Supplementary material
Latitude-dependent underestimation of microbial extracellular enzyme activity in soils
2. Materials and Methods
2.1. Samples
Samples were collected from different environments in sterile, 50-ml disposable
polycarbonate tubes and transported on ice to the laboratory for further analysis. Sampling
site A corresponds to a clay soil in Southwestern Spain, near Seville (location 37º 21.02’ N 5º
59.34’ W, altitude 5 m), which has been previously described (Ordoñez-Fernández et al.
2007). Soil temperatures at this location fluctuate from minimum values around 10ºC in
winter up to maxima over 60ºC during the summer period. Sandy sediment were collected
from a wetland at Doñana National Park (DNP; Southwestern Spain) (Díaz-Paniagua et al.
2010). DNP has been designated a Biosphere Reserve in 1980 and a Natural World
Heritage site in 1995. These samples were collected from the edge of three freshwater
ponds: “La Dulce” (Sampling site B) (location 36º 58.84’ N 6º 29.23’ W, altitude 3 m), “Santa
Olalla” (Sampling site C) ( location 36º 58.84’ N 6º 28.91’ W, altitude 5 m) and “Zahillo”
(Sampling site D)(location 36º 59.21’ N 6º 30.43’ W, altitude 2 m) were the three ponds visited
in DNP. Sediment samples were collected from the edge of the ponds where sediments
were not covered by water. Sandy soil (Sample site E) from DNP around “Santa Olalla”
Pond was also collected. The samples collected at the lowest temperature in this study were
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from a meadow in the vicinity of Cambridge (United Kingdom) during winter (Sampling
site F) (location 52º 11.92’ N 0º 7.07’ E, altitude 37 m). Triplicate samples were collected and
ice-cold preserved until arrival to the laboratory. Soil samples were collected from the
surface except when reported otherwise.
2.2. Enzymatic assays
In situ protease and glucosidase activities in natural samples were determined using
fluorogenic substrates without additional treatments. These substrates were the EnzCheck
green fluorescence protease assay kit (E6638; Invitrogen, Carlsbag, California, USA) and
fluorescein diglucoside (F2881; Invitrogen). Natural samples were suspended in phosphate
buffer (pH 7.0). Bacterial isolates were grown overnight, harvested by centrifugation
(5000xg 5 min) and suspended in phosphate buffer (pH 7.0). All samples were processed in
triplicate and controls without sample, controls without fluorogenic dye and autoclaved
controls were carried out. The reactions (50 µl) were prepared in 96-well microplates
according to the manufacturer’s recommendations. A final substrate concentration of 0.1
mM was used in this study (Marx et al. 2001). Preliminary studies showed maximum
activities around pH 7 in the assayed samples in agreement to other authors (Niemi and
Vepsäläinen 2005). Reaction times were 5 min. A final weight of 1-2 mg of sample per
reaction was used. The amount of samples was corrected by their dry weights. Reactions
were incubated in an optical thermocycler (iQ iCycler, Bio-Rad, Hercules, California,
USA) were fluorescence measurements were obtained. Final activity was expressed as the
slope of the linear increase of fluorescence produced during the reaction time per dry
weight at each temperature. Temperatures were assayed at 5ºC intervals from 5ºC to 95ºC.
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The proteolytic substrate was stable within this range. The glucoside substrate showed
certain instability and forced to discard numerous experiments due to increasing
fluorescence produced at increased temperatures in controls. Other fluorogenic substrates
targeting different enzymatic activities, such as phosphatases (using borate buffer) and
esterases, were tested but they were unstable at elevated temperatures.
Bacterial isolates were used to evaluate enzymatic activity along the full range of
temperatures tested (from 5ºC to 95ºC) from known bacteria. Escherichia coli K12 was
chosen as a mesophilic microorganism. E. coli was grown in nutrient broth at 30ºC. Two
thermophilic bacteria isolated from soils at 50ºC were selected as models to compare the
patterns of enzymatic activity by moderate thermophiles against mesophilic
microorganisms along the studied temperature range. These soil isolates were classified as
Brevibacillus sp. strain 10 (CECT7629) and Ureibacillus sp. strain 12 (CECT7628) and
were isolated from Seville (Andalusia, Southwestern Spain) and Teruel (Aragon,
Northeastern Spain), respectively (Portillo et al. 2012). Optimum growth temperatures were
55ºC and 60ºC for strains 10 and 12, respectively. These two thermophilic strains were
grown in nutrient broth at 50ºC. Nutrient broth was composed by beef extract (3 g l-1),
peptone (5 g l-1) and sodium chloride (5 g l-1).
2.3. Temperature sensitivity
Temperature sensitivity was studied using the parameter Q10 which was estimated according to
Stone et al. (2012) as Q10 = exp(slope x 10). The slopes represent the change of activity for each
sample enzymes at the temperature range being considered (Stone et al. 2012). Q10 constitutes the
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change of activity resulting from a temperature variation of 10ºC and it is generally expected to
show values around 2 in biological systems (Madigan et al. 2003). Q10 is probably the most
frequently used parameter to evaluate the effect of temperature on biological processes and it is
widely used in global modelling efforts (Conant et al. 2011; Davidson and Janssens 2006; Stone et
al. 2012). Q10 is probably the most frequently used parameter to evaluate the effect of
temperature on biological processes and it is widely used in global modelling efforts
(Conant et al. 2011; Davidson and Janssens 2006; Stone et al. 2012). ANOVA tests were
used to determine significant differences among samples (Sokal and Rohlf 1995).
2.4. Temperature record
The maximum daily temperature reached at locations over a full range of latitudes was
analyzed during the last forty years. Temperature records were obtained at the National
Oceanic and Atmospheric Administration (Washington DC, USA;
http://www7.ncdc.noaa.gov/CDO/cdoselect.cmd). The number of days which maximum
recorded temperature was 30ºC or above was counted for each year at a number of
locations covering roughly from 70ºN to 70ºS. An air temperature of 30ºC warranties that
soil temperature reaches 40ºC according to our measurements.
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