Exploration of Pectin – Utilizing Yeast from Soil of Bogor and Wleri Fruit Orchards

There is a high demand on pectin–utilizing yeasts for industrial, agricultural and environmental purposes. Further exploration of yeast from various sources are important to enrich yeast culture collections. Nine yeast strains were isolated from various soil sources sampled based on biological sampling in Bogor and Central Java. Enriched media containing pectin as carbon sources was employed for isolation of the yeast. The isolated yeast were identified according to the methods described in monographs by Kreger – Van Rij (1984), Barnett et al. (2000), Guilliermond and Tanner (2006) . The strains isolated were taxonomically separated into 3 groups. Group I contains 3 strains, and this group is closely related to Candida tropicalis. Group II contains 4 strains, and this group is included in this genus Rhodotorula. Group III contain 2 strains, and this group is closely related to Williopsis saturnus, which is a synonym of Hansenula saturnus. Pectinolytic enzymes (Polygalacturonase) were produced by all of the tested strains. Polygalacturonase was produced as high as 1.7 U.ml -1 by strain no. 111 of group I, 1.7 U.ml -1 by strains no. 123 of group II, and 1.0 U.ml -1 by strain no. 211 of group III.


INTRODUCTION
Yeasts are eucaryotic microorganism, also may be defined as uni cellular fungi reproduced by budding or fission.Flagel (1977) has pointed the advantages of this morphological definition which leaves out others such as historical consideration discussed by Lodder and Kreger-Van Rij (1952).Budding yeast cells may be stage in the life cycle of multicellular fungi (Kreger-Van Rij, 1984).Yeasts are widely distributed in nature, can be found in the soil, water, fermented food, plant materials such as leaves and fruits, and also can be found in coastal water and sediments (Kimura et.al., 1985).
Exploration of yeast from various sources is important to increase yeast collection (Hazra, 2005).Some workers tend to use available yeasts for their study.On the other hand, there is a high demand on yeasts for industrial, agricultural, and environmental purposes.Bacteria and fungi are commonly used by many workers to study pectolytic enzymes.However, studies on the enzymes production in yeast was quite limited.Some studies have been conducted from yeast of Kluyveromyces fragilis (Sakai et al., 1984), from Galactomyces reesei (Sakai and Yoshitake, 1984), from Trichosporon penicillatum (Sakai et al., 1982;Sakai and Okushima, 1982), from Saccharomyces fragilis (Lim et al., 1980).
Isolated yeasts are likely to produce pectinolytic enzymes (Kaur et al., 2004;Leizerson and Shimoni, 2005), as they are able to utilize pectin as a sole carbon source.Pectinolytic enzymes were reported to be produced extracellularly (Schomburg and Salzmann, 1991).Improved strains are important to be developed.For that purpose, it is necessary to increase various strains by appropriate methods such as isolation which is parallel to genetic engineering.
Isolates of yeasts were identified based on their morphology, physiology, and biochemical (Kreger-Van Rij, 1984;Barnett et al., 2000).The result can be compared with the taxonomic describtion of pectin or pectic substance utilizing yeast identified previously.In this study, Production of pectinolytic enzyme (polygalacturonase) was investigated.

Source and Sampling
Soil as sources of yeasts used for this experiment was obtained from Bogor and Central Java.The sample of soil was collected from fruit plantation (mango, papaya, and banana), soil, and soil from garden.

Isolation of Yeasts
Yeasts were isolated from soil samples by using a medium containing (per litre) 5g pectin, 4 g (NH 4 ) 2 SO 4 , 2 g KH 2 PO 4 , 1 g K 2 HPO 4 , 0.4 g MgSO 4 .7H 2 O, 3 mg Fe citrate, 0.5 mg MnSO 4 .4H 2 O, 0.05 mg CuSO 4 .5H 2 O, 0.4 mg ZnSO 4 .7H 2 O, 0.2 g yeast extract, pH of 5.0 (isolated medium).Enrichment culture were carried out for the isolation of pectin assimilating yeasts.Large test tubes containing 15 ml of enriched medium were inoculated aseptically with approximately one gram of sample soil.This culture was shaked and incubated aerobically at 28 o C for four days, then transferred to another test tube containing 1ml enriched medium.
Afterward, enriched cultures were streaked on plate of isolation medium containing 2% agar, and then incubated at 28 o C for three days.Isolated yeast strains were maintained using YM medium containing 2% agar.Pure culture of the yeasts were obtained by means of streaking techniques.The culture of each soil sample was repeated three times.

Identification of Yeasts
The strains used for this study are listed in The yeasts isolated were tested for their characteristics of vegetative reproduction, sexual characteristics by the methods described by Kreger-Van Rij (1984), Barnett et al. (2000), and Guilliermond and Tanner (2006).

Investigation of Pectinolytic Enzyme
One strain were selected from each group (strain no. 111, 123, and 211), and determined for production of pectinolytic enzyme (polygalacturonase).The test was based on the method previously described by Endo (1961), Kozaki et al. (1980) and Kaur et al. (2004), with some modification.Media containing 2% pectin (sigma), 0.6% peptone, 0.2% yeast extract were used for the production of the pectinolytic enzyme.One unit of Polygalacturonase (PGase) is defined as the amount of enzyme that liberates 1 µmol of galacturonic acid min -1 ml -1 under the assay conditions.

RESULTS AND DISCUSSION
Nine yeast strains have been isolated from soil.Soil sampled in fruit plantation and soil from garden in west Java and Central Java.Most isolated yeasts were obtained from medium containing pectin with enrichment culture.The source and location of strains were listed in Table 1.
The first identification test comprised Urease test, DNase test (Sen and Komagata, 1979), DBB test, and YFG test.The result indicated that there are some yeasts that incapable of fermenting Dglucose (Table 2).
Test of morphological characteristics by using the method previously described by Kreger-Van Rij (1984) indicate that there were differences in colour of colony and cell shape, and the preserce of pseudo or true mycelium also presence.These characteristics could be used in a yeast grouping (Table 2 and Table 3).
Based on their physiological and biochemical characteristics identified with involved the method previously describe by Barnett et al. (2000), Guilliermond and Tanner (2006) suggested that the isolated strains could be devided into three groups (Table 4).
From three groups, one strain representing the groups were selected to test whether they have pectinolytic enzyme or not.The presence of pectinolytic enzyme such as polygalacturonase was investigated.
Based on their physiological and biochemical characteristics identified with involved the method previously describe by Barnett et al. (2000), Guilliermond and Tanner (2006) suggested that the isolated strains could be devided into three groups (Table 4).
From three groups, one strain representing the groups were selected to test whether they have pectinolytic enzyme or not.The presence of pectinolytic enzyme such as polygalacturonase was investigated.
w : weak vw : very weak Group I consisted of 3 strains, group II had 4 strains, and group III had 2 strains (Table 3 and  Table 4).Each group is discussed separately as follows:

Group I
All strains in this group had negative respons to urease test, DBB test, and extracelluler DNase test (Table 2).The yeast culture growth liquid medium produced pellicle, except strain no.131 that produced a ring on the surface of the medium.The strains produced pseudomycelia and were likely to form septate hyphae.Comparing the observed yeast characteristics with that described by Banett et al. (2000), group I was closely related to Candida tropicalis.The yeasts formed a septate hyphae, had a similar fermentation pattern and grew well at a temperature of 40 o C.
Physiological and biochemical characteristics of group I were similar to those of C. tropicalis.However, the growth of strains in group I in lactose medium were positive, in starch were negative, in glucosamine (N) were positive or weak, and in 50% D-glucose were negative or very weak or weak.There were a small differences among the strain in group I in assimilating L-rhamnose, salicin, and galactitol (Table 4).The strain no. 111 exhibited a very weak response, whereas the other strains showed a negative response.The strains in group I were interesting in the growth at a temperature of 40 0 C. Their adaptability to high temperature seemed to be related to sampling site, as the sources were obtained from tropical region.C. tropicalis as reported by Barnett et al. (2000), was found in rotten fruit, soil, and soil from fruit plantation, where all strains in group I were selected.

Group II
The strains in this group showed a negative response on fermentation test and a similar result in aerobic utilization test.There was an exception result found in starch assimilation test in no.121, which had a weak response.Strain formed a multilateral budding and might have capsulated Comparing result of morphological and physiological characteristization with those described by Kreger-Van Rij (1984) and Barnett et al (2000), the strains in group II were similar with genus Rhodotorula.The spesies in genus Rhodotorula Harrison had characteristics as follow : multilateral budding, the cultures were red or yellow due to carotenoid pigments, the cultures were often mucous, had no fermentation, nitrat was assimilated or not, inositol was not assimilated, starch-like compounds were not produced, urease test was positive.Strain in group II had speciality over the other groups as they assimilated lactose and inositol as carbon sources.However, some species in genus Rhodotorula assimilated lactose, but did not assimilated inositol.

Group III
Isolated strains included into group III had different characteristic when they were compared with other groups, especially in nitrate and nitrite assimilation showed by a positive reaction.The strains in this group had a similar morphological and physiological characteristics.Inulin test failed to demonstrate wether they assimilated the inulin or not.Kreger-Van Rij (1984) describe that genus Hansenula H. and P. Sydow had characteristics as follow : multilateral budding; pseudo or true mycelium might be present; ascospore hat-saturnshaped or hemispherical were generally liberated; fermentation was present or absent; and assimilated nitrate.These description were very close to the morphological and psychological characteristics of isolated strain in group III, therefore the could be classified as genus Hansenula.
The isolated strains had also evanescent and persistent asci, containing 1 to 4 saturn-shape ascospore.This result showed that the strains were closely related to species Williopsis saturnus (Barnett et.al., 2000) which was the synonyms of Hansenula saturnus var.saturnus and Hansenula saturnus var.subsufficiens.Characteristics of isolated strains in group III differed slightly from species Williopsis saturnus.The strains in group III fermented cellobiose but the response was very weak (Table 4).The other characteristics were similar.

Pectinolytic Enzyme
The enzymes that hydrolaise pectic substances are broadly known as pectinolytic enzymes or pectinases, which include polygalacturonase (PGase), pectin esterase, pectin lyase, and pectate lyase on the basis of their mode of action (Alkorta et al. 1998).Pectinase have widespread application in food and textile industries (Henriksson et al. 1999).
The activity of polygalacturonase enzyme was observed in group I, II, III.These groups produced polygalacturonase, if pectin was used as a carbon source for enzyme production.Strains tested exhibited low activity (1.0-1.7 U.ml -1 ).Serrat et al. (2004) reported the activity of poligalacturonase of 4.19 U.ml -1 in YNB (Difco)glucose medium from the Kluyveromyces marxianus CCEBI 2011 yeast strain.However, polygalacturonase production by the thermophilic mould Sporotrichum thermophile Apinis was high (about 30 U.ml -1 ) in pectin 2% (Kaur et al., 2004).

CONCLUSIONS
The strains isolated were taxonomically separated into 3 groups.Group I contains 3 strains, and this group is closely related to Candida tropicalis.Group II contains 4 strains, and this group is included in genus Rhodotorula.Group III contains 2 strains, and this group is closely related to Williopsis saturnus, which is a synonym of Hansenula saturnus.Pectinolytic enzymes (Polygalacturonase) were produced by all of the tested strains.Polygalacturonase was produced as high as 1.7 U.ml -1 by strain no. 111 of group I, 1.7 U.ml -1 by strains no.123 of group II, and 1.0 U.ml -1 by strain no.211 of group III, respectively.

Table 1 .
Source of isolates

Table 4 .
Physiological and biochemical characteristic of isolated strains in group I, group II, and group III

Table 4 .
Physiological and biochemical characteristic of isolated strains in group I, group II, and group III (continous…) cells, the culture on solid media are generally mucous.Color of the colony was brownish-red.There was no fermentation, and no utlization of nitrate and nitrite.Positive reaction were found for urease, DBB reaction, and DNase tests (Table2).