NOTE Studies of the Biological Characteristics of Some Halophilic and Halotolerant Actinomycetes Isolated from Saline and Alkaline Soils

Actinomycetol. (2002) 17:06–10 VOL. 17, NO. 1 NOTE Studies of the Biological Characteristics of Some Halophilic and Halotolerant Actinomycetes Isolated from Saline and Alkaline Soils

Shu-Kun Tang, Wen-Jun Li, Wang Dong, Yong-Guang Zhang, Li-Hua Xu and Cheng-Lin Jiang*

The Key Laboratory for Microbial Resources of Ministry of Education, P. R. China, Yunnan Institute of Microbiology, Yunnan University, Kunming 650091,Yunnan, China (Received Nov. 11, 2002 / Accepted Feb. 19, 2003)

This study investigated the biological characteristics of 43 actinomycete isolates from saline and alkaline soils in Xinjiang, Hebei and Qinghai and representative strains of four genera under differing conditions of pH and varying concentrations of the mineral salts Na+, K+, Mg2+ and Ca2+. The results indicated that halotolerant actinomycetes have extensive adaptability to Na+, K+ and Mg2+, but that few strains can grow + even in low concentrations of CaCl2. Halophilic actinomycetes have extensive adaptability to Na ; for most, Na+ may be replaced by K+ or Mg2+, but not by Ca2+. Certain halophilic actinomycetes require Na+ to grow. It was also clear that the growth of all halophilic actinomycetes is dependent on different concentrations of Na+, K+ or Mg2+. We believe that only kaliumophilic or magnesiumophilic or calciumphilic actinomycetes may be found in high salt environments. In addition, the range of pH values in which growth occurred was 6.0~10.0; optimum pH values were 7.0~8.0 for both halophilic and halotolerant actinomycetes. The distribution of halophilic actinomycetes was also found to be related to sample sources.

INTRODUCTION mechanisms that provide tolerance to high concentrations of NaCl, whether having addiction with different ions, how Microorganisms found in extreme environments have pH and nutrition material of medium affect its growth and attracted a great deal of attention, due to the production by metabolic product. Answers to these problems would such microorganisms of various natural compounds and advance efforts to isolate, classify, and apply halophilic their specialized mechanisms for adaption to extreme envi- actinomycetes. ronments1-4). But relatively little research work has been Halophilic bacteria and actinomycetes have been done on actinomycetes found in extreme environments5). grouped according to salinity requirements by microbiolo- For example, the nutritional requirements of actinomycetes gists such as Kushner (1978)6), Lasern (1986)7), Vreeland under extreme environments (including halophilic actino- (1987)8), Ramos- Cormenzana (1989)9). Among these mycetes) remain unknown. It is also not known whether methods, the method of classification proposed by Kushner halophilic actinomycetes are similar to bacteria in their (1978)6) (see Table 1) appears to be the most popular. mechanisms for adapting to high salt conditions. In short, But, in fact, NaCl is not the only salt found in high salt our current understanding and ability to use halophilic environments such as saline soils and seawater. For exam- Actinomycetes is greatly circumscribed. Nevertheless, a ple, most saline soils in Xinjiang10) and Inner Mongolia11), study of such physiological problems is essential for China, also have high concentrations of Na+, K+, Mg2+, achieving an overall understanding of halophilic Ca2+. Accordingly, studies of halophilic actinomycetes Actinomycetes. focusing only on NaCl concentrations are inadequate for Scientists have known that halophilic bacteria could gaining an overall understanding of these actinomycetes. endure and grow at high salt concentrations. However, This paper reports on studies of the biological charac- they were unaware of the existence of halophilic actino- teristics of 43 actinomycete strains isolated from mycetes until the 1970s. Gochnauer6) first discovered a Xinjiang, Hebei and Qinghai provinces, P. R. China, and strain of halophilic actinomycetes from a contaminated of strains representative of four genera, including plate and named it Actinopolyspora halophila. Thereafter, Prauserell rugosa (DSM43194T), Saccharomonospora research efforts focused on finding new halophilic actino- halophila (DSM44411T), Nocardiopsis kunsanensis mycetes. However, in contrast to halophilic bacteria, there (KCTC9831T), Nocardiopsis halotolerans (DSM44410T), are very few reports in the literature on halophilic actino- in environments of varying pH levels and varying concen- mycetes. We still do not understand the physiological trations of the salts Na+, K+, Mg2+, and Ca2+.

* Corresponding author. +86-871-5034139. Fax: +86-871-5173878. E-mail: [email protected], [email protected] 6 ACTINOMYCETOLOGICA VOL. 17, NO. 1

MATERIALS AND METHODS NaOH. The liquid cultures were cultivated in tubes at 28 ˚C for 30 days. Test Strains For endurance experiments involving NaCl concentra- Forty three halophilic and halotolerant actinomycete tions, ISP5 was used as the basic medium. The following strains were isolated using modified glycerol-asparagine NaCl concentrations (w/v) were used: 0%, 1%, 3%, 5%, agar (ISP5 medium, adding 20% sodium chloride [w/v], 10%, 15%, 20%, 25%, 30%. The slants were cultivated at same as below) from soils collected from Xinjiang, Hebei 28 ˚C for 30 days. and Qinghai Provinces, P. R. China (See Table 2). Most For experiments involving dependence on Na+, K+, soil samples had compositions of 10% to 30% sodium Mg2+, Ca2+, the basic medium was ISP5 of pH in the range chloride at pH values of 7.0–10.0, with varying amounts of 7.0~8.0. The following NaCl concentrations (w/v) were other salts such as KCl, MgCl2·6H2O, CaCl2, etc., but used: 0%, 1%, 3%, 5%, 10%, 15%, 20%, 25%, 30%. The often containing NaCl as the major component. Four following KCl concentrations (w/v) were used: 0%, 2%, strains representative of other genera, including 5%, 10%, 15%, 20%, 25%, 30%; MgCl2.6H2O concentra- Prauserella rugosa (DSM43194T), Saccharomonospora tions (w/v) were: 0%, 5%, 10%, 15%, 20%, 25%, 30%; T halophila (DSM44411 ), Nocardiopsis kunsanensis CaCl2 concentrations (w/v) were: 0%, 1%, 3%, 5%, 10%, (KCTC9831T), Nocardiopsis halotolerans (DSM44410T), 15%, 20%, 25%, 30%. The plates were cultivated at 28 ˚C were collected from DSMZ and KTCC. for 30 days.

Identification of Actinomycete Isolates RESULTS AND DISCUSSION Morphological observations were carried out by the methods and media proposed by Shirling and Gottlieb12). Identification Results The chemical compositions of cell walls and whole-cell Some halophilic isolates (YIM90001 to YIM90007) had hydrolysates were analyzed by the methods of Lechevalier been identified to the species level by Polyphasic and Lechevalier13). Polar lipids were extracted, examined Taxonomic Methods, based on their morphology, physio- by two-dimensional thin-layer chromatography, and iden- logical and biochemical characteristics, DNA G + C tified by procedures, accounts of which have been previ- mol%, and 16S rDNA sequences analysis, etc. The other ously published14). Menaquinones were isolated by the isolates (YIM90008-YIM90045) were identified only to methods of Minnikin et al.14) and separated by HPLC15). the genus level, based on morphology and chemotaxonom- Analysis of cellular fatty acid compositions was performed ic characteristics (See Table 2). Some details of the identi- as described by Sasser16). fication results for certain strains have been or will be pub- Media and Cultivating Conditions for Biological lished in Int. J. Syst. Evol. Microbiol17–22). Characteristics For pH endurance experiments, the basic medium was NaCl tolerance of actinomycetes

ISP5 (g/l): Asparagine 1.0, Glycerol 10, K2HPO4·3H2O Our research results show that the tolerance of actino- 1.0, NaCl 100, trace salt solution 1 ml. The following mycetes to NaCl differs with soil source and kind of buffer solutions were used: pH6.0: NaOH-KH2PO4 ; actinomycetes. 32, 8 and 3 actinomycete strains (See in pH7.0: NaOH-KH2PO4; pH8.0: NaOH-KH2PO4; pH9.0: Table 3) were isolated, respectively, from Qinghai, Borax-Boric acid; pH10.0: Borax-NaOH; pH11.0: Xinjiang, and Hebei Province; 15 strains are halophilic

Na2HPO4-NaOH; pH12.0:KCl-NaOH; pH13.0:KCl- actinomycetes, while the other 38 strains are halotolerant

Table1 Types of microorganisms by Kushner (1978)6) Microorganism Optimum NaCl concentration Non-halophilic <0.2 mol/L ( 1.17%NaCl) Weak halophilic 0.2~0.5 mol/L (1.17~2.93%NaCl) Moderately halophilic 0.5~2.5 mol/L (2.93~14.63%NaCl) Extremely halophilic 2.5~5.2 mol/L (14.63~30.4%NaCl) Halotolerant Tolerant range 0.2~2.5 mol/L (1.17~30.45%NaCl)

Table2 Comparison of chemical properties of three source saline and alkaline soils

+ 2+ + 2+ − 2− 2− − Chemical compositions (mg/L) Na Mg K Ca Cl SO4 CO3 HCO3 Soil source Xinjiang Province (pH8.0-10.0) 84234 9848 1873 723 133434 33363 2041 3871 Qinghai Province (pH7.5-10.0) 68360 35130 5978 4241 204209 22290 171 127 Hebei Province (pH7.0-10.0) 10500 1350 380 400 19000 2967 140 140

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Table3 Impact on the growth of halophilic and halotolerant actinomycete strains of varying Na+, K+, Mg2+, Ca2+ concentrations (w/v%)

Type Strains no. Isolation Genus* Different Ion Concentraion

source NaCl KCl MgCl2·6H2OCaCl2 YIM 90001 Xinjiang Prauserella 3%~20% 2%~25% 5%~25% _** YIM 90002 Xinjiang Streptomonospora 5%~20% _ 20% _ YIM 90003 Xinjiang Streptomonospora 1%~20% 5%~20% _ _ YIM 90004 Xinjiang Nocardiopsis 3%~20% 5%~25% 5%~20% _ YIM 90006 Xinjiang Nocardiopsis 3%~20% 2%~25% 5%~25% _ YIM 90007 Xinjiang Saccharomonospora 5%~20% _ _ _ YIM 90008 Hebei Nocardiopsis 1%~20% 2%~25% 5%~15% _ YIM 90009 Hebei Nocardiopsis 3%~20% 2%~25% 5%~20% _ YIM 90010 Xinjiang Nocardiopsis 3%~20% 2%~25% 5%~20% _ YIM 90011 Hebei Nocardiopsis 3%~20% 2%~25% 5%~20% _ YIM 90014 Qinghai Nocardiopsis 1%~20% 2%~25% 5%~20% _ Halophilic actinomycetes YIM 90015 Qinghai Nocardiopsis 3%~20% 5%~25% 15%~20% _ YIM 90016 Qinghai Nocardiopsis 3%~20% 5%~25% 15%~25% _ KCTC9831 Korea Nocardiopsis 5%~20% 5%~20% _ _ DSM44411 Kuwait Saccharomonospora 5%~20% _ 20%~25% _ YIM 90005 Xinjiang Prauserella 0%~20% 0%~25% 0%~25% 0%~15% YIM 90012 Qinghai Nocardiopsis 0%~20% 0%~15% 0%~25% 0%~1% YIM 90013 Qinghai Nocardiopsis 0%~20% 0%~20% 0%~20% _ YIM 90017 Qinghai Streptomyces 0%~15% 0%~5% 0%~20% 0%~1% YIM 90018 Qinghai Streptomyces 0%~15% 0%~5% 0%~25% 0%~1% YIM 90021 Qinghai Nocardiopsis 0%~20% 0%~20% 0%~25% _ YIM 90022 Qinghai Nocardiopsis 0%~20% 0%~20% 0%~25% _ YIM 90023 Qinghai Nocardiopsis 0%~20% 0%~25% 0%~25% _ YIM 90024 Qinghai Nocardiopsis 0%~20% 0%~20% 0%~25% _ YIM 90025 Qinghai Nocardiopsis 0%~20% 0%~25% 0%~25% _ YIM 90026 Qinghai Nocardiopsis 0%~20% 0%~25% 0%~25% _ YIM 90027 Qinghai Nocardiopsis 0%~20% 0%~25% 0%~25% _ YIM 90028 Qinghai Nocardiopsis 0%~20% 0%~25% 0%~25% _ YIM 90029 Qinghai Nocardiopsis 0%~20% 0%~25% 0%~25% _ YIM 90030 Qinghai Nocardiopsis 0%~20% 0%~20% 0%~25% _ YIM 90031 Qinghai Nocardiopsis 0%~20% 0%~25% 0%~25% _ YIM 90032 Qinghai Nocardiopsis 0%~20% 0%~25% 0%~25% _ YIM 90033 Qinghai Nocardiopsis 0%~20% 0%~20% 0%~25% _ YIM 90034 Qinghai Nocardiopsis 0%~20% 0%~20% 0%~25% _

Halotolenant actinomycetes YIM 90035 Qinghai Nocardiopsis 0%~20% 0%~20% 0%~25% _ YIM 90036 Qinghai Nocardiopsis 0%~20% 0%~20% 0%~20% _ YIM 90037 Qinghai Nocardiopsis 0%~20% 0%~20% 0%~25% _ YIM 90038 Qinghai Nocardiopsis 0%~20% 0%~20% 0%~25% _ YIM 90039 Qinghai Nocardiopsis 0%~20% 0%~20% 0%~25% _ YIM 90040 Qinghai Nocardiopsis 0%~20% 0%~20% 0%~20% _ YIM 90041 Qinghai Nocardiopsis 0%~20% 0%~20% 0%~25% _ YIM 90042 Qinghai Nocardiopsis 0%~20% 0%~20% 0%~20% _ YIM 90043 Qinghai Nocardiopsis 0%~20% 0%~25% 0%~20% _ YIM 90044 Qinghai Nocardiopsis 0%~20% 0%~25% 0%~25% _ YIM 90045 Qinghai Nocardiopsis 0%~20% 0%~25% 0%~20% _ DSM44410T Kuwait Nocardiopsis 0%~20% 0%~25% 0%~25% _ DSM43194T unknown Prauserella 0%~20% 0%~25% 0%~25% 0%~10% Note: * : The isolates YIM90001-YIM90007 had been identified to the species level and have thus been determined to belong to distinct species. The other isolates were identified only to the genus level, based on their morphology and chemical characteristics. * * : “_”means no growth

8 ACTINOMYCETOLOGICA VOL. 17, NO. 1 actinomycetes. These strains belong to the following five KCl; Strain YIM90007 grew only in media with NaCl and genera: Nocardiopsis, Streptomonospora, Prauserella, Na+ but were unable to accept the substitution of K+ or Saccharomonospora, Streptomyces. Most actinomycete Mg2+. No halophilic actinomycetes strain was able to grow strains from Qinghai soils were halotolerant and belong to in media containing more than 1% CaCl2. the genus Nocardiopsis. Most actinomycetes strains from All halophilic actinomycetes isolated from Qinghai and Xinjiang and Hebei soils were halophilic and belong to Hebei soils were able to grow in media containing only four genera: Streptomonospora, Prauserella, NaCl, KCl or MgCl2·6H2O, except that no growth occurred Saccharomonospora, Streptomyces. None of the actino- in media containing only CaCl2. Among 7 isolates from mycetes strains (including halophilic and halotolerant Xinjiang soils, 4 were able to grow in media containing actinomycetes) were able to grow in NaCl concentrations only NaCl, KCl or MgCl2·6H2O. With respect to the other exceeding 25%. Optimal NaCl concentrations were 3 isolates, YIM 90002 was unable to grow in media con- 5%~15%. taining only KCl; YIM 90003 was unable to grow in media

containing only MgCl2·6H2O; and YIM 90007 was unable pH range of growth to grow without Na+. These results show that the halophilic All tested strains, including halophilic and halotolerant actinomycete strains isolated from Qinghai and Hebei have actinomycetes, were able to grow in the pH range of different requirements for Na+, K+, Mg2+than those from 6.0~10.0, with an optimal pH of 7.0~8.0. This result under- saline and alkaline soils in Xinjiang. mines the popular notion that isolates from saline and alka- Based on this study of halophilic and halotolerant actin- line soils would be halo-alkalophilic actinomycetes22). omycetes isolated from saline and alkaline soils collected from Xinjiang, Qinghai and Hebei Province, China, we can Addiction of actinomycetes to salt with Na+, K+, Mg2+, conclude that the relationship of actinomycetes (especially Ca2+ halophilic actinomycetes strains) to positive ions such as Halotolerant actinomycetes can grow extensively in Na+, K+ and Mg2+ is very complex. In past studies of media containing Na+, K+, and Mg2+. All 34 halotolerant halophilic and halotolerant actinomycetes researchers have actinomycetes can grow at certain concentrations of NaCl, used media in which only concentrations of NaCl were

KCl, MgCl2·6H2O. 38 of 43 strains were unable to grow in varied. This approach limits our potential understanding media containing over 1% CaCl2. Adaptations of only two and discovery of new and unknown halophilic actino- Streptomyces strains, YIM90017 and YIM90018 to Na+, mycetes. In addition to numerous generalized halophilic K+, Mg2+ were different. They were able to grow in high actinomycetes capable of adapting to different concentra- + + 2+ concentrations of NaCl (0%~15%) and MgCl2·6H2O tions of Na , K and Mg in high salt environments, there (0%~20%), but not in concentrations of KCl over 5%. exist sodiumphilic, kaliumphilic, magnesiumphilic, and Strains YIM 90012, YIM 90017, YIM 90018 were able to calciumphilic actinomycete strains. Based on the findings grow in 1% CaCl2. Strain YIM 90005 and Prauserell of this study, we believe we can design new media to iso- T rugosa (DSM43194 ) can grow in 15% and 10% CaCl2, late other kaliumphilic, or magnesiumphilic, or calci- respectively. The two strains of Prauserella may be called umphilic actinomycete strains that may be found in hyper- broad spectrum halotolerant actinomycetes saline environments (Table 4). It is worth noting that For most halophilic actinomycetes (except YIM 90003 calciumphilic actinomycetes can be found in high calcium and Nocardiopsis kunsanensis (KCTC9831T), we can environments. replace Na+ with K+ or Mg2+. The two strains can grow in media with NaCl or KCl, but failed to grow in media ACKNOWLEDGEMENTS containing only MgCl2·6H2O; strains YIM 90002 and Saccharomonospora halophila (DSM44411T) were able to The authors wish to express their gratitude to Prof. Sang- grow in 5%~20% NaCl or high concentrations of MgCl2 Jin Kim for kindly providing the strains Nocardiopsis kun- T ·6H2O (20%), but failed to grow in media containing only sanensis (KCTC9831 ), Prof. Kroppenstedt providing type

Table 4 New grouping criterion for halophilic actinomycetes based on positive ions proposed by the authors

Generalized halophilic actinomycetes Na+ can be substituted by K+ or Mg2+ Halophilic actinomycetes Sodiumphilic actionmycetes No growth or worse growth without Na+ Kaliumphilic actionmycetes No growth or worse growth without K+ Magnesiumphilic actionmycetes No growth or worse growth without Mg2+ Calciumphilic actinomycetes No growth or worse growth without Ca2+

Halotolerant actinomycetes Endurance range 0 ~ 0.2-2.5 mol/L (NaCl, KCl or MgCl2·6H2O)

Non-halophilic actinomycete <0.2 mol/L (NaCl, KCl or MgCl2·6H2O)

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