohydrates, and similar sugars (Zahner and Ettlinger 1957), Glu, Glucose; Gal, Galactose; Ara,
Arabinose; Man, Mannose; Sor, Sorbitol; Suc, Sucrose.; Manni, Mannitol; Raf, Raffinose; Fru, Fructose; Lac,
Lactose; Mel, formation of Melanin ((Shirling and Gottlieb 1966), UA, Utilization of Organic acids (Nitsch and
Kutzner 1969); UO, Utilization of oxalate (Nitsch and Kutzner 1969); FO, Formation of organic acids (Ziegler and
Kutzner 1973); HEA, Hydrolysis of esculin and arbutin (Kutzner et al. 1986); HUA, Hydrolysis of urea and allantoin
(Gordon 1967); LV, lecithovitellin reaction (Nitsch and Kutzner 1969); HT, Hemolysis test (Kutzner et al. 1978); LY,
Resistance to lysozyme (Kutzner et al. 1986); NaCl, Resistance to NaCl (Tresner et al. 1968)
(-) negative results; (+) moderately positive; (++) strongly positive; d, days
*Data similar to this and the study by Zhang et al. (2003) for Nocardia caishijiensis and Park et al. (2008), Huang et
al. (2002) for Pseudonocardia carboxydivorans; ** Data different to this and the study by Zhang et al. (2003) for
Nocardia caishijiensis and Park et al. (2008), Huang et al. (2002) for Pseudonocardia carboxydivorans
The 16S sequences obtained were checked for the quality of the sequencing by FinchTV 1.4.0 (Geospiza, Inc. USA)
and chimeras were removed manually by alignment of the forward and reverse complementary of the reverse
sequences using NCBI nucleotide BLAST (Altschul et al. 1990). Since chimeric sequences are becoming a problem
on public databases due to their frequency therefore chimeric sequences were also checked for alignment using SINA
alignment service of SILVA RNA database (Quast et al. 2013). The regions of both sequences were refined and
manually joined together to get completed sequence of the gene.

Table 2 Cytotoxic activity of N. caishijiensis (SORS 64b) and P. carboxydivorans

(AGLS 2) in the brine shrimp cytotoxicity assay

Strain code
DMSO (1% in seawater)
SORS64b
AGLS2

10
0
33.6
25.6

% mortality under the concentration studied (g/mL-1)

50
100
150
500
0
0
0
0
38
40
50
53.3
26.6
30
30
36.6

LC50 (g/mL-1)
1000
0
65.3
42.7

0
570
1100

Score for LC50: Highly toxic - <20(g/mL-1), toxic upto 1000 g/mL-1 and non toxic - >1000g/mL-1; Values are
means of triplicate studies

Table 3 Antioxidant activity of N. caishijiensis (SORS 64b) and P. carboxydivorans

(AGLS 2) in DPPH antioxidant assay

Strain code
Ascorbate standard
SORS64b
AGLS2

% activity under the concentration studied (g/mL-1)

100
500
1000
5000
100
100
100
100
28.3
37.1
38.7
41.4
40.5
74.1
76.0
76.7

Values are means of triplicate studies; (-) not determined

EC50 (g/mL-1)
10000
100
43.7
98.3

0.338
0.552
0.670

Table 4 Antimicrobial activity of N. caishijiensis (SORS 64b) and P. carboxydivorans (AGLS 2)

Strain Code
SORS 64b
AGLS 2

B.
subtilis
ATCC
6051
11
21

Ent.
spp.

Pseudo.
spp.

11
15

13
15

S.
aureus
ATCC
25923
11
17

E.
coli
ATCC
25922
14

K.
pneumonia
ATCC
706003
13

MRSA

E.
coli
K12

14
17

16

S.
cerevisisae
ATCC
9080

C.
tropicalis

C.
vulgaris

E.coli
DDRT

20
20

10

20

B.subtilis Bacillus subtilis; S. aureus Staphlococcus aureus; E.coli Escherichia coli; K. pneumoniae Klebsiella pneumoniae; MRSA Methicillin Resistant Staphlococcus aureus; C. tropicalis
Candida tropicalis, C.vulgaris Chlorella vulgaris; E.coli DDRT, Escherichia coli DNA Damage Repair Test; (-) no inhibition; values are means of triplicate studies; zones of inhibition were
measured in mm(s)