Marine Environmental Research 119 (2016) 156e160

Contents lists available at ScienceDirect

Marine Environmental Research

journal homepage: www.elsevier.com/locate/marenvrev

Isolation and characterization of agar-digesting Vibrio species from the

rotten thallus of Gracilariopsis heteroclada Zhang et Xia
Joval N. Martinez a, *, Philip Ian P. Padilla b
a
Department of Natural Sciences, College of Arts and Sciences, University of St. La Salle, 6100 Bacolod City, Philippines
b
Division of Biological Sciences, College of Arts and Sciences, University of the Philippines Visayas, 5128 Miag-ao, Iloilo, Philippines

a r t i c l e i n f o a b s t r a c t

Article history: Gracilariopsis heteroclada Zhang et Xia (Gracilariaceae, Rhodophyta) is one of the most studied marine
Received 4 December 2014 seaweeds due to its economic importance. This has been cultivated extensively on commercial scale in
Received in revised form the Philippines and other Asian countries. However, sustainable production of G. heteroclada in the
17 May 2016
Philippines could not be maximized due to the occurrence of rotten thallus disease. Thus, isolation and
Accepted 23 May 2016
Available online 27 May 2016
characterization of agar-digesting bacteria from the rotten thalli of G. heteroclada was conducted. A total
of seven representative bacterial isolates were randomly selected based on their ability to digest agar as
evidenced by the formation of depressions around the bacterial colonies on nutrient agar plates sup-
Keywords:
Seaweed
plemented with 1.5% NaCl and liquefaction of agar. Gram-staining and biochemical characterization
Gracilariopsis heteroclada revealed that isolates tested were gram-negative rods and taxonomically identified as Vibrio para-
Rotten thallus haemolyticus (86e99.5%) and Vibrio alginolyticus (94.2e97.7%), respectively. It is yet to be confirmed
Vibrio whether these agar-digesting vibrios are involved in the induction and development of rotten thallus
Agar-digesting bacteria disease in G. heteroclada in concomitance with other opportunistic bacterial pathogens coupled with
adverse environmental conditions.
© 2016 Elsevier Ltd. All rights reserved.

1. Introduction
Seaweeds provide a large amount of oxygen in the marine
environment that sustains the growth of many organisms in the
sea. These are being used in biomonitoring and bioremediation
because of their ability to absorb heavy metals from the water (Stirk
and Van Staden, 2000). They also have the high capacity to with-
stand extreme conditions and other environmental stresses (Chan
et al., 2006) thus, they are widely used in farming. Seaweeds
have been used as a source of food for both human and animals.
These are also used by many industries as raw materials to
manufacture different products such as cosmetics, toothpaste and
other seaweed-containing products.
With the rise of ecological and economic importance of sea-
weeds, world-wide interest in harvesting seaweeds from the wild
has also been rapidly increasing. This has prompted many scien-
tists, researchers and even farmers to develop farming methods to
improve the quality of the products. However, the occurrences of
seaweed diseases (Largo, 2002) arising from the plant itself or from
* Corresponding author.
E-mail address: j.martinez@usls.edu.ph (J.N. Martinez).
the environment where they grow have depleted the wild stocks
thereby lowering the number of strains of some marine
macroalgae.
The relationship between the seaweeds and the pathogen
affecting the seaweeds (Correa, 1996) has been given much atten-
tion these days due to the greater demand in the market especially
in the Philippines. Many etiological agents are being considered by
most experts in order to better understand the occurrence of dis-
eases in seaweeds. The presence of bacteria, fungi, endophytes and
nematodes has shown to affect many seaweed species such as the
fronds of Chondrus crispus (Craigie and Correa, 1996), branches of
Kappaphycus alvarezii and Eucheuma denticulatum (Largo et al.,
1995), Gracilaria spp. (Lavilla-Pitogo, 1992; Weinberger et al.,
1994; Musa and Wei, 2008) and some other seaweed species.
Gracilaria spp. (Gracilariaceae, Rhodophyta) is one of the most
studied taxon in Asian countries due to its economic importance.
Better known as “gulaman” in the Philippines, this species is the
source of natural products such as fatty acids, pigments, poly-
saccharides and protein. Raw gulaman is used for human con-
sumption and also used as fertilizer, animal feed and processed as
medicine. Agar yield and gel strength of Gracilariopsis heteroclada
Zhang et Xia were investigated to determine the seasonality of the

http://dx.doi.org/10.1016/j.marenvres.2016.05.023
0141-1136/© 2016 Elsevier Ltd. All rights reserved.
 J.N. Martinez, P.I.P. Padilla / Marine Environmental Research 119 (2016) 156e160
biomass of this species (Luhan, 1992; Hurtado-Ponce, 1995). How-
ever, due to the effects of deteriorating agents to the quality of
seaweeds, a thorough investigation of bacteria present on seaweed
thalli should be done.
In the present study, bacteria associated with ‘rotten thallus’ of
G. heteroclada Zhang et Xia were investigated to isolate and char-
acterize bacteria from the ‘rotten thallus’. Specifically, the current
study aimed to: (1) isolate bacteria associated with rotten thallus of
G. heteroclada; (2) characterize and identify the isolates based on
their morphological characteristics and biochemical activity.
2. Materials and methods
2.1. Culture media preparation
Nutrient Agar (NAþ) with 1.5% NaCl was used to culture and
isolate bacteria from thallus of G. heteroclada. These were prepared
by dissolving 23 g of nutrient agar powder in 1 L sterile distilled
water containing 15 g of NaCl. The media was then autoclaved at
121 C at 15psi in 15 min. After autoclaving, the NA þ medium was
cooled to 50  C. About 25e35 ml of this medium was poured to
each sterile petri plates and solidified prior to culture and isolation.
Three (3) replicates of petri plates containing NA þ medium were
prepared to culture bacteria from both healthy and unhealthy
seaweed thalli separately. All procedures were performed
aseptically.
2.2. Preparation of seaweed materials and bacterial culture
Healthy and unhealthy G. heteroclada showing ‘rottening
thallus’ (Fig. 1) were taken from the seaweed tank cultures at the
Seaweed Wet Lab, Southeast Asian Fisheries Development Center-
Aquaculture Department (SEAFDEC-AQD). These seaweed mate-
rials were freshly collected from Carles, Iloilo. These were placed in
a sterile plastic bag and immediately brought in Algal Production
Laboratory of SEAFDEC-AQD for processing. About 1 cm of wet algal
materials from both healthy and rottening thalli were separately
cut using a sterile scalpel and directly rubbed on prepared
NA þ plates for growth and isolation of bacteria. Cultures were
incubated at ambient temperature (28  C) for 24 h.
Fig. 1. Samples of Gracilariopsis heteroclada with rotten thalli (red arrow).(For inter-
pretation of the references to colour in this figure legend, the reader is referred to the
web version of this article.)
157
2.3. Bacterial isolation and characterization
2.3.1. Bacterial isolation
Bacteria showing agar-digesting characteristics (liquefaction of
agar) (Goresline, 1933) were considered in this study. Bacterial
growth was examined after 24 h incubation of plates containing
bacteria from healthy and unhealthy seaweed thalli. Various col-
onies showing different morphological characteristics were classi-
fied and marked for reculturing. Only bacterial isolates that liquefy
agar were selected and recultured in a new set of media (NAþ) to
obtain a pure culture. Representative colonies were randomly
picked and were subsequently streaked on new set of agar media
(NAþ).
2.3.2. Gram-staining
Pure culture of each isolate was subjected to Gram-staining.
Briefly, a loopful of bacteria from pure culture was smeared in a
sterile glass slide containing a drop of sterile distilled water. The
smear was covered with crystal violet, which served as the primary
stain for 1 min, and Grams iodine was added as a mordant. This was
then decolorized using 95% ethanol and was counterstained with
safranin for about 2 min. This was briefly washed with distilled
water and blotted-dry for microscope examination. Gram-stained
isolates was viewed under the microscope (Olympus BX51) at
1000  magnification and photographed using Digital Camera
(Samsung).
Fig. 2. Bacterial isolates coming from rotten G. heteroclada thalli grown on NAþ plates.
(A) whitish small irregular colonies of isolate 01 (V. parahaemolyticus) and; (B) whitish
circular colonies of Isolate 02 (V. alginolyticus).
158 J.N. Martinez, P.I.P. Padilla / Marine Environmental Research 119 (2016) 156e160
2.3.3. Biochemical characterization
API 20E kit (BioMerieux) was used for the characterization of
bacteria based on biochemical activity and identified using the
programmed software for identification. Pure culture (24-h culture)
of bacterial isolates was prepared for biochemical tests mentioned
above. Each of the isolate was inoculated in 5 ml of sterile saline
water separately and transferred in the cupule following pro-
cedures in the kit. Color change was observed after 24-h of incu-
bation at ambient temperature (28  C). A reading table for positive
and negative results of the different tests was referred to the kit.
3. Results and discussion
3.1. Isolation of bacteria
Isolation of bacteria from G. heteroclada thalli was initially car-
ried out based on the work of researchers working on seaweed
diseases such as ice-ice (Largo et al., 1995) and rotten thalli (Lavilla-
Pitogo, 1992). Characteristic of bacteria found to be common to
these diseases was the formation of depression around bacterial
colonies or liquefaction on agar medium. In the present study,
bacterial colonies from unhealthy (rotten) and healthy thalli of
G. heteroclada were isolated separately. Bacterial samples revealed
various morphological and colonial characteristics. A total of 28
colonies (15 from whitening thalli and 13 from unaffected thalli)
were selected and isolated as pure culture. Two (2) groups of iso-
lates liquefy the agar (Fig. 2) and were considered the most
dominant among the bacterial cultures taken from the seaweed
material. These were identified as agar-digesting bacteria. These
bacteria have long been studied and described by Gran (1902) (in
Goresline, 1933) as group of bacteria that can liquefy seaweed agar
Fig. 3. Representative gram-negative bacteria (1000) isolated from whitening (A,C) and healthy (B,D) G. heteroclada thalli. (A) Isolate 01 identified as V. parahaemolyticus (B) Isolate
07 identified as V. parahaemolyticus (C) Isolate 02 identified as V. alginolyticus, (D) Isolate 06 identified as V. alginolyticus.
through extracellular enzymes. These were the basis of character-
ization of the isolates in this study.
3.2. Characterization of isolates
Morphological characterization of the isolated bacteria was
done based on colony and pigmentation on NA þ agar (Fig. 2).
Majority of the bacterial cultures developed a whitish pigmentation
on NA þ agar. One group of isolates (01, 04, 05, 07) were observed as
whitish irregular colonies with filamentous margin while another
group (02, 03, 06) exhibited small whitish circular colonies with
smooth margin. Most of the agar-digesting bacteria isolated from
other diseased seaweeds such as Kappaphycus alvarezii and
Eucheuma denticulatum (Largo et al., 1995) and Gracilaria spp.
(Lavilla-Pitogo, 1992) exhibited the same colonial characteristics
with the present study. Interestingly, most of the isolates liquefied
the agar medium. The same results support the previous findings of
Lundestad (Goresline, 1933) where organisms isolated from
seawater had the capacity to dissolve seaweed agar and liquefied
fish-agar slants. Agar polymer chains could have been cleaved by
enzymes and digested by organisms. With shorter polysaccharide
chains and digested agar molecules, the agar matrix could have
been loosely packed and altered. Thus, water might have been
“released” from the matrix as observed in the agar plates.
Results of the Gram-staining showed that all isolates were
gram-negative (Fig. 3). These were morphologically small curved
rods of approximately 0.5e1.0 mm in size. Although there are
relative abundance of these gram-positive bacteria (Jensen and
Fenical, 1995) that can be found from many tropical marine algae,
the present findings were consistent with the result of other studies
(Lavilla-Pitogo, 1992; Largo et al., 1995; Musa and Wei, 2008) that
 J.N. Martinez, P.I.P. Padilla / Marine Environmental Research 119 (2016) 156e160 159

Table 1
Biochemical characteristics of seven isolates from G. heteroclada thalli.

Characteristics Isolates code Ma Wb

01 02 03 04 05 06 07 VP VA VP VA

Cell morphology Rod Rod Rod Rod Rod Rod Rod Rod Rod Rod Rod
Gram reaction e e e e e e e e e e e
b-galactosidase e e e e e e e nd nd v e
Arginine dihydrolase e e þ e e e e e e e e
Lysine decarboxylase þ þ e þ þ þ þ þ þ v v
Ornithine decarboxylase þ þ e þ þ e þ þ þ þ v
Citrate utilization þ þ e þ þ e þ þ þ þ þ
H2S production e e e e e e e e e e e
Urease e e e e e e e e e v e
Tryptophane DeAminase þ þ e þ þ e þ nd nd nd nd
Indole production e e e e e e e þ þ þ þ
Acetoin production e e e e e e e e þ v þ
Gelatinase þ þ þ þ þ þ þ þ þ þ þ
Glucose þ þ þ þ þ þ þ þ þ þ þ
Mannitol þ þ þ þ þ þ þ þ þ þ þ
Inositol e e e e e e e nd nd e e
Sorbitol e e e e e e e nd nd e e
Rhamnose e e e e e e e nd nd nd nd
Saccharose e þ þ e e þ e e e nd nd
Melibiose e e e e e e e nd nd v e
Amygdalin e e e e e e e nd nd v e
Arabinose þ e e e þ e þ þ þ v e
Oxidase þ þ þ þ þ þ þ þ þ þ þ

Identification by API20E VP VA VA VP VP VA VP
Percent identification 95.5 94.2 96.6 86 99.5 97.7 99.5
Source of isolates W W H H H H H

VA- Vibrio alginolyticus; W- whitening thalli; VP- Vibrio parahaemolyticus; H - Healthy thalli; nd- no data; v- variable.
a
Molitoris et al. (1985).
b
West et al. (1986).

have isolated and identified most gram-negative bacteria from red
algae. These are group of bacteria that possessed thin cell wall
containing peptidoglycan layer and a periplasmic space that houses
a variety of hydrolytic enzymes for metabolism (Holt et al., 2000).
Biochemical characterization using API20E kit (Biomerieux)
identified 2 dominant species of vibrio from both rotten and
healthy thalli: Vibrio alginolyticus (94.2e97.7%) and Vibrio para-
haemolyticus (99.5%). Twenty biochemical tests using this kit are
shown in Table 1. The two identified bacteria were consistently
positive in eight biochemical tests: lysine decarboxylase, ornithine
decarboxylase, citrate utilization, tryptophan deaminase, gelati-
nase, glucose, mannitol and oxidase test. Other tests, which showed
positive result, were saccharose test for V. alginolyticus and arabi-
nose test for V. parahaemolyticus. All isolates from unhealthy thalli
consistently showed positive result for gelatinase test. This
biochemical characteristic of the isolates in the present investiga-
tion suggests an enzymatic activity that can be observed in the
liquefaction of the agar plates. Similar types of agar-digesting lu-
minous bacteria were isolated by Fukasawa et al. (1987) in which
isolate FLB-17 produces gelatinase, an exoenzyme that hydrolyses
gelatin.
Vibrios have been isolated from different species of seaweeds
(Vugia et al., 1997; Lakshmanaperumalsamy and Purushothaman,
1982). These are associated with most red algae such as Gracilaria
species (Lavilla-Pitogo, 1992; Musa and Wei, 2008; Wang et al.,
2009) and Kappaphycus and Eucheuma (Largo et al., 1995). It is
not known, however, if the same species of vibrio isolated by
Lavilla-Pitogo (1992) occured in G. heteroclada used in this study.
Musa and Wei (2008) reported and identified V. alginolyticus in
Gracilaria changii branches, which was isolated also from
G. heteroclada thalli in the present study. The presence of
V. parahaemolyticus in seaweeds was also reported by Mahmud
et al. (2007) and found the correlation of their occurrence to
temperature as it changes with season.
These characteristics of V. parahaemolyticus and V. alginolyticus
isolated and identified through biochemical characterization
consistently conform to the characteristics of vibrio isolated from
the study of Molitoris et al. (1985) and West et al. (1986) (Table 1).
4. Conclusions
From the foregoing, isolation and characterization of bacteria
from rotten thallus of G. heteroclada has been demonstrated based
on morphological and biochemical characteristics. Vibrios isolated
from rotten thallus of Gracilaria were considered agar digesters
since it formed depression around the colonies and liquefied the
nutrient agar. Infection bioassay should be done to ascertain the
role of the bacteria in the physical and cellular appearance of the
thalli in terms of necrosis or rottening.
Acknowledgments
This study was supported by the University of the Philippines
Visayas Research and Extension Office and University of St. La Salle
Bacolod, Philippines under the faculty development program. We
are grateful to Ms. Ma. Rovilla J. Luhan, Hananiah Sollesta and
Dianne Aster Yungque of SEAFDEC-AQD for the supervision during
the lab work. Thank you to Dr. Rolando Pakingking and Dr. Resur-
reccion Sadaba for helpful comments on this paper.
References
Chan, C., Hong, C., Phang, S., 2006. Trends in seaweed research. In: Trends in Plant
Research, pp. 165e166.
Correa, J.A., 1996. Diseases in seaweeds: an introduction. Hydrobiologia 326e327
(1), 87e88.
Craigie, J.S., Correa, J.A., 1996. Etiology of infectious diseases in cultivated Chondrus
crispus. Hydrobiologia 326e327 (1), 97e104.
160 J.N. Martinez, P.I.P. Padilla / Marine Environmental Research 119 (2016) 156e160
Fukasawa, S., Dunlap, V., Baba, M., Osumi, M., 1987. Identification of an agar-
digesting, luminous bacterium. Agric. Biol. Chem. 51 (1), 265e268.
Goresline, H.E., 1933. Studies on Agar-digesting Bacteria. Iowa State College.
Holt, J., Kreig, N., Sneath, P., Staley, J., Williams, S., 2000. Bergey’s Manual of
Determinative Bacteriology. Lippincott-Williams & Wilkins, Philadelphia, USA.
Hurtado-Ponce, A., 1995. Research on seaweeds and mollusks. In: Bagarinao, T.,
Flores, E. (Eds.), Towards Sustainable Aquaculture in Southeast Asia and Japan,
ADSEA ’94 Proceedings. SEAFDEC Aquaculture Department, Iloilo City,
pp. 199e208.
Jensen, P.R., Fenical, W., 1995. The relative abundance and seawater requirements of
gram-positive bacteria in near-shore tropical marine samples. Microb. Ecol. 29
(3), 249e257.
Lakshmanaperumalsamy, P., Purushothaman, A., 1982. Heterotrophic bacteria
associated with seaweed. Proc. Indian Acad. Sci. 91, 487e493.
Largo, D.B., 2002. Recent developments in seaweed diseases. In: Hurtado, A.Q.,
Guanzon Jr., N.G., de Castro-Mallare, T.R., Luhan, M.R.J. (Eds.), Proceedings of the
National Seaweed Planning Workshop. SEAFDEC Aquaculture Department,
Tigbauan, Iloilo, pp. 35e42.
Largo, D.B., Fukami, K., Nishijima, T., 1995. Occasional pathogenic bacteria pro-
moting ice-ice disease in carageenan-producing algae Kappaphycus alvarezii
and Eucheuma denticulatum (Solieriaceae, Gigartinales, Rhodophyta). J. Appl.
Phycol. 7, 545e554.
Lavilla-Pitogo, C.R., 1992. Agar-digesting bacteria associated with “rotten thallus
syndrome” of Gracilaria sp. Aquaculture 102, 1e7.
Luhan, M.R.J., 1992. Agar yield and gel strength of Gracilaria heteroclada collected
from Iloilo, Central Philippines. Bot. Mar. 35, 169e172.
Mahmud, Z.H., Neogi, S.B., Kassu, A., Wada, T., Islam, M.S., Nair, G.B., Ota, F., 2007.
Seaweeds as a reservoir for diverse Vibrio parahaemolyticus populations in
Japan. Int. J. Food Microbiol. 118, 92e96.
Molitoris, E., Joseph, S.W., Krichevsky, M.I., Sindhuhardja, W., Colwell, R.R., 1985.
Characterization and distribution of Vibrio alginolyticus and Vibrio para-
haemolyticus isolated in Indonesia. Appl. Environ. Microbiol. 50 (6), 1388e1394.
Musa, N., Wei, L.S., 2008. Bacteria attached on cultured seaweed Gracilaria changii at
Mengabang Telipot, Terengganu. Acad. J. Plant Sci. 1 (1), 01e04.
Stirk, W.A., Van Staden, J., 2000. Removal of heavy metals from solution using dried
brown seaweed material. Bot. Mar. 43 (5), 467e473.
Vugia, D.J., Shefer, A.M., Douglas, J., Greene, K.D., Bryant, R.G., Werner, A.S., 1997.
Cholera from raw seaweed transported from the Philippines. J. Clin. Microbiol.
35 (1), 284e285.
Wang, Z., Xiao, T., Pang, S., Liu, M., Yue, H., 2009. Isolation and identification of
bacteria associated with the surfaces of several algal species. Chin. J. Oceanol.
Limnol. 27 (3), 487e492.
Weinberger, F., Friedlander, M., Gunkel, W., 1994. A bacterial facultative parasite of
Gracilaria conferta. Dis. Aquat. Org. 18 (2), 135e141.
West, P.A., Brayton, P.R., Bryant, T.N., Colwell, R.R., 1986. Numerical Taxonomy of
Vibrios isolated from aquatic environments. Int. J. Syst. Bacteriol. 36 (4),
531e543.