ACTINOMYCETE DIVERSITY IN UNUSUAL HABITATS

Actinomycetes
University of Udine, Mycology Department
ISSN: 0732-0574
Vol. 4, Num. 2, 1993
Actinomycetes, Vol. 4, Part 2, 47-57, 1993 ACTINOMYCETE DIVERSITY IN UNUSUAL HABITATS C.JIANG and L.XU

Yunnan Institute of Microbiology, 650091 Kunming, China

Code Number: AC93007
File Sizes:
Text: 34K
Graphics: Line Drawing (Gif) - 33K

ABSTRACT. Results of a 10-year investigation on actinomycetes from unusual habitats of Yunnan, China, are reported. The study deals with alkalophilic, acidophilic, thermophilic and psychrophilic actinomycetes isolated from the different environments of the region. Strategies for studying actinomycetes in unusual habitats are discussed.

This paper is an abridged version of a note presented at the ICCC, Beijing, October 1992

Actinomycetes play quite an important role in natural ecological systems. In the past research work was mainly concentrated on actinomycetes present in common habitats, while comparatively less attention was devoted to those from unusual ones. The latter should have distinctive biological activities because of the adaptation to these habitats. Such activities can be of economic importance and in addition represent ideal materials to study evolution, phylogeny and adaptation mechanisms.

In Yunnan there are extremely complex climates including, from Helong River to Hailan Island, wide tropical, subtropical and frigid forests, alkaline soils and lakes, acid soils, and hundreds of hot springs. The present note summarises an over ten year-research work carried out on actinomycete diversity in unusual habitats of Yunnan.

Alkalophilic actinomycetes

Actinomycetes in an alkaline lake. Lake Chenghai, located in Yongsheng, Yunnan, 1,503m above sea level, is 20 km long and 4.3km wide and covers an area of 79 sq.km. The maximum depth is 37m (average 15m) and the lake is one of the largest on the Yunnan Plateau.

Forests around lake Chenghai were destroyed 300 years ago and since water input was scarce it became a "dead lake" with alkaline water (up to pH 9). The number of actinomycetes isolated from mud and water samples are given in Table 1.

Micromonospora species are dominant (88.3%) with streptomycetes contributing 7.3% of the total, while other actinomycete genera are represented by almost 4% of the isolates. The total number is lower than that in fresh water lakes (Jiang and Xu, 1985/6). Actinomycete frequency in shallow water plots (plot B) is not high, however the population is rather complex since eight genera are represented. The actinomycete composition is relatively uniform in deep water plots (plots C and D). No actinomycetes were isolated from the water samples.

Tests on 251 strains isolated from Lake Chenghai showed that 71.7% could grow in broth with 5.0% NaCl, nearly all grew at pH 9.0 and about 70% were able to develop at pH 10.5. However most of the isolates from Chenghai Lake should be considered alkaline-resistant organisms, with an optimum growth occurring between pH 7.0 and 9.0. Only 0.8% of them are true alkalophilic with an optimum growth between pH 9 and 10 and no growth at pH 7.0.

Actinomycetes in alkaline soils. In Xiangyun, Yunnan, there is a number of mineral springs rich in NaCO3 and farmers have been making alkali with spring water for 300 years. The soil around the springs is alkaline (pH 1012).

Two-hundred and eighty-four strains were isolated from soil samples. Forty-nine isolates and 10 reference cultures of Streptomyces obtained from ATCC and CCCCM subjected to cluster analysis by using 82 unit characters. All tested strains were recovered in two clusters (Fig. 1) at 66% similarity level (S-level) by using the S[sm] coefficient (UPGMA algorithm).

Cluster I. All isolates from alkaline soil fall into this cluster and can be grouped into three subclusters defined at 76% S-level.

Subcluster IA contains 19 strains defined at 83% S-level. Nine strains, with pH 12 as the upper limit for growth, were identified as Nocardiopsis species. The remaining 10, which cannot grow or only very weakly at pH 6.0, but grow well at pH 10-11, were classified as members of the genus Streptomyces.

Subcluster IB consists of 11 strains, with an optimum growth at pH 9-10, defined at 83% S-level. Six isolates are not able to grow at pH 6.0.

Subcluster IC is made up of 19 strains defined at 79% S-level. Growth is good at pH 9-10, scarce at pH 6.0, except for strain 13867 which does not grow at pH 6.0.

Cluster II. This cluster contains reference cultures of the genus Streptomyces obtained from ATCC and CCCCM.

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ORGANISM PLOT MEAN %
A B C D E
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Micromonospora
aurantiaca 97.4 121.6 315.3 205.1 363.6 220.6 56.9
Micromonospora

nigrofusca 59.7 56.5 153.2 179.5 272.7 144.3 37.3 Micromonospora violacea 22.0 3.4 49.5 4.3 17.7 19.7 5.0 Micromonospora viridis 3.1 1.7 4.3 5.1 2.8 0.7 ------------------------------------------------------------------- SUBTOTAL 182.2 183.2 518.0 393.2 659.1 387.4 88.3 ------------------------------------------------------------------- Streptomyces 6.3 85.6 22.5 12.8 32.8 32.0 7.3 Actinosporangium 5.1 1.0 0.2 Actinomadura 9.4 3.4 2.6 0.6 Thermopolyspora 1.7 0.3 0.1 Microtetraspora 13.0 2.6 0.6 Saccharopolyspora 2.5 0.5 0.1 Promicromonospora 3.4 0.7 0.2 Nocardia 3.1 1.7 15.2 4.0 0.9 Rhodococcus 5.1 9.0 15.2 5.9 0.9 Unidentified 5.1 2.5 1.5 0.3 ------------------------------------------------------------------- TOTAL 201.0 294.3 549.5 406.0 740.3 439.5 100.0

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Table 1. Actinomycetes (no. of propagules x 10 /g dry wt) isolated from the mud of Lake Chenghai

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>Figure 1. A simplified dendrogram showing relationships between isolates from alkaline soil samples.

Biological characteristics of alkalophilic actinomycetes. No streptomycetes with spiral spore chains and red or blue aerial mycelium were isolated from alkaline soil. The pattern of carbon utilisation of alkalophilic strains tested is relatively narrow. They do not, or weakly, utilise D-mannose, L-rhamnose, xylose, galactose and D-mannitol. Cellulolytic and starch hydrolytic activities are low. Most isolates are sensitive to 0.1% phenol.

On the basis of pH requirements, they can be divided into three groups: extreme alkalophiles, with an optimum growth at pH 10 and 11.0, and no growth at pH 7.0; moderate alkalophiles, with an optimum growth at pH 9.0 and 10.0, but poor growth at pH 7.0; alkaline-resistant actinomycetes growing at pH 6.011.0

Chemical composition. Cell wall composition of 26 strains of Micromonospora isolated from Lake Chenghai was analysed. Nine strain contained both meso- and L-diaminopimelic acid (DAP). Interestingly enough, two of these strains, Y84-4341 and Y84-4343, are dominant among the isolates, representing about 20% of the actinomycete population.

Thirty-two streptomycetes isolated from Lake Chenghai were also subjected to cell wall analysis. Ten strains contain meso-DAP.

Nine of 49 alkalophilic strains from alkaline soil contain meso-DAP and MK-10 (H4, H6) and, according to Mikami (1985/6), should be identified as Nocardiopsis species. However morphological characteristics are typical of streptomycetes and whole cell wall hydrolysates show the presence of galactose or ribose. In addition a few strains with streptomycete-like morphology were found to contain L-DAP, meso-DAP and glycin. These "streptomycetes with an abnormal cell wall chemotype" were previously isolated from alkaline habitats (Mikami et al., 1985/6).

DNA homology. Results of DNA homology tests carried out on strains belonging to different clusters (Fig. 1) are shown in the Table 2.

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DNA Source Relative DNA bound (%)

Strain 13617-B S.griseus CCCCM
4.139
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13617-B 100.0 36.3
13655 107.1 5.8
13612 74.2 63.1
13629 38.5 52.4
13666 30.1 58.9
13688 29.1 67.0
13608 39.1 63.6
CCCCM 4.139 36.3 100.0
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Table 2. DNA reassociation of isolates with reference strains Nocardiopsis sp. 13617-B and S.griseus CCCCM 4.139

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Reassociation rate of the DNA of reference strain 13617-B (Nocardiopsis sp.) with DNA of strain 13655 and 13612 (Nocardiopsis sp.) which belong to the same cluster (IA) are 107.1 and 74.2% respectively: 38.5 and 30.1% with reference to strains 13629 and 13666 (Streptomyces, Cluster IB); and 29.1 and 39.1% to strains 13688 and 13608 (Streptomyces, cluster IC) and 36.3% with S.griseus (CCCCM 4.139). When DNA of S.griseus is used as a reference, the reassociation pattern is reversed.

Acidophilic actinomycetes

Unlike fungi and other bacteria, most actinomycetes cease to grow at pH 5.0. In highly acid soils they often make up less than one percent of the total microbial count. Since the 20-year-old studies of Williams and collaborators (Davies and Williams, 1970; Williams et al., 1971) on the distribution and pH requirement of streptomycetes from acid soils, research work in this area has received comparatively little attention.

Acidophilic actinomycetes were isolated from soil and mud samples from 18 areas and from 17 hot (50 C) springs (pH 2.0 to 5.0) across Yunnan, using three media adjusted at pH 4.5.

The mean frequency of acidophilic actinomycetes in soil was: 90% for pine forests, 63% for evergreen broad-leaf forests, 33% for farmland and 12% for waste land in dry hot valleys. No actinomycetes were isolated from soils 3,500m above sea level. 7,400 cfu/g acidophilic actinomycetes were isolated from soil samples of the horizon A0 (0-5 cm) with pH 3.6-4.6 on the Ailao mountain. The deeper the soil horizon and the higher the soil pH, the lower was the frequency of acidophilic actinomycetes. No acidophilic actinomycetes were isolated from hot springs. All isolates were streptomycetes (Table 3).

On the basis of growth at various pH, isolates could be grouped into three types. Type I is characterised by optimum growth at pH 4.5, pH 3.0 to 6.5 growth range and no growth at pH 7.0. Strains of this type represent 70% of the isolates from the Ailao Mountain A0 horizon samples and less than 1% from other areas. Type II, with growth at pH 4.5 to 9.0, consists of common actinomycetes. Type III is represented just by one strain with growth at pH 3.0 to 9.0 and optimum growth at 4.5 to 6.5.

Thermophilic actinomycetes

Thermophilic actinomycetes as producers of antibiotics, enzymes and other bioactive metabolites are attractive because of their rapid growth rate and mycelium autolysis (Moreira et al. , 1981; Mutsue et al., 1982; Waldon and Eveleigh, 1986). They are also useful materials for studying evolutionary and adaptation mechanisms to extreme environments. In the past research work dealt mainly with isolation methods, taxonomy and distribution in some habitats (Henssen, 1957; Kuster and Locci, 1963; Cross, 1968; Nonomura and Ohara, 1971; McCarthy and Cross, 1984; Williams et a1., 1985; Goodfellow et a1., 1987; Tseng et a1., 1990). Thermophilic actinomycetes in hot springs and lakes have received little attention.

Several thermophilic actinomycetes were isolated from hot springs, lakes and soils in Yunnan and their distribution, physiological and biochemical characters, cell composition and DNA homology investigated. Seventy thermophilic strains from hot springs and soil were compared through 82 unit characters with ten representative mesophilic streptomycetes. The results of this research are presented here.

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AREA ALTITUDE VEGETATION SOIL No.(1) of
(m) pH
streptomycetes
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Wanding 1030 EBF(2) 5.5 10
Zhefang 1040-1080 EBF 5.5 16
Longlin 1140-1620 EBF 5.5 9
Tengchong 1460 EBF 5.5 26
Baoshan 1600-1800 Farmland 5.7 1
Zijinshan 2400-2450 EBF 5.5 26

Ailao EBF
Mountain 0-5 cm depth 3.6-4.6 74
2450-2600 10-20cm depth 4.0-5.1 21
30-70 cm depth 4.5-5.4 0
100 cm depth 4.8-5.5 0
Yuangmei 800 Farmland 6.0 2
Kunming 1890-2050 Pine forest 5.0-5.5 52
Baima Snow Mt. 3100 Pine forest 4.8-6.0 32
3500-4800 Conifer forest 5.0 0
and lawn

Zhongdian 3100 Pine forest 6.0 6 Jinsha River 1800 Secondary shrub 6.0-6.5 8 2100 Secondary shrub 7.0 0 Jianchuan 1800-2200 Pine forest 5.0-6.0 152 Dali 1900 Farm land 6.0 38 Yongsheng 1550-1700 Pine forest 6.0-7.0 71 Yongshan 700-1200 Pine forest 5.0-5.5 10 Qingshui 1200-1450 Pine forest 5.5 20 -------------------------------------------------------------------

Table 3. Distribution of acidophilic actinomycetes in soil of Yunnan

(1) 10^2/g dry wt,

(2) Evergreen broadleaf forest

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Distribution of thermophilic actinomycetes. Yunnan is famous for its earth-heat resources. There are about five hundred hot springs and the water temperature ranges between 40 and 100 C with pH 2-10. These hot springs are located in Western Yunnan (Tengchong and Dali).

Streptomyces (550 cfu/g mud), Micromonospora (110), Actinomadura (5), Saccharomonospora (5) and Thermoactinomyces (50) were found in 10 mud samples of 23 hot springs in Dali. Water temperatures of these springs varied from 43 to 74 C. However no thermophilic actinomycetes were isolated from a hot spring at 74 C.

Streptomyces (230 cfu/g mud), Saccharomonospora (30) and Thermoactinomyces (250) were isolated from 18 out of 45 hot springs at 45-77 C in Tengchong.

Thermoactinomyces were isolated in four hot springs at 70-75 C. Frequency and generic diversity of thermophiles isolated from hot springs of Tengchong were lower than those of Dali (Table 4).

The distribution of thermophilic actinomycetes in plateau lakes was very wide.

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GENUS Dali ho Tengchong
springs hot springs
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Streptomyces 550 230
Micromonospora 110
Actinomadura 5
Saccharomonospora 5 30
Thermoactinomyces 50 250
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Table 4. Distribution of thermophilic actinomycetes in hot springs of Yunnan (cfu/g mud)

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Streptomyces, Micromonospora, Microbispora, Microtetraspora, Saccharomonospora, Nocardia and Thermoactinomyces were isolated.

In Lake Chenghai (pH 9.0) thermophiles were present as 53.8 cfu/g dry mud. Lakes Yilong and Jian dried up for several months in 1981 and thermophiles increased to 7,350 and 4,850 cfu/g dry mud respectively (Table 5).

In Xishuangbanna total counts of soil thermophilic actinomycetes averaged 23.3 (cfu/g dry soil) in primeval forests, 46.6 in secondary forests, 3,650 in uncultivated land, 11,031 in cultivated land and 340 in paddy fields.

Most probably the reason of the lower frequency of thermophiles in primeval forest soil than in soil of non-forests in the tropical area of Yunnan is due to the fact that the soil of primeval forest is never irradiated by the sun and soil temperature is very constant. In addition in non-forest soil the range of temperatures is quite wide, with maxima up to 40-50 C. No thermophilic actinomycetes were isolated from soil samples of primeval forest in Baoshan (Table 6). Thus it can be assumed that, as a regular pattern, levels of soil thermophilic actinomycetes decrease progressively according to the sequence: cultivated land, grass land, uncultivated land, secondary forest and primeval forest.

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Lake Altitude Depth S. Mim. Mib. Mit. Sm. N. The.
Total
(m) (m)
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Xingyun 1723 9.0 100 50 150
Cibi 2150 5.0 50 50 100
Qilu 1731 4.0 100 100 50 250
Yilong 1411 3.5 3950 3400 7350
Datun 1280 1.3 50 150 200
Jian 2015 3.5 4150 50 300 350 4850
Chenghai 1503 15.0 18 1 1 35 55
Fuxian 1721 89.0 233 8 58 299
Yangzhong 1770 20.0 900 50 50 50 100 1150
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Table 5. Thermophilic actinomycetes in sedimental samples from nine lakes on Yunnan Plateau (cfu/g dry wt S.: Streptomyces; Mim.: Micromonospora; Mib.: Microbispora; Mit.: Microtetraspora; Sm.: Saccharomonospora; N.: Nocardia; Tha.: Thermoactinomyces).

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Vegetation type Streptomyces Saccharo- Thermo- Total
monospora actinomyces
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Primeval forest 0.0 0.0
Secondary forest 3.7 3.7
Uncultivated land 21.7 21.7
Vegetable land 64.0 40.3 384.7 489.0
Cultivated land 24.0 474.0 124.7 622.7
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Table 6.

Thermophilic actinomycetes in soil samples from
various vegetations in Baoshan
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Area Altitude Yearly S. Min.
(m) temp. ( C)
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Mengna 690-890 20.9 4014.3
Menglun 550-950 21.2 5351.1
Yuangjiang 450-800 23.8 12952.0 42.9
Jinsha River 2100 16.2 980.0 5.0
Baoshan 620-2300 15.5 858.3
Kunming 1900-2100 14.7 2276.6
Northeast of Yunnan 550-1400 13.3 50.2
Yulong Snow Mt. 2600-3000 6.6 85.0 105.0
Zhongdian 3100-3300 5.2 680.0 290.0
Baima Snow Mt. 3000-3500 10.0

Table 7 continued
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Area Am. Sm. Tha. Total
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Mengna 6.7 4021.0
Menglun 58.8 5409.1
Yuangjiang 1285.7 14280.6
Jinsha River 90.0 1075.0
Baoshan 86.3 247.4 1192.0
Kunming 240.0 400.0 2916.6
Northeast of Yunnan 0.9 833.6 884.7
Yulong Snow Mt. 15.0 205.0
Zhongdian 60.0 10.0 1040.0
Baima Snow Mt. 45.0 55.0
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Table 7. Thermophilic actinomycetes in soil samples from ten areas of Yunnan (cfu/g dry wt)

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The distribution of soil thermophilic actinomycetes in ten areas of Yunnan is shown in Table 7.

Streptomyces and Thermoactinomyces species represent nearly 97% of the total population of soil thermophilic actinomycetes. Thermophilic Micromonospora, Actinomadura and Saccharomonospora counts are lower in these areas. Yuangjiang is a dry-hot valley, with an annual average temperature of 23.8 C and the number of thermophilic actinomycetes reaches up to 14,280 cfu/g dry soil. In general, the higher the altitude, the lower the number of thermophiles; the higher the average annual temperature, the more the thermophiles. The upper limit of the distribution of thermophiles is 3,500m above sea level at the latitude of 25 degrees north of Yunnan. No thermophiles were found in soil above 3,500m (Table 7).

Cluster analysis. Eighty tested strains were grouped into five clusters at 63% similarity level (S-level) by using the S[sm] coefficient (UPGMA).

Cluster I consists of 29 strains defined at 73% S-level. Nineteen strains (65%) were isolated from hot springs and the other ten from soil. 90% strains of the cluster grow well at 60 C . 93% of the isolates do not grow at 20 C.

Cluster II is made up by 12 strains defined at 73% S-level. Three strains were isolated from hot spring, 4 strains from soil and 5 strains were obtained from ATCC. 83% of the cultures grow well at 45 C.

Cluster III contains 7 strains with an optimum growth at 28 to 37 C. They are represented by ATCC cultures and by hot spring isolates and are mesophilic actinomycetes.

Cluster IV consists of 13 strains with an optimum growth of 55 to 60 C. Nine organisms (69%) were isolated from soil and 4 from hot springs and do not grow at 28 C.

Strains of clusters I to IV were identified as streptomycetes.

Cluster V is made up of 19 strains defined at 71% S-level. They grow optimally at 55 C. Members of this cluster belong to Streptomyces (4 strains), Actinomadura (11), Saccharomonospora (2) and Thermoactinomyces (1). This cluster shows a 63% S-level with clusters I to IV.

Biological characteristics. Most thermophilic streptomycetes either from hot springs or from soil form Retinaculiaperti spore chains. Strains producing spiral spore chains are less frequent than in mesophilic streptomycetes. No strains producing red or green aerial mycelium were found among thermophiles.

The pattern of carbon and nitrogen utilisation of thermophiles both from soil and hot springs is narrower and activities of some enzymes of thermophiles are lower than those of mesophilic streptomycetes. However keratinase, chitinase and utilisation of galactose by thermophiles are higher. Possibly highly active producers of keratinase and chitinase can be found in both hot springs and soil thermophilic actinomycetes.

Nitrate reduction, H2S production, activities of keratinase and chitinase and antibiotic resistance of soil thermophiles are higher than those of thermophilic actinomycetes from hot springs. However, the temperature growth range of hot spring thermophiles is wider than that of soil thermophiles.

With reference to temperature requirements, thermophilic actinomycetes can be divided into three group: strict thermophiles with an optimum at 55 to 60 C, growth between 37 to 65 C and no growth at 28 C; moderate thermophiles with an optimum at 50 to 55 C, growth between 28 to 60 C (some strains of Actinomadura tested during this study can be incorporated into this group); thermotolerant actinomycetes with a growth temperature range from 15 to 55 C.

Chemical composition. Cell walls of both hot spring and soil thermophilic streptomycetes contain L-diaminopimelic acid. No characteristic sugars are present.

DNA homology. Strain 4009 (optimum growth temperature 60 C, isolated from soil, Cluster IV) and S.griseus (Cluster III) were selected as References. Reassociation rate of DNA of strain 4009 with DNA of strain 4027 (cluster IV) is 63.2%. The reassociation rates of the DNA of strain 4009 or of S.griseus with DNA of strain 4011 (Cluster I), 14055 (Cluster I), 14045 (Cluster II). 4023 (Cluster IV) and 14046 (Cluster V) are below 35%.

Psychrophilic actinomycetes

Baima Snow-Mountain is situated in Yunnan's north-western area. The highest point is 5,137m above-sea level. Forests there have been well protected.

Soil samples were collected from various altitudes. Actinomycetes were isolated at 8,14 and 55 C .

Psychrophilic actinomycetes increase gradually with the rise in the altitude and are reduced at 4,400m. A opposite pattern was observed for thermophiles. No thermophiles were isolated from the samples taken at 3,500m. Possibly in Yunnan 3,500m represents a line of demarcation for the distribution of both psychrophilic and thermophilic actinomycetes at a latitude of about 25 degrees.

Psychrophilic actinomycetes isolated from Beima Snow Mountain samples were represented by Streptomyces (58%), Micromonospora (12%) and nocardioform (30%) species.

On the basis of growth at various temperatures, psychrophilic actinomycetes isolated from Baima Snow Mountain could be grouped into three types.

Type I organisms are characterised by growth at 0 C , optimum growth at 10 to 28 C and poor growth at 37 C. These organisms belong to a transitional group between mesophiles and psychrophiles and can be defined as facultative psychrophiles.

Type II with growth at 0 C, no growth at 37 C and optimum growth at 10 to 28 C. Organisms of this group can be defined as moderate psychrophiles.

Type III actinomycetes show optimum growth at 10 to 14 C, no growth at 28 C and moderate growth at 0 C. These may be considered true psychrophiles.

It appears from these data that the range of growth temperatures of psychrophiles isolated from soil is wider than that of psychrophilic bacteria in the bottom of sea where temperature is usually constant at about 5 C (Grent et a1., 1981).

One hundred and eight cultures of psychrophilic actinomycetes were selected for evaluating their physiological activities. Eighty-three strains utilised cellulose, 17 hydrolysed starch, 5 hydrolysed pectin, 48 hydrolysed casein, 47 liquefied gelatine, 10 showed lysozyme activity and 8 were active against Bacillus subtilis and Mucor sp.

In the forest soil of Baima Snow Mountain actinomycetes represented only 2 to 12% of the total microbial population. Therefore their activity in the decomposition of organic substances seems to be lower than that of bacteria and fungi.

Strategies for studying actinomycetes from unusual habitats

It was stressed above that the study of actinomycetes in unusual or extreme environments is important both in theoretical and economic terms. We may at present be dazzled by the enormous progress in molecular studies of actinomycete biology made during the last few years, but the knowledge of actinomycetes in extreme habitats is still not satisfactory.

A lot of questions have not yet been answered. We still do not know how to isolate actinomycetes from extreme habitats.

Are there basic differences between actinomycetes living in usual and unusual habitats? How do actinomycetes adapt themselves to extreme environments? What is their economic potential?

Lack of this knowledge has hampered microbiologists so far. The strategies outlined in this context can be viewed as an initial attempt to face these problems.

ACKNOWLEDGEMENTS. Support for this work was provided by NFSC and International Cooperative Science Foundation Or Yunnan. We are grateful for the assistance of Drs.Yang, G.Guo and Y.Yang.

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