the formation of spores is a survival mechanism of microorganisms when exposed to unfavorable environmental conditions ( e.g. , heavy metal stress , nutrient limitations ) leading to a  dormant  or  resting
 growth state [ 1 , 2 ] . a variety of bacteria identified in diverse habitats including soil is able to form endospores .
these physiological groups include aerobic heterotrophs ( e.g. , bacillus , paenibacillus , brevibacillus , geobacillus , thermoactinomyces , and sporolactobacillus ) , anaerobes ( clostridium , anaerobacter , and   desulfotomaculum ) , microaerophiles ( sporolactobacillus ) , halophiles ( sporohalobacter ) , and phototrophs ( heliobacterium , heliophilum ) [ 3 , 4 ] . bacterial spores are characterized by a series of unique chemical features which can facilitate their identification in natural environments . besides the high content of minerals ( particularly calcium ) , spores contain high amounts of dipicolinic acid , dpa .
dpa is uniquely found in bacterial spores in amounts of up to 25% of the spore dry weight and depends on the bacterial species [ 6 , 7 ] . in solution ,
a complex is formed in the presence of terbium which shows a very strong and distinctive fluorescence spectrum .
originally , dpa was used to detect very low concentrations of terbium ( iii ) . on this basis ,
methods for the detection of bacterial endospores have been developed [ 1013 ] : by the addition of terbium , the dpa content was determined .
however , terbium - dpa fluorescence might be interfered by a series of compounds , especially when dpa has to be determined in complex samples such as sediments or soils .
it has been reported that the presence of phosphorus compounds ( especially ortho - phosphate ) reduced terbium fluorescence by as much as 98% .
the addition of aluminium compounds ( especially aluminium chloride , alcl3 ) , however , ameliorated the interference caused by the quenching substances . from a series of organic compounds (
benzoate , tryptophan , tyrosine , phenylalanine , glucose , malate , riboflavin , nad , and tryptone ) only the latter two ( especially tryptone ) reduced fluorescence significantly .
carbohydrates ( e.g. , starch , dextrine ) were reported not to interfere with the terbium fluorescence .
inorganic compounds such as calcium carbonate , sodium chloride , potassium chloride , ammonium sulphate , ammonium nitrate , and sodium nitrate did not lead to a reduction of the fluorescence , but only dipotassium phosphate did .
the aim of this study was to adopt the fluorescence - based method to determine the spore content in soils sampled from various locations .
in particular , we were interested in the differentiation between different types of soil such as grasslands ( pasture , meadow ) , allotment gardens , and forests , as well as fluvial sediments , the relationship of soil parameters ( carbon - to - nitrogen ratio ) on the occurrence of bacterial spores , and the distribution of spores in relation to sampling depth .
different bacillus species ( b. megaterium , b. subtilis ) were cultivated in liquid medium containing ( in g / l ) : glucose ( 3.6 ) , ammonium chloride ( 2.5 ) , magnesium sulfate ( 0.2 ) , calcium chloride ( 0.07 ) , iron sulfate ( 0.01 ) , edta ( 0.01 ) , potassium dihydrogen phosphate ( 0.6 ) , dipotassium hydrogen phosphate ( 0.9 ) , and yeast extract ( 1.0 ) .
erlenmeyer flasks ( 250  ml ) containing 100  ml of growth medium were inoculated and incubated for 10 to 15 days ( 150  rpm , 30c ) . to initiate and stimulate sporulation ,
bacteria were subsequently transferred to a sporulation medium ( identical composition , but without glucose and less ammonium chloride [ only 1  g / l ] ) .
after additional 30 days of incubation  until vegetative cells were not present anymore after inspection by microscopy  spores were harvested by centrifugation , immediately frozen in liquid nitrogen followed by lyophilization .
samples from different locations were collected using a stainless steel soil corer ( 15  mm in diameter ) , which was sterilized before each sampling .
cores with a maximum length of 25  cm were obtained , cut in sections of 5  cm , transferred to sterile screw cap falcon tubes ( 20  ml ) , and stored on dry ice .
after return to the laboratory , samples were immediately lyophylized or stored at 80c until further processing .
sampling sites were located in the surroundings of zurich ( switzerland ) : grassland soil , meadow ( municipalities of mnnedorf ; uerikon ; stfa ; and dbendorf ) , allotment garden ( university of zurich , irchel campus ) , pasture ( university of zurich , irchel campus ; municipality of wdenswil ) , forest soil ( municipality of stfa ) , and aquatic sediments ( river glatt in dbendorf ) . lyophilized aliquots of approximately 1  g were transferred to an eppendorf micro test tube ( 2  ml ) and ground ( by adding a 6  mm glass bead ) in tissuelyser ( retsch , haan , germany ) for 5  1 min .
elemental composition ( carbon , hydrogen , nitrogen ) of soil was performed with a chn-932 elemental analyzer ( leco corp . , st .
composition ( in % of dry soil ) varied between 2.2 and 15.4 , 0.2 and 1.4 , and 0.2 and 2.0 for total carbon , total hydrogen , and total nitrogen , respectively .
phosphate in aqueous soil extracts ( 250  mg soil in 5  ml sodium acetate buffer ; 0.2 m , ph 5 ) was determined using commercially available kits ( lck 348 and 349 ; hach lange ag , hegnau , switzerland ) .
10  mg of dry spore powder was resuspended in 10  ml sodium acetate buffer ( 0.2  m , ph 5 ) .
soil samples were thawed and 50  mg were suspended in 0.9  ml sodium acetate buffer and 0.1  ml aluminium chloride ( alcl3 , 0.5  m ) .
samples were microwaved ( berghof microwave digester mws-1 , with built - in in situ infrared temperature control ) in teflon tfm screw cap digestion vessels .
temperature and power were set to 140c and approximately 680 w ( 80% ) , respectively .
alternatively , dpa was released from spores by autoclaving the samples in screw cap glass test tubes for 15 minutes at 121c .
the identical dpa extraction protocol was applied for soil samples . however , microscopy was not possible due to the presence of mineral particles interfering the observation .
after cooling for 30 minutes , 100  l of the spore suspensions were mixed with 100  l terbium chloride solution ( tbcl3 , 30  m ) in white 96-well microtiter plates ( in 8 replicates ) .
fluorescence was immediately measured using a plate reader ( spectramax m2 , bucher biotec , basel , switzerland ) with the following settings : time - resolved fluorescence ( delay 50  s , interval 1200  s ) at an excitation wavelength of 272  nm , emission wavelength of 545  nm , and 10 endpoint readings per sample at 30c .
the number of spores in the soil samples was determined using standard addition method with spores of b. subtilis .
microwave treatment of spore suspensions and soil samples led to a fast release of dpa ( figure 1 ) . within two minutes , maximum release was obtained .
highest spore numbers up to 4   10 spores per gram dry soil were found in agriculturally used land ( meadow , pasture ) , less in forest soil .
the interference of different compounds present in soil ( e.g. , phosphate ) might lead to quenching of the fluorescence signal .
this drawback has been overcome by the addition of aluminium chloride as already shown for the determination of bacterial spores in aquatic sediments .
concentration of ortho - phosphate in soil extracts ( 22.5  m ) was reduced by the addition of aluminium chloride to concentrations below the detection limit ( < 1.2  m ) .
concomitantly , a de - colorization of the extract was observed suggesting the removal of humic acids which have also the potential to form complexes with terbium and quench the fluorescence signal .
the method based on terbium fluorescence for the detection of total numbers of bacterial endospores in soils is fast and easy .
a transect ( approximately 100  m in length ) through a grass field with different land use management ( unused meadow , allotment garden , and pasture ) gave spore numbers in the range of 5 to 9  10 spores per gram of dry soil ( figure 3 ) .
spore counts were not related to the type of land use : in allotment garden soil , counts were not significantly different from soil samples taken from a pasture ( p = 0.423 ; t - test ) . regarding the different sampling sites ,
our results show that grassland soils ( meadow , allotment garden , and pasture ) contains much more bacterial spores than forest soils and fluvial sediments .
spore content was related to the carbon - to - nitrogen ratio ( figure 4 ) . at c / n ratios > 20 only low spore counts ( 0.5  10 spores per gram of dry soil ) were detected as compared to c / n ratios < 20 .
it has been demonstrated in pure cultures of bacillus thuringiensis in a stirred bioreactor that low carbon - to - nitrogen ratios of 4  :  1 resulted in high spore counts .
in contrast however , spore formation in streptomyces coelicolor was stimulated under nitrogen - limiting conditions .
in particular , c / n ratios between 50 and 100 promoted sporulation , whereas c / n rations < 40 did not allow spore formation .
our results showed that in soils with extremely high c / n ratios , spore content was low .
the importance of c / n ratio was stressed by gao and coworkers regarding the sporulation of fungi , although fungal spores do not contain dpa . a carbon - to - nitrogen ( c / n ) ratio of 20 stimulated spore formation by fungi such as penicillium camembertii .
the fungus colletotrichum coccodes produced highest spore counts at a c / n ratio of 5 to 10 , whereas at a ratio of 40 , spore formation was significantly lower .
similarly , in plectosporium tabacinum optimal spore formation was found when c / n rations were between 5 and 10 .
the distribution of spores in marine sediments ( determined as dpa ) showed only a low correlation with the content of total organic carbon and varied with the sediment type .
highest numbers have been found in organic - rich black sediments , lowest number in sandy sediments .
it was hypothesized from anthrax outbreaks , that the high numbers of bacillus spores might be related to soils rich in organic matter that is , to a high c / n ratio .
these soil environmental conditions are suggested to support the presence and viability of b. anthracis spores .
depth distribution of spores from an area currently used as allotment garden ( cultivation of flowers and vegetables ) showed highest numbers in a horizon of 5 to 10  cm ( figure 5 ) .
the two methods evaluated ( microwaving , autoclaving ) for the mobilization of dpa from bacterial spores gave similar results .
however , microwaving was less time - consuming , whereas autoclaving allowed faster throughput of samples .
in summary , microwave treatment of soil samples followed by the measurement of fluorescence after addition of terbium proved to be a fast and easy method to assess the content of bacterial spores .
our study might provide a basis for the detection of  hot spots  of endospores in soil .
 spore formation is a survival mechanism of microorganisms when facing unfavorable environmental conditions resulting in  dormant  states . 
 we investigated the occurrence of bacterial endospores in soils from various locations including grasslands ( pasture , meadow ) , allotment gardens , and forests , as well as fluvial sediments . 
 bacterial spores are characterized by their high content of dipicolinic acid ( dpa ) . in the presence of terbium , dpa forms a complex showing a distinctive photoluminescence spectrum . 
 dpa was released from soil by microwaving or autoclaving . 
 the addition of aluminium chloride reduced signal quenching by interfering compounds such as phosphate . 
 the highest spore content ( up to 109 spores per gram of dry soil ) was found in grassland soils . 
 spore content is related to soil type , to soil depth , and to soil carbon - to - nitrogen ratio . 
 our study might provide a basis for the detection of  hot spots  of bacterial spores in soil . 
1. Introduction
2. Materials and Methods
3. Results and Discussion
4. Conclusions