BACKGROUND
the bacillus rrna group  <dig> which comprised thermophilic bacillus strains was transferred into new genus geobacillus which represented a phenotypically and phylogenetically coherent group of thermophilic bacilli with high levels of 16s rrna sequence similarity   <cit> .
the members of this genus are widespread in various thermophilic and mesophilic geographic areas on the earth such as oilfields, hay compost, hydrothermal vent or soils  <cit> .
at present, the members of this genus included geobacillus stearothermophilus, geobacillus thermocatenulatus, geobacillus thermoleovorans, geobacillus kaustophilus, geobacillus thermoglucosidasius, geobacillus thermodenitrificans, geobacillus subterraneus, geobacillus uzenensis, geobacillus caldoxylosilyticus, geobacillus toebii, geobacillus vulcani, geobacillus lituanicus, geobacillus tepidamans, geobacillus gargensis, geobacillus jurassicus, geobacillus caldoproteolyticus, geobacillus pallidus and geobacillus debilis with growth temperatures ranging from  <dig> to 78°c  <cit> .
recently, microorganisms such as bacillus sp.
rsj- <dig>  <cit> , bacillus thermoleovorans id- <dig>  <cit> , bacillus sp.
thl <dig>  <cit> , bacillus sp.
strain a30- <dig>  <cit> , bacillus sp.
strain  <dig>  <cit> , bacillus thermocatenulatus  <cit> , bacillus spp.
<cit>  were reported as thermostable lipase producers.
as thermophilic bacterial strains have an optimum growth temperature of 65–70°c, lipases isolated from such strains are good candidates for lipid modifications  <cit> .
the stability of biocatalysts is an important criterion when dealing with bioprocesses at high temperature for sustainable operation.
enzyme stability is dictated by its three-dimensional configuration, which in turn is determined by genetic and environmental factors  <cit> .
therefore, thermophiles are promising sources of heat-stable enzymes.
in addition to higher thermostability, proteins from thermophiles often showed higher stability toward organic solvents and higher activity at elevated temperature  <cit> .
in addition, genetic engineering in altering the stability of enzymes is a difficult task and laborious processes.
therefore, efforts have been focused on the screening of microorganisms harboring intrinsically stable biocatalysts.
putative lipase producers were screened quantitatively across various basal media to check for lipase production and the stability of lipase.
the highest lipase producing strain have been identified and characterized intensively.
screening and isolation of heat-stable lipase producers are important to fulfill industrial requirements for the desired characteristics.
identification of industrially important enzyme producer is conducted to determine its phylogenetic position in systematic microbiology.
RESULTS
screening and isolation of thermophilic lipolytic bacteria
rubbish dump sites and palm oil mill effluent are potential sites containing thermophilic lipolytic bacteria because these sites served as sewage discharge areas for household waste and palm oil factory.
the oily environment may provide a good environment for lipolytic microorganisms to flourish.
samples were collected from the rubbish dump site and palm oil mill effluent at different treatment areas.
samples were enriched with enrichment medium  containing olive oil as the sole carbon source at 60°c for  <dig> days under shaking condition to promote the growth of thermophilic lipolytic bacteria.
twenty nine putative lipase producers gave positive results on triolein agar plate by forming an intense blue color around the colonies .
of these,  <dig> and  <dig> isolates were isolated from rubbish dump site and palm oil mill, respectively.
further confirmation test was performed quantitatively on the  <dig> putative lipase producers that gave positive results on triolein agar plates.
various basal media m <dig>  m <dig>  tyem and bm- <dig> were tested for lipase production during the selection.
low lipase activities ranging from  <dig> ~  <dig>  u/ml were detected colorimetrically in culture supernatant, except for isolate t1t, which showed high lipase activity  after  <dig> h incubation .
note: , indicates formation of intense blue colour on triolein agar plate
on the basis of relatively higher lipase activity detected for isolate t1t, the effect of temperature on the activity and stability of crude t <dig> lipase was further investigated.
the effect of temperature on the lipase activity and stability was examined from  <dig> to 80°c.
as shown in fig.
<dig>  the crude enzyme from isolate t1t manifested its maximal activity at 70°c with olive oil as substrate.
crude t <dig> lipase was fairly stable up to 60°c for  <dig> min and gradually decreased upon prolonged temperature treatment.
this is due to t <dig> lipase tended to lose its native conformation as a result of breaking of the intrinsic interaction above its stable range.
this is a discrepancy between temperature activity  and stability , as the enzyme is tend to be protected by heat denaturation in the presence of olive oil.
in addition, the enzyme thermostability is greatly influenced by the presence of water, because denaturation is linked to its conformational mobility in aqueous mixture  <cit> .
the crude enzyme of isolate t1t was fairly active at higher temperature as compared to other thermostable lipases from bacillus spp.
<cit>  which exhibited maximal activity at 60°c.
high temperature activity and stability of enzyme offer great potentials in industrial applications, and hence attempts have been made to identify isolate t1t.
identification of isolate t1t
during the characterization of organism isolated from palm oil mill effluent, strain t1t was recovered on nutrient broth at 60°c.
the growth condition for strain t1t was 50–70°c and between ph  <dig> and  <dig> with the optimum growth temperature and ph of 65°c and ph  <dig> , respectively, in nutrient broth.
these met the criteria of thermophilic bacteria, which grew at temperatures above 50°c  <cit> .
to verify the systematic position of this bacterium, a study of morphological and physiological characteristics, 16s rrna analysis, cellular fatty acids analysis, dna composition, dna/dna hybridization, riboprint analysis, lipase gene analysis and protein profile were undertaken.
the cellular morphology of isolate t1t is rod-shaped,  <dig> – <dig>  μm width and  <dig> – <dig>  μm length, gram positive bacteria.
the terminal spore is oval/cylindrical in shape and swollen the sporangium.
the dna base composition of strain t1t is around  <dig> % mol g + c. the partial sequencing of the 16s rdna shows  <dig> % similarity to validly described geobacillus kaustophilus  and geobacillus thermoleovorans .
the 16s rrna sequence of strain t1t is a continuous stretch of  <dig> bp .
construction of phylogenetic trees using the neighbour-joining method in determining the evolutionary relationship among a group of validly described closely related species is indicated in fig.
<dig>  a comparison of biochemical, morphological and physiological properties of strain t1t with its closest phylogenetic neighbors is presented in table  <dig>  strain t1t can be distinguished from geobacillus thermoleovorans  phenotypically by oxidase test, arabinose, mannitol, inositol, lactose and casein hydrolysis.
however, strain t1t differs from geobacillus kaustophilus  by lysozyme test, arabinose, mannitol, ribose, adonitol, lactose, gelatin and casein tests.
since the sequencing result and physiological data did not allow strain t1t to be identified with one of the above mentioned species, further analysis need to be carried out to verify its phylogenetic position.
taxa  <dig>  g. zalihae t1t;  <dig>  g. kaustophilus ;  <dig>  g. thermoleovorans .
characteristics are scored as: +, positive; -, negative; w, weak; v, variable within the group; nd, not determined.
data were obtained from the present study , nazina et al.
<cit> , sunna et al.
<cit> , kuisiene et al.
<cit>  and markossian et al.
<cit>  .
all strains were negative for anaerobic growth, growth at 30°c, vp reaction, indole production and acid from l-rhamnose, sorbitol.
all strains were positive for catalase, hydrolysis of starch, use of citrate and acid from d-fructose and d-glucose.
result of chemotaxonomic analyses is given in the species description.
the fatty acid profile of strain t1t is typical for the bacillus rrna-group  <dig> .
the major content of cellular fatty acids of strain t1t is iso-fatty acids.
among them, iso-branched pentadecanoic acid , hexadecanoic acid  and heptadecanoic acid  making up  <dig> % of the total fatty acids for strain t1t.
strain t1t can be differentiated from geobacillus thermoleovorans dsm 5366t based on percent composition of iso-fatty acids .
there was only  <dig> % of iso-c <dig> for strain t1t but 21% for geobacillus thermoleovorans dsm 5366t .
note: data were obtained from the present study , nazina et al.
<cit>  .
although fame is not always a reliable tool for preliminary identification of bacilli, it could be used in combination with other identification methods  <cit> .
dna/dna hybridization experiments were performed in dsmz  with strain t1t and its type strains of closest phylogenetical neighbors.
the genomic dna/dna relatedness between strain t1t and its type strains geobacillus kaustophilus  geobacillus thermoleovorans  were  <dig> % and  <dig> %, respectively.
whereas, the type strains geobacillus kaustophilus  and geobacillus thermoleovorans  showed a dna/dna similarity of  <dig> %.
the dna/dna reassociation values were below the threshold value of 70% dna/dna similarity for definition of species  <cit>  between strain t1t and geobacillus thermoleovorans dsm 5366t but above the threshold between strain t1t and geobacillus kaustophilus dsm 7263t.
neither the species geobacillus kaustophilus dsm 7263t and geobacillus thermoleovorans dsm 5366t can be differentiated from one another nor strain t1t can be differentiated at the species level from its closest phylogenetic neighbors by dna/dna hybridization.
however, dna/dna hybridization tests between geobacillus kaustophilus dsm 7263t and geobacillus thermoleovorans dsm 5366t were 84% and 54% as reported by sunna et al.
<cit>  and nazina et al.
<cit> .
this disagreement may be due to the absence of adequate hybridization controls in the experiments.
therefore, further tests need to be carried out to accurately place the strain t1t phylogenetically.
the riboprint analysis was carried out for the decision on the affiliation of strain t1t.
however, the riboprint pattern of strain t1t was not identified by the dupont identification library to give rise to the identification at the species level .
its riboprint pattern showed the highest similarity to geobacillus kaustophilus dsm 7263t .
the similarity to the pattern of geobacillus thermoleovorans dsm 5366t was somewhat lower .
the patterns between type strains geobacillus kaustophilus dsm 7263t and geobacillus thermoleovorans dsm 5366t show a binary similarity of  <dig> .
further analysis was also carried out by amplifying full-length thermostable lipase gene using primers as described in materials and methods  <cit> .
fig.
<dig> showed amplified full-length lipase genes of strain t1t and its type strains.
the amplified lipase gene of strain t1t was around  <dig> kb but  <dig>  kb for its type strains geobacillus kaustophilus dsm 7263t and geobacillus thermoleovorans dsm 5366t.
a stretch of about  <dig> bp insertion could be seen at downstream of the open reading frame of thermostable lipase gene .
in addition, the intracellular protein profiles were determined by sds-page.
samples  were separated on 12% sds-polyacrylamide gel and stained using coomassie blue.
strain t1t showed obvious different protein profile as compared to its type strains at region between  <dig> to  <dig> kda .
description of geobacillus zalihae sp.
nov
geobacillus zalihae .
the novel species is isolated from palm oil mill effluent in selangor, malaysia, with the type strain t1t .
cells are rod-shaped,  <dig> – <dig>  width and  <dig> – <dig>  length, gram positive bacteria.
the terminal spores are oval/cylindrical and swollen the sporangium.
growth occurs at 50–70°c with an optimum temperature of 65°c.
growth at 65°c occurs between ph  <dig> and  <dig> with maximal growth at ph  <dig> .
the dna base composition of strain t1t was around  <dig> % mol g + c. the iso-fatty acids were in major amount according to cellular fatty acid profile in which iso-c <dig> , and iso-c <dig>  were in abundant .
growth is aerobic and tolerant up to 2% nacl.
it can not perform anaerobic growth.
it shows positive in catalase test but not oxidase test.
it is able to hydrolyze starch but not gelatin and casein.
acids are produced from l-arabinose, d-lactose but not d-mannitol.
CONCLUSIONS
as a consequence, the strain t1t merits recognition as a member of a novel species through morphological and physiological studies, cellular fatty acids composition, dna composition, dna/dna hybridization, riboprint analysis.
sizes of full-length lipase genes and protein profiles were additional evidences.
the geobacillus zalihae strain t1t was deposited in dsmz  and nite .

 the bacillus rrna group which comprised thermophilic bacillus strains was transferred into new genus geobacillus which represented a phenotypically and phylogenetically coherent group of thermophilic bacilli with high levels of 16s rrna sequence similarity. the members of this genus are widespread in various thermophilic and mesophilic geographic areas on the earth such as oilfields, hay compost, hydrothermal vent or soils. at present, the members of this genus included geobacillus stearothermophilus, geobacillus thermocatenulatus, geobacillus thermoleovorans, geobacillus kaustophilus, geobacillus thermoglucosidasius, geobacillus thermodenitrificans, geobacillus subterraneus, geobacillus uzenensis, geobacillus caldoxylosilyticus, geobacillus toebii, geobacillus vulcani, geobacillus lituanicus, geobacillus tepidamans, geobacillus gargensis, geobacillus jurassicus, geobacillus caldoproteolyticus, geobacillus pallidus and geobacillus debilis with growth temperatures ranging from to 78°c. recently, microorganisms such as bacillus sp. rsj-, bacillus thermoleovorans id-, bacillus sp. thl, bacillus sp. strain a30-, bacillus sp. strain, bacillus thermocatenulatus, bacillus spp. were reported as thermostable lipase producers. as thermophilic bacterial strains have an optimum growth temperature of 65–70°c, lipases isolated from such strains are good candidates for lipid modifications. the stability of biocatalysts is an important criterion when dealing with bioprocesses at high temperature for sustainable operation. enzyme stability is dictated by its three-dimensional configuration, which in turn is determined by genetic and environmental factors. therefore, thermophiles are promising sources of heat-stable enzymes. in addition to higher thermostability, proteins from thermophiles often showed higher stability toward organic solvents and higher activity at elevated temperature. in addition, genetic engineering in altering the stability of enzymes is a difficult task and laborious processes. therefore, efforts have been focused on the screening of microorganisms harboring intrinsically stable biocatalysts. putative lipase producers were screened quantitatively across various basal media to check for lipase production and the stability of lipase. the highest lipase producing strain have been identified and characterized intensively. screening and isolation of heat-stable lipase producers are important to fulfill industrial requirements for the desired characteristics. identification of industrially important enzyme producer is conducted to determine its phylogenetic position in systematic microbiology.  screening and isolation of thermophilic lipolytic bacteria rubbish dump sites and palm oil mill effluent are potential sites containing thermophilic lipolytic bacteria because these sites served as sewage discharge areas for household waste and palm oil factory. the oily environment may provide a good environment for lipolytic microorganisms to flourish. samples were collected from the rubbish dump site and palm oil mill effluent at different treatment areas. samples were enriched with enrichment medium containing olive oil as the sole carbon source at 60°c for days under shaking condition to promote the growth of thermophilic lipolytic bacteria. twenty nine putative lipase producers gave positive results on triolein agar plate by forming an intense blue color around the colonies. of these, and isolates were isolated from rubbish dump site and palm oil mill, respectively. further confirmation test was performed quantitatively on the putative lipase producers that gave positive results on triolein agar plates. various basal media m m tyem and bm- were tested for lipase production during the selection. low lipase activities ranging from ~ u/ml were detected colorimetrically in culture supernatant, except for isolate t1t, which showed high lipase activity after h incubation. note:, indicates formation of intense blue colour on triolein agar plate on the basis of relatively higher lipase activity detected for isolate t1t, the effect of temperature on the activity and stability of crude t lipase was further investigated. the effect of temperature on the lipase activity and stability was examined from to 80°c. as shown in fig. the crude enzyme from isolate t1t manifested its maximal activity at 70°c with olive oil as substrate. crude t lipase was fairly stable up to 60°c for min and gradually decreased upon prolonged temperature treatment. this is due to t lipase tended to lose its native conformation as a result of breaking of the intrinsic interaction above its stable range. this is a discrepancy between temperature activity and stability, as the enzyme is tend to be protected by heat denaturation in the presence of olive oil. in addition, the enzyme thermostability is greatly influenced by the presence of water, because denaturation is linked to its conformational mobility in aqueous mixture. the crude enzyme of isolate t1t was fairly active at higher temperature as compared to other thermostable lipases from bacillus spp. which exhibited maximal activity at 60°c. high temperature activity and stability of enzyme offer great potentials in industrial applications, and hence attempts have been made to identify isolate t1t. identification of isolate t1t during the characterization of organism isolated from palm oil mill effluent, strain t1t was recovered on nutrient broth at 60°c. the growth condition for strain t1t was 50–70°c and between ph and with the optimum growth temperature and ph of 65°c and ph, respectively, in nutrient broth. these met the criteria of thermophilic bacteria, which grew at temperatures above 50°c. to verify the systematic position of this bacterium, a study of morphological and physiological characteristics, 16s rrna analysis, cellular fatty acids analysis, dna composition, dna/dna hybridization, riboprint analysis, lipase gene analysis and protein profile were undertaken. the cellular morphology of isolate t1t is rod-shaped, – μm width and – μm length, gram positive bacteria. the terminal spore is oval/cylindrical in shape and swollen the sporangium. the dna base composition of strain t1t is around % mol g + c. the partial sequencing of the 16s rdna shows % similarity to validly described geobacillus kaustophilus and geobacillus thermoleovorans. the 16s rrna sequence of strain t1t is a continuous stretch of bp. construction of phylogenetic trees using the neighbour-joining method in determining the evolutionary relationship among a group of validly described closely related species is indicated in fig. a comparison of biochemical, morphological and physiological properties of strain t1t with its closest phylogenetic neighbors is presented in table strain t1t can be distinguished from geobacillus thermoleovorans phenotypically by oxidase test, arabinose, mannitol, inositol, lactose and casein hydrolysis. however, strain t1t differs from geobacillus kaustophilus by lysozyme test, arabinose, mannitol, ribose, adonitol, lactose, gelatin and casein tests. since the sequencing result and physiological data did not allow strain t1t to be identified with one of the above mentioned species, further analysis need to be carried out to verify its phylogenetic position. taxa g. zalihae t1t; g. kaustophilus; g. thermoleovorans. characteristics are scored as: +, positive; -, negative; w, weak; v, variable within the group; nd, not determined. data were obtained from the present study, nazina et al., sunna et al., kuisiene et al. and markossian et al.. all strains were negative for anaerobic growth, growth at 30°c, vp reaction, indole production and acid from l-rhamnose, sorbitol. all strains were positive for catalase, hydrolysis of starch, use of citrate and acid from d-fructose and d-glucose. result of chemotaxonomic analyses is given in the species description. the fatty acid profile of strain t1t is typical for the bacillus rrna-group. the major content of cellular fatty acids of strain t1t is iso-fatty acids. among them, iso-branched pentadecanoic acid, hexadecanoic acid and heptadecanoic acid making up % of the total fatty acids for strain t1t. strain t1t can be differentiated from geobacillus thermoleovorans dsm 5366t based on percent composition of iso-fatty acids. there was only % of iso-c for strain t1t but 21% for geobacillus thermoleovorans dsm 5366t. note: data were obtained from the present study, nazina et al.. although fame is not always a reliable tool for preliminary identification of bacilli, it could be used in combination with other identification methods. dna/dna hybridization experiments were performed in dsmz with strain t1t and its type strains of closest phylogenetical neighbors. the genomic dna/dna relatedness between strain t1t and its type strains geobacillus kaustophilus geobacillus thermoleovorans were % and %, respectively. whereas, the type strains geobacillus kaustophilus and geobacillus thermoleovorans showed a dna/dna similarity of %. the dna/dna reassociation values were below the threshold value of 70% dna/dna similarity for definition of species between strain t1t and geobacillus thermoleovorans dsm 5366t but above the threshold between strain t1t and geobacillus kaustophilus dsm 7263t. neither the species geobacillus kaustophilus dsm 7263t and geobacillus thermoleovorans dsm 5366t can be differentiated from one another nor strain t1t can be differentiated at the species level from its closest phylogenetic neighbors by dna/dna hybridization. however, dna/dna hybridization tests between geobacillus kaustophilus dsm 7263t and geobacillus thermoleovorans dsm 5366t were 84% and 54% as reported by sunna et al. and nazina et al.. this disagreement may be due to the absence of adequate hybridization controls in the experiments. therefore, further tests need to be carried out to accurately place the strain t1t phylogenetically. the riboprint analysis was carried out for the decision on the affiliation of strain t1t. however, the riboprint pattern of strain t1t was not identified by the dupont identification library to give rise to the identification at the species level. its riboprint pattern showed the highest similarity to geobacillus kaustophilus dsm 7263t. the similarity to the pattern of geobacillus thermoleovorans dsm 5366t was somewhat lower. the patterns between type strains geobacillus kaustophilus dsm 7263t and geobacillus thermoleovorans dsm 5366t show a binary similarity of. further analysis was also carried out by amplifying full-length thermostable lipase gene using primers as described in materials and methods. fig. showed amplified full-length lipase genes of strain t1t and its type strains. the amplified lipase gene of strain t1t was around kb but kb for its type strains geobacillus kaustophilus dsm 7263t and geobacillus thermoleovorans dsm 5366t. a stretch of about bp insertion could be seen at downstream of the open reading frame of thermostable lipase gene. in addition, the intracellular protein profiles were determined by sds-page. samples were separated on 12% sds-polyacrylamide gel and stained using coomassie blue. strain t1t showed obvious different protein profile as compared to its type strains at region between to kda. description of geobacillus zalihae sp. nov geobacillus zalihae. the novel species is isolated from palm oil mill effluent in selangor, malaysia, with the type strain t1t. cells are rod-shaped, – width and – length, gram positive bacteria. the terminal spores are oval/cylindrical and swollen the sporangium. growth occurs at 50–70°c with an optimum temperature of 65°c. growth at 65°c occurs between ph and with maximal growth at ph. the dna base composition of strain t1t was around % mol g + c. the iso-fatty acids were in major amount according to cellular fatty acid profile in which iso-c, and iso-c were in abundant. growth is aerobic and tolerant up to 2% nacl. it can not perform anaerobic growth. it shows positive in catalase test but not oxidase test. it is able to hydrolyze starch but not gelatin and casein. acids are produced from l-arabinose, d-lactose but not d-mannitol.  as a consequence, the strain t1t merits recognition as a member of a novel species through morphological and physiological studies, cellular fatty acids composition, dna composition, dna/dna hybridization, riboprint analysis. sizes of full-length lipase genes and protein profiles were additional evidences. the geobacillus zalihae strain t1t was deposited in dsmz and nite. 
thermophilic bacillus strains of phylogenetic bacillus rrna group were described as a new genus geobacillus. the results of physiological and biochemical tests, dna/dna hybridization, riboprint analysis, the length of lipase gene and protein pattern allowed genotypic and phenotypic differentiation of strain t1t from its validly published closest phylogenetic neighbors. the strain t1t was identified as geobacillus zalihae t1t as it differs from its type strains geobacillus kaustophilus and geobacillus thermoleovorans on some physiological studies, cellular fatty acids composition, riboprint analysis, length of lipase gene and protein profile. the members from the genus geobacillus have a growth temperatures ranging from to 78°c and contained iso-branched saturated fatty acids as the major fatty acids. the quest for intrinsically thermostable lipases from thermophiles is a prominent task due to the laborious processes via genetic modification. strain t1t therefore represents a novel species, for which the name geobacillus zalihae sp. on the basic of 16s rdna analysis, strain t1t was shown to belong to the bacillus rrna group related to geobacillus thermoleovorans and geobacillus kaustophilus. nov. is proposed, with the type strain t1t. their geographical distribution included oilfields, hay compost, hydrothermal vent or soils. chemotaxonomic data of cellular fatty acids supported the affiliation of strain t1t to the genus geobacillus. of these, isolate t1t was chosen for further study as relatively higher lipase activity was detected quantitatively. twenty-nine putative lipase producers were screened and isolated from palm oil mill effluent in malaysia. strain t1t was a gram-positive, rod-shaped, endospore forming bacterium. strain t1t was able to secrete extracellular thermostable lipase into culture medium. thermophiles harboring intrinsically stable enzymes are suitable for industrial applications.
 thermophilic bacillus strains of phylogenetic bacillus rrna group were described as a new genus geobacillus. their geographical distribution included oilfields, hay compost, hydrothermal vent or soils. the members from the genus geobacillus have a growth temperatures ranging from to 78°c and contained iso-branched saturated fatty acids as the major fatty acids. the members of geobacillus have similarity in their 16s rrna gene sequences. thermophiles harboring intrinsically stable enzymes are suitable for industrial applications. the quest for intrinsically thermostable lipases from thermophiles is a prominent task due to the laborious processes via genetic modification.  twenty-nine putative lipase producers were screened and isolated from palm oil mill effluent in malaysia. of these, isolate t1t was chosen for further study as relatively higher lipase activity was detected quantitatively. the crude t lipase showed high optimum temperature of 70°c and was also stable up to 60°c without significant loss of crude enzyme activity. strain t1t was a gram-positive, rod-shaped, endospore forming bacterium. on the basic of 16s rdna analysis, strain t1t was shown to belong to the bacillus rrna group related to geobacillus thermoleovorans and geobacillus kaustophilus. chemotaxonomic data of cellular fatty acids supported the affiliation of strain t1t to the genus geobacillus. the results of physiological and biochemical tests, dna/dna hybridization, riboprint analysis, the length of lipase gene and protein pattern allowed genotypic and phenotypic differentiation of strain t1t from its validly published closest phylogenetic neighbors. strain t1t therefore represents a novel species, for which the name geobacillus zalihae sp. nov. is proposed, with the type strain t1t.  strain t1t was able to secrete extracellular thermostable lipase into culture medium. the strain t1t was identified as geobacillus zalihae t1t as it differs from its type strains geobacillus kaustophilus and geobacillus thermoleovorans on some physiological studies, cellular fatty acids composition, riboprint analysis, length of lipase gene and protein profile. 