Actinomycetes Community from Starch Factory Wastewater
The aim of the research was to study the biodiversity, antimicrobial activity and ability to degrade starch among selected actinomycetes isolates from starch wastewater of rice vermicelli factory. Thirty distinct actinomycetes were identified using their 16S rDNA sequences. Twenty eight strains were classified as Streptomyces spp. and two other as Norcardia spp. Each of the actinomycetes isolates was tested for their ability to inhibit the growth of other indicator strains, Staphylococcus aureus ATCC25923, Escherichia coli ATCC25922 and other two and three phytopathogenic bacteria and fungi. Only one isolate designated WPS132, displayed inhibitory properties against both tested phytopathogenic bacteria and fungi. Phylogenetic analysis showed that this strain was genetically closely related to S. antibioticus strain HBUM 174911. The isolates were also tested for their ability to degrade starch. All strains were capable of degrading starch but strain WPS005 showed the highest amylase production at 32.4 U mL-1. We concluded that actinomycetes can be found abundantly from wastewater of the rice vermicelli factory, however, isolates with antimicrobial activities were observed at a low frequency.
to cite this article:
Parichat Saenna, Thomas Gilbreath, Nuttapol Onpan and Watanalai Panbangred, 2011. Actinomycetes Community from Starch Factory Wastewater. Research Journal of Microbiology, 6: 534-542.
Received: March 04, 2011;
Accepted: May 02, 2011;
Published: June 23, 2011
The microbial taxonomic composition in each environmental community is an important
indicator of their ecology and function. The biodiversity of bacteria in particular
environments can provide insight into what conditions accommodate microorganisms
of interest. This information can lead to a better understanding of their function
and the environment in which they inhabit (Ghosh et al.,
The actinomycetes represent a well-known and extremely diverse group of Gram-positive,
filamentous bacteria belonging to the order Actinomycetales. Most actinomycetes
produce a diverse mixture of hydrolytic enzymes that permit the utilization
of various kinds of organic compounds such as starch, cellulose and hemicelluloses.
They are recognized as a source of a wide variety of bioactive compounds. Actinomycetes,
especially Streptomyces, have been most widely studied for the versatility
and diversity of useful metabolites they can produce (Berdy,
2005). Thus, most study on actinomycetes was focused on their potential
in producing biological compounds (Kumar et al.,
2011; Kitouni et al., 2005) rather than their
function in any specific environment (Bensultana et al.,
2010; Rintala et al., 2002).
Thailand is an agricultural country with many economically important crops
such as rice, corn and cassava which are grown and exported. Not only are these
agricultural products exported as raw materials but processed products, especially
tapioca and rice starches, are produced and exported in large quantities in
each year (Sriroth et al., 2000). Thus every
day, factories processing these agricultural products generate tons of by-products
and other wastes. Industrially occurring starch waste is collected in aerobic
ponds and prior to the treatment steps required for disposal and may serve as
a good source for beneficial bacteria. Generally wastewater discharge from vermicelli
factories contain suspended organic pollutants in much concentrations than domestic
wastewater and are especially high in starch (Hu, 1989).
The accumulation of these compounds in an open-pond system may pose a risk to
the health of people living in close proximity to the factory, both due to the
nature of the pollutants as well as the potential for the growth of dangerous
aquatic organisms. In contrast, these same environments may be capable of supporting
several types of bacteria that produce many useful metabolites using high levels
of starch as a carbon source (Thavasi et al., 2006).
Several groups have isolated actinomycetes producing valuable compounds from
varying sources (El-Shirbiny et al., 2007). The
richness of actinomycetes in an environment appears to reflect the abundance
of the resources as well as the waste treatment process. Recently, actinomycetes
from a sand bed wastewater filter were screened for the production of antibacterial
compounds to investigate their role in removing wastewater-associates pathogens
in this environment. Several other actinomycetes have also been reported to
effectively control bacterial and fungal pathogens of agriculturally important
crops (Prapagdee et al., 2008). However, there
are only a few reports on the testing of these actinomycetes strains against
agricultural pathogens (Rizk et al., 2007).
In this study, actinomycetes from the open-pond lagoon wastewater system of
a rice vermicelli factory were isolated and identified. In order to estimate
the diversity of isolated actinomycetes, an analysis of 16S rDNA sequence which
has been proven to be an effective tool for actinomycetes discrimination, was
employed (Thampayak et al., 2008). The objective
of the present study was to analyze their biodiversity, testing their capacity
to degrade starch, as well as their inhibitory activity against phytopathogenic
MATERIALS AND METHODS
The study was conducted during October 2009-April 2010 at Faculty of Science, Mahidol University, Bangkok, Thailand.
Actinomycetes strains and culture conditions: Actinomycetes strains
in this study were isolated from 4 wastewater samples containing blackish muddy
soil. The samples were collected from a single aerobic pond containing a large
amount of starch waste. Three procedures were used to obtain variety of actinomycetes
strains from the wastewater, filtration of water samples, serial dilution of
sediment and heating of sediment followed by serial dilution. (1) Water samples
were filtered through a 0.45 μm pore size membrane and the membrane was
placed on screening medium and removed after 4 days of incubation (Hirsch
and Christensen, 1983), (2) 1 g of sediments and soil sample were diluted
with 9 mL of 0.85% NaCl and dilutions of this mixture were then spread on the
screening medium, (3) Sediment samples were heated at 80°C for 60 min prior
to being serially diluted and spread on to the screening medium. Screening for
actinomycetes was performed using water-proline selective medium (1% proline,
1.2% agar and tap water) and Pridhams agar (1% glucose, 1% glucose, 0.2%
(NH4)SO4, 0.3% CaCO3, 0.1% K2PHO4,
0.1% NaCl, 1.2% agar and deionized water). Media were supplemented with cycloheximide
(50 μg mL-1) and nalidixic acid (20 μg mL-1)
to inhibit the growth of fungi and other bacteria, respectively. Plates were
incubated at 30°C for up to one month. Each isolate was purified by streaking
on Waksmans agar (Waksman et al., 1946)
(1% glucose, 0.5% peptone, 0.5% meat extract, 0.3% NaCl, 1.2% agar and deionized
water) and were maintained at 30°C for 7 days for colony development and
general morphology examination. Strains were maintained on slants of Waksmans
agar and Seino agar (1% starch, 0.3% N-Z amine, 0.3% yeast extract, 0.1% meat
extract, 1.2% agar and deionized water) and long term storage was in 20% glycerol
Characterizationf and molecular identification of actinomycetes: The
number of actinomycetes species presented in an extremely polluted open-pond
wastewater system of a starch factory was investigated, using a plate-culturable
method (Zin et al., 2010). The actinomycetes
isolates were characterized according to their general macromorphology, growth
characteristics, colors of aerial spore mass, substrate mycelium and the soluble
pigments production (Williams et al., 1983) in
both Waksmans and Seino agar plates. The 30 distinct actinomycetes isolates
were then further characterized by examining the sequences of the 16S rRNA gene.
Chromosomal DNA used as the template for PCR was prepared by a simple boiling
method using 16S rRNA gene universal primers UFUL and URUL as forward and reverse
primers (Avaniss-Aghajani et al., 1996), respectively.
The 16S rDNA sequences of actinomycetes isolates were deposited in GenBank under
the accession numbers GU581285-GU581314.
Phylogenetic tree analysis: Ribosomal gene sequences from the actinomycetes
isolates were compared with those available in Genbank database (http://www.ncbi.nih.gov).
Multiple sequence alignments and phylogenetic tree construction was performed
by using the MEGA 4 program (Tamura et al., 2007).
Determination of genetic distances among the sequences was carried out by the
Neighbor-Joining method (Saitou and Nei, 1987). Bootstrap
analysis with the resembling method was performed as previously described (Felsenstein,
1985) with 1,000 replications.
Detection of antibacterial and antifungal activities: The actinomycetes
isolates were also tested for antibacterial and antifungal activities against
both standard strains and phytopathogenic strains using co-cultivation method
(Bredholdt, et al., 2007) as shown in Table
|| Indicator bacteria and Fungi
|aATCC: American Type Culture Collection, DOA: Department
of Agriculture, Ministry of Agriculture and Co-operatives, Thailand
Bacterial (Xanthomonas campestri and Erwinia carotovora) and
fungal (Colletotrichum gloeosporioides DOA d0762, C. gloeosporioides
DOA c1060 and C. capsici DOA c1511) pathogens were purchased from Bacterial
and Fungal laboratories, Plant Pathology and Microbiology Division, Department
of Agriculture, Ministry of Agriculture and Co-operatives. Antifungal activity
was evaluated as the distance from the edge of colonies (Bredholdt
et al., 2007).
Starch degradation and determination of α-amylase activity: The
capacity of the actinomycetes isolates to degrade starch was monitored by observing
starch hydrolysis (Seibold et al., 2006). Isolates
producing clear zone were picked and further analyzed for amylase activity in
α-Amylase activity in culture supernatants was assayed by measuring the
release of reducing sugars (Miller, 1959; Seibold
et al., 2006).
RESULTS AND DISCUSSION
Morphology and genetic assignment of actinomycetes isolates: Different
isolation methods coupled with selective media and culturing conditions were
used to segregate actinomycetes species. In such polluted wastewater, 135 actinomycetes
isolates were successively recovered from different samples, a large number
of which were obtained from slightly basidic conditions (pH 8, data not shown).
These results confirm that many actinomycetes can be found in neutral or slightly
alkaline environments (Basilio et al., 2003;
Selyanin et al., 2005). In addition, the filamentous
in nature of bacteria in this group, increasing the probability of discovering
actinomycetes from soil or sediment samples compared with water samples (Bensultana
et al., 2010). In this study, more numbers of isolates were found
in sediment (117/135) than in wastewater (18/135) which coincided with the above
report. Based on the present investigation of colony morphology, such as shape,
edge and elevation characteristics, as well as colony color, 30 distinct actinomycetes
isolates were selected for further characterization by sequencing the 16S rRNA
gene. Based on this analysis, 28 isolates were identified as belonging to Streptomyces
spp. (96-100% similarity) and 2 were Norcardia spp. (99.5% similarity)
(Table 2). Streptomyces spp. were also found predominantly
among actinomycetes isolated from starch wastewater in other study (Tapia
and Simoes, 2008). Phylogenetic tree relationship of the isolates and other
well-characterized actinomycetes species was shown in Fig. 1.
The 28 isolates belonging to Streptomyces were highly diverse and affiliated
into 10 different cluster groups. The major cluster of Streptomyces isolates
was classified into the following groups, S. massasporreus (9 isolates,
cluster I), S. griseoloalbus (5 isolates, cluster IV), S. fradiac
(3 isolates, cluster VI), S. tritolerans (3 isolates, cluster IX)
and S. thermolineatus (3 isolates, cluster X), whereas 5 isolates were
singly affiliated into clusters (cluster II, III, V, VII and VIII, respectively).
The dispersion of isolates in various clusters may reflect the abundance of
Streptomyces which can be found in any environments (Kitouni
et al., 2005), including the open-pond wastewater system examined
in this study. Thus, the high frequency of Streptomyces in the waste
environment may indicate that they are well adapted to live in diverse conditions.
Of interest to concern regarding potential hazards to human health was the identification
of two isolates of Norcardia that were closely related to N. brasiliensis
(Fig. 1) an opportunistic human pathogen known to cause inflammatory
disease (Fukuda et al., 2008).
Antibacterial and antifungal activities: Seventeen of the actinomycetes
isolates failed to show any inhibitory activity against the indicator microorganisms
and none of the isolates displayed inhibitory activity against E. coli.
||Neighbor-joining tree based on distance analysis representing
relationship between the 16S rDNA sequences of 30 isolates derived from
waste reservoir of starch factory. Thirteen sequences of actinomycetes were
obtained from Genbank. Bootstrap values generated from 1,000 replicates
are shown at the nodes (values over 50% were shown). The sequence from Bacillus
cereus strain ATCC 10987 was used as the out-group species. Saenna et
However, four isolates were capable of inhibiting the growth of S. aureus
and five were inhibitory to both S. aureus and fungi (DOA d0762 and DOA
c1511). In addition, 7 isolates were active against at least one of indicator
fungi (Table 2). Only the WPS 132 isolate showed the activity
against both indicator phytopathogenic bacteria and fungi but though its inhibitory
activity was rather weak.
|| Actinomycete isolates and their amylase and antimicrobial
activities against indicator microorganisms
|1Activity was measured in cell free culture broth.
2a, S. aureus ATCC 25923; b, E. coli ATCC 25922;
c, X. campestris pv campestris; d, E.carotovota pv
carotovora; e, C. gloeosporioides DOA c1060; f, C. gloeosporioides
DOA d0762; g, C. gloeosporioides DOA c1511. The number in parenthesis
indicates the zone of inhibition (in mm) including diameter of cork borer
(for bacteria a-d) or distance between edges of fungal colony and actinomycetes
agar block (in mm) at day 7 (for fungi e-g) (see Materials and Methods).
3No activity against all indicator microorganisms
The compound(s) which is involved in the inhibitory activity was not determined
but the nature of the antibacterial and antifungal compounds may be distinct
in each actinomycetes strain (Bredholdt et al., 2007;
Egan et al., 2001). Interestingly, one of the
actinomycetes strain isolated falls into the same cluster as S. antibioticus
(Fig. 1) which is capable of producing actinomycin (Fawaz
and Jones, 1988). Since very few of the actinomycetes isolates displayed
any antagonistic activity against indicator microorganisms, it is suggested
that these strains have most likely adapted themselves to live in the constrained
environment of the open-pond wastewater system where competition with other
microorganisms is limited (Bensultana et al., 2010).
The similar finding of low proportion of actinomycetes or other bacteria with
antimicrobial activity from waste reservoirs were also reported (Schomburg
and Muller, 1984; Bal and Dhagat, 2001). Furthermore,
all of these isolates showed low activity in starch degradation even though
they were isolated from conditions containing high level of starch deposition.
The composition of waste and pH had an effect on the activity of amylase (Arotupin,
2007). In S. rimosus both α-amylase and glucoamylase activities
were lower in submerged culture when compared with solid state cultivation (Yang
and Wang, 1999). The low amylase activity of actinomycetes from starch wastewater
in this study might be due to both culturing medium composition and culture
condition. In addition, bacteria in this group are known to have decreased amylase
production in liquid cultures due to fragmentation of mycelium which generally
occurs in Streptomyces (Simpson and McCoy, 1953).
This is the first report in Thailand to show that actinomycetes can be isolated from an open-pond waste system. The majority of the actinomycetes isolated are Streptomyces though some are Nocardia and the Streptomyces isolates show a high level of diversity. However, a few of the isolates displayed antagonistic activity against indicator microorganisms. The results suggest that actinomycetes from starch waste pond have most likely adapted themselves to live in the polluted environment instead of producing compounds to inhibit the growth of other microorganisms. An important aspect of this finding is the identification of Nocardia in the factory wastewater. The result of this investigation highlights the need for awareness of the microorganism present to facilitate the waste treatment process and show the importance of increasing the understanding of the relationship of these bacteria in the waste environment.
Parichat Saenna is a recipient of the Promotion of Science and Mathematics Teacher Project. This work was supported in part by a research grant from Mahidol University. We wish to thank Saiyawit Voravinit and the officers from Cho Heng Rice Vermicelli factory for their kind assistance in sample collection and sharing information pertaining to the physiolochemical properties of the wastewater effluent. We also thanks Laran Jensen, Department of Biochemistry, Mahidol University, for critically read-proof of the manuscript.
Arotupin, D.J., 2007.
Evaluation of microorganisms from cassava waste water for production of amylase and cellulase. Res. J. Microbiol., 2: 475-480.CrossRef | Direct Link |
Avaniss-Aghajani, E., K. Jones, A. Holtzman, T. Aronson and N. Glover et al
Molecular technique for rapid identification of mycobacteria. J. Clin. Microbial., 34: 98-102.Direct Link |
Bal, A.S. and N.N. Dhagat, 2001.
Upflow anaerobic sludge blanket reactor-a review. Ind. J. Environ. Health, 43: 1-82.Direct Link |
Basilio, A., I. Gonzalez, M.F. Vicente, J. Gorrochategui, A. Cabello, A. Gonzalez and O. Genilloud, 2003.
Patterns of antimicrobial activities from soil actinomycetes isolated under different conditions of pH and salinity. J. Applied Microbiol., 95: 814-823.CrossRef | PubMed |
Bensultana, A., Y. Ouhdouch, L. Hassani, N.E. Mezrioui and L. Rafouk, 2010.
Isolation and characterization of wastewater sand filter actinomycetes. World J. Microbiol. Biotechnol., 26: 481-487.CrossRef |
Berdy, J., 2005.
Bioactive microbial metabolites: A personal view. J. Antibiot., 58: 1-26.CrossRef | Direct Link |
Bredholdt, H., O.A. Galatenko, K. Engelhardt, E. Fjrvik, L.P. Terekhova and S.B. Zotchev, 2007.
Rare actinomycete bacteria from the shallow water sediments of the Trondheim fjord, Norway: Isolation, diversity and biological activity. Environ. Microbiol., 9: 2756-2764.CrossRef |
Egan, S., P. Wiener, D. Kallifidas and E.M.H. Wellington, 2001.
Phylogeny of Streptomyces species and evidence for horizontal transfer of entire and partial antibiotic gene clusters. Antonie Van Leeuwenhoek, 79: 127-133.CrossRef |
El-Shirbiny, S.A., M.F. Ghaly, Y.M. El-Ayoty and N.S. Fleafil, 2007.
Niromycin a: An antialgal substance produced by Streptomyces endus
N40. Res. J. Microbiol., 2: 606-608.CrossRef | Direct Link |
Fawaz, F. and G.H. Jones, 1988.
Actinomycin synthesis in Streptomyces antibioticus
. Purification and properties of a 3-hydroxyanthranilate 4-methyltransferase. J. Biol. Chem., 263: 4602-4606.PubMed |
Felsenstein, J., 1985.
Confidence limits on phylogenies: An approach using the bootstrap. Evolution, 39: 783-791.CrossRef | Direct Link |
Fukuda, H., A. Saotome, N. Usami, O. Urushibata and H. Mukai, 2008.
Lymphocutaneous type of nocardiosis caused by Nocardia brasiliensis
: A case report and review of primary cutaneous nocardiosis caused by N. brasiliensis
reported in Japan. J. Dermatol., 35: 346-353.PubMed |
Ghosh, A., B. Maity, K. Chakrabarti and D. Chattopadhyay, 2007.
Bacterial diversity of East Calcutta Wet land area: Possible identification of potential bacterial population for different biotechnological uses. Microbiol. Ecol., 54: 452-459.CrossRef | PubMed |
Hirsch, C.F. and D.L. Christensen, 1983.
Novel method for selective isolation of actinomycetes. Applied Environ. Microbiol., 46: 925-929.Direct Link |
Hu, T.L., 1989.
Treatment of vermicelli wastewater by an acid-tolerant, starch-degrading yeast. Biol. Wastes, 28: 163-174.CrossRef |
Kitouni, M., A. Boudemagh, L. Oulmi, S. Reghioua and F. Boughachiche et al
Isolation of actinomycetes producing bioactive substances from water, soil and tree bark samples of the North-East of Algeria. J. Med. Mycol., 15: 45-51.CrossRef | Direct Link |
Kumar, K.S., R. Haritha, Y.S.Y.V.J. Mohan and T. Ramana, 2011.
Screening of marine actinobacteria for antimicrobial compounds. Res. J. Microbiol., 6: 385-393.CrossRef |
Miller, G.L., 1959.
Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal. Chem., 31: 426-428.CrossRef | Direct Link |
Prapagdee, B., C. Kuekulvong and S. Mongkolsuk, 2008.
Antifungal potential of extracellular metabolites produced by Streptomyces hygroscopicus
against phytopathogenic fungi. Int. J. Biol. Sci., 4: 330-337.CrossRef | PubMed | Direct Link |
Rintala, H., A. Nevalainen and M. Suutari, 2002.
Diversity of streptomycetes in water-damaged building materials based on 16S rDNA sequences. Lett. Applied Microbiol., 34: 439-443.CrossRef | Direct Link |
Rizk, M., T. Abdel-Rahman and H. Metwally, 2007.
Screening of antagonistic activity in different Streptomyces
species against some pathogenic microorganisms. J. Boil. Sci., 7: 1418-1423.CrossRef | Direct Link |
Saitou, N. and M. Nei, 1987.
The neighbor-joining method: A new method for reconstructing phylogenetic trees. Mol. Biol. Evol., 4: 406-425.CrossRef | PubMed | Direct Link |
Selyanin, V.V., E.G. Oborotov, G.M. Zenova and D.G. Zvyagintsev, 2005.
Alkaliphilic soil actinomycetes. Microbiology, 76: 729-734.CrossRef |
Seibold, G., M. Auchter, S. Berens, J. Kalinowski and B.J. Eikmanns, 2006.
Utilization of soluble starch by a recombinant Corynebacterium glutamicum
strain: Growth and lysine production. J. Biotech., 124: 381-391.CrossRef |
Schomburg, I. and H.E. Muller, 1984.
Significance of antibiotic-forming microorganisms in biological waste water clarification. Zentralbl. Bakteriol. Mikrobiol. Hyg. B, 179: 162-169.PubMed |
Simpson, F.S. and E. McCoy, 1953.
The amylases of five streptomycetes. Applied Microbiol., 1: 228-236.Direct Link |
Sriroth, K., K. Piyachomkwan, S. Wanlapatit and C.G. Oates, 2000.
Cassava starch technology: The Thai experience. Starch, 52: 439-449.CrossRef | Direct Link |
Tamura, K., J. Dudley, M. Nei and S. Kumar, 2007.
MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Mol. Biol. Evol., 24: 1596-1599.CrossRef | PubMed | Direct Link |
Tapia, D.M.T. and M.L.G. Simoes, 2008.
Production and partial characterization of keratinase produced by a microorganism isolated from poultry processing plant wastewater. Afr. J. Biotechnol., 7: 296-300.Direct Link |
Thampayak, I., N. Cheeptham, W. Pathom-Aree, P. Leelapornpisid and S. Lumyong, 2008.
Isolation and identification of biosurfactant producing actinomycetes from soil. Res. J. Microbiol., 3: 499-507.CrossRef | Direct Link |
Thavasi, R., S. Jayalakshmi, T. Balasubramanian and I.M. Banat, 2006.
Biodegradation of crude oil by nitrogen fixing marine bacteria Azotobacter chroococcum
. Res. J. Microbiol., 1: 401-408.CrossRef | Direct Link |
Waksman, S.A., A. Schatz and H.C. Reilly, 1946.
Metabolism and the chemical nature of Streptomyces griseus
. J. Bact., 51: 753-759.PubMed |
Williams, S.T., M. Goodfellow, G. Alderson, E.M.H. Wellington, P.H.A. Sneath and M.J. Sackin, 1983.
Numerical classification of Streptomyces
and related genera. J. Gen. Microbiol., 129: 1743-1813.CrossRef | PubMed | Direct Link |
Yang, S.S. and J.Y. Wang, 1999.
Protease and amylase production of Streptomyces rimosus
in submerged and solid state cultivations. Bot. Bull. Acad. Sin., 40: 259-265.Direct Link |
Zin, N.M., C.S. Loi, N.M. Sarmin and A.N. Rosli, 2010.
Cultivation-dependent characterization of endophytic actinomycetes. Res. J. Microbiol., 5: 717-724.CrossRef | Direct Link |