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International Journal of Speleology                  40 (1)           17-21       Tampa, FL (USA)                       January 2011



                         Available online at www.ijs.speleo.it & scholarcommons.usf.edu/ijs/

                            International Journal of Speleology
                                  Official Journal of Union Internationale de Spéléologie



In situ acetylene reduction activity of Scytonema julianum in Vapor cave
(Spain)
Antonia Dolores Asencio1 and Marina Aboal2

Abstract:
Asencio A.D., Aboal M. 2011. In situ acetylene reduction activity of Scytonema julianum in Vapor cave (Spain). International Journal of
Speleology, 40 (1), 17-21. Tampa, FL (USA). ISSN 0392-6672. DOI: 10.5038/1827-806X.40.1.3

Nitrogen fixation was measured in situ for the first time by acetylene reduction for a greyish mat composed of Scytonema julianum in cave-
like environments. Mat-specific rates (129.9-215.7 nmol C2 H4 m-2 s-1 for daytime fixation and 65.1-120.6 nmol C2 H4 m-2 s-1 for nighttime
fixation) recorded in the Vapor cave differed considerably due to the energy reserves stored during photosynthesis being exhausted and
used in the dark phase. The most influential environmental parameter for nitrogen fixation in the Vapor cave is temperature in the daytime
and nighttime fixations. Nitrogen fixation by cyanobacteria may contribute considerably to the overall nitrogen cycle in harsh environments
such as caves. Nitrogenase activity in Scytonema julianum was roughly 30 times higher than that of Scytonema mirabile, which also grew
in cave environments, which is due to the characteristics of each site. The entrance of Vapour cave (Spain) faces SE, measures 0.75 x
0.6 m and opens to shafts of a total depth of 80 m. Its dimensions and environmental conditions (relative humidity up to 100%; maximum
temperature, 43ºC) imply that it is isolated from external influences, and that the microclimate differs substantially from that experienced
externally. Nitrogen fixation, photon flux density, relative humidity and temperature in the Vapor cave were taken hourly over a 24-hour
period in winter.
Keywords: caves, cyanobacteria, nitrogen fixation, Scytonema julianum, SE Spain
Received 14 August 2010; Revised 28 September 2010; Accepted 12 October 2010


                     INTRODUCTION                                        branched, heterocysted species characterized by the
  Nitrogen is a nutrient that limits the growth                          presence of calcium carbonate crystals on sheaths.
of microorganisms in all kinds of environments,                          Most authors consider this species as typical of
especially N-poor ones, like caves where nutrients are                   lit walls close to cave openings (Hoffmann, 1989),
provided fundamentally thanks to the visits of certain                   whereas others have detected it in poorly lit conditions
animals that use caves as shelters (Hill, 1987).                         (Aboal et al., 1994).
  Cyanobacteria are the most abundant phototrophic                          Scytonema julianum has been studied from
microorganisms to grow in caves. Most of them have                       morphological (Aboal et al., 1994; Couté & Bury, 1988;
structures called heterocysts in which the enzyme                        Friedman, 1973; Hoffmann, 1992) and biochemical
responsible for nitrogen fixation, nitrogenase, is                       (Bellezza et al., 2006; Antonopoulou et al., 2002,
found to protect the enzyme from oxygen since this                       2005) viewpoints, but nothing is known about its
gas irreversibly inhibits this enzyme (Bothe, 1982).                     atmospheric nitrogen fixation capacity.
The capacity to fix atmospheric nitrogen allows                             There are very few previous studies on in situ nitrogen
cyanobacteria to play an important role in extreme                       fixation by cyanobacteria in caves (Griffiths et al.,
environments (Houssman et al., 2006; Zielke et al.,                      1987; Asencio & Aboal, 2010), despite its importance
2005).                                                                   owing to the fact that certain environmental factors,
  Scytonema julianum (Meneghini ex Franck) Richter                       which cannot be reproduced in the laboratory,
is one of the most abundant cyanobacteria in caves                       intervene in this process. This research work focuses
(Hoffmann, 1989). It is an aerophytic, filamentous,                      on investigating the in situ nitrogen fixation capacity of
                                                                         a typical cave species, Scytonema julianum, in special
1
  División Botánica. Departamento de Biología Aplicada.                  environmental conditions (maximum temperature,
Facultad de Ciencias Experimentales. Universidad Miguel                  43ºC and relative humidity up to 100%) where it
Hernández. Campus de Elche. Avenida Universidad, s/n.                    grows.
03202 Elche, Alicante, Spain.                                                                     STUDY AREA
e-mail: aasencio@umh.es (corresponding author)                             The Vapor cave (Fig. 1) is located in Alhama (Murcia
2
  División Botánica. Departamento de Biología Vegetal.                   Region, SE Spain), a municipal area crossed by the
Facultad de Biología. Universidad de Murcia. Campus de                   Alhama Fault. This Fault is considered one of the most
Espinardo. 30100 Murcia, Spain.                                          active in the Iberian Peninsula, and is responsible for
e-mail: maboal@um.es                                                     heating ground waters in this region.
18                                                      Antonia Dolores Asencio and Marina Aboal




Fig. 1. The Vapor cave entrance in Murcia (SE Spain).

  This enclave is located on the Castillo Hill at an
altitude of 295 m (UTM 30SXG389912) and calcite
is the dominant mineral (95%). At the bottom of this
hill we find a warm water spring, used by different                            Fig. 3. Wall of Vapor cave showing the Scytonema julianum mat.
civilizations for centuries. The phreatic level of the
region has dropped by more than 100 m over time,
and currently the Alhama warm-water spring does
not actually flow; but when approaching the Vapor
cave a constant steam flow can be observed (Fig. 2).




Fig. 2. Panoramic view of Vapor cave with the steam flowing (arrow) from
the entrance.

  The entrance to this cave, facing SE, is oval-shaped
and the axes measure 0.6 x 0.75 m. It opens to form
a diaclase with the strata of conglomerated Triassic
limestone edges. Then a small 3.5 m deep vertical
                                                                               Fig. 4. Drawing showing the different passages and shafts in the Vapor
shaft is found in which only plant organisms have been
                                                                               cave.
registered. The initial 0.5 m are covered by bryophytes
giving it a greenish tone whereas the following 3 m are                        two descents commence with an uneven level at
made up of cyanobacteria that create a greyish hue                             approximately -20 m, which places the cavity on a
(Fig. 3).                                                                      final uneven level of -80 m.
  The cave continues over approximately 50 m along a                             The relative humidity inside the cavity is 100%.
sharp downward sloping passage, which runs unlevel                             Temperatures vary and increase with depth, ranging
at -30 m in a SE-NW direction until it reaches the                             between 27ºC and 43ºC. The carbon dioxide and
top of the Agobio shaft (Fig. 4). This shaft is split in                       oxygen concentrations inside the cave are 1.8% for CO2
two sections: one measuring 18.30 m that reaches the                           and 18.5% for O2 as opposed to 0.03% and 20.93%,
Chopard shelf and the other is the bottom measuring                            respectively, recorded in the atmosphere. Given these
13.70 m. The total shaft length comes to 32 m where                            environmental conditions, the visitors only can access
the uneven level lies at -61.41 m. From this point,                            the cave for short periods of time.

                                   International Journal of Speleology, 40 (1), 17-21. Tampa, FL (USA). January 2011
In situ acetylene reduction activity of Scytonema julianum in Vapor cave (Spain)                                        19


           MATERIALS AND METHODS
  Material was collected aseptically from the wall near
the Vapor cave opening where Scytonema julianum
grows. We were careful not to remain inside too long
to avoid any ill effects to our organisms because of the
high temperature and high relative humidity of this
cave (Ruíz de Almirón, 1998).
  Non-hydrated samples were incubated in situ from
sunrise to sunset in triplicate inside transparent and
opaque sterile vials to reproduce the light and dark
conditions, respectively. Then 10% of the gaseous
phase of these vials was replaced with acetylene by
placing syringes through the rubber vial stoppers.
Gaseous samples were collected on an hourly basis
over a 24-hour period in winter between sunrise and
sunset. The following values were recorded hourly
throughout the incubation period: photon flux
density (PAR - photosynthetically active radiation),
air temperature and relative humidity. A LI-1400
datalogger model (LICOR) with a LI-192 sensor and
a Delta Ohm HD 8501 H thermohygrometre were
used. Electrodes were placed on the rock surface.
The concentrations of CO2 and O2 in the cave air were
measured with a hand pump (Gastec Corporation,
Japan) and detector tubes (measurement range: CO2,
300-5,000 ppm; O2, 3-24%). Nitrogen fixation was
quantified by acetylene-ethylene reduction and was
subsequently analyzed in a Shimadzu GC 14 A gas
chromatograph.

                       RESULTS
  Nitrogen fixation by Scytonema julianum (Fig. 5), a
species growing in the Vapor cave forming a greyish
covering, was measured for the first time.
  During the daytime, nitrogenase activity ranged
between 129.9 and 215.7 nmol of C2 H4 m-2 s-1 (Fig.
6). It reached its peak value after a series of high
values followed by low values. Thus, the graphic
representation of nitrogenase activity is a zigzag
pattern. The highest values were recorded at 11:00h,
13:00h and 15:00h at 201.2, 215.7 and 213.0 nmol
of C2 H4 m-2 s-1, respectively, while the lowest values
were observed at 08:00h, 10:00h, 12:00h, 14:00h and
17:00h at 125.1, 129.9, 157.8 and 130.4 nmol of C2
H4 m-2 s-1, respectively.                                            Fig. 5. Light micrographs [scale bar: 10 µm] of Scytonema julianum.
  The PAR values in the Vapor cave ranged between 5.2
and 57.7 μE.m-2.s-1 (Fig. 7). From 08:00h to 10:00h,                 27.5, 28.1, 29.1 and 27.4ºC noted at 08:00h, 10:00h,
these values increased from 7.5 to 18.9 μE.m-2.s-1.                  12:00h, 14:00h and 18:00h, respectively.
Then at this time, a substantial increase took place at                Nighttime nitrogen fixation varied from 65.1 to
11:00h reaching 48.6 μE.m-2.s-1. Finally, these values               120.6 nmol of C2 H4 m-2 s-1 (Fig. 6) and nitrogenase
continued rising until a peak value of 57.7 μE.m-2.s-1               activity was graphically depicted as a zigzagging line.
was noted at 13:00h, to drop later.                                  At 08:00h, a value of 79.2 nmol of C2 H4 m-2 s-1 was
  The minimum value of relative humidity (Fig. 7)                    noted, which rose to 120.6 nmol of C2 H4 m-2 s-1 at
was 72% recorded at 08:00h. Relative humidity                        10:00h. After this time, it dropped to 78.3 nmol of C2
progressively increased to a maximum value of 100%                   H4 m-2 s-1 at 11:00h. Nitrogenase activity started rising
at 11:00h, and remained so until 17:00h when it                      again to reach 94.4 nmol of C2 H4 m-2 s-1 at 12:00h and
dropped slightly to 99.2% at 18:00h.                                 then dropped to 75.8 C2 H4 m-2 s-1 at 13:00h. Finally,
  Temperatures in the Vapor cave ranged between                      at 14:00h it rose to 97.3 C2 H4 m-2 s-1 at 15:00h, from
27.2ºC, recorded at 08:00h, to 31.0ºC noted at 13:00h                which time nitrogenase activity descended to reach a
(Fig. 7). The highest temperatures were 28.9, 31.0                   minimum value of 65.1 to 120.6 nmol of C2 H4 m-2 s-1
and 29.5ºC recorded at 11:00h, 13:00h and 15:00h,                    at 17:00h.
respectively, while the lowest temperatures were 27.2,

                         International Journal of Speleology, 40 (1), 17-21. Tampa, FL (USA). January 2011
20                                                                                               Antonia Dolores Asencio and Marina Aboal


                                              250                                                                       the U.S.A. (Jeffries et al., 1992), we notice a vast
                                                                                                                        difference, despite previously rehydrating samples,
                                                                                                                        which confirms the importance of a high, constant
                                              200
  Nitrogenase activity (nmol C 2H4 m -2s-1)




                                                                                                                        relative humidity in the nitrogen fixation process,
                                                                                                                        which also occurs in the Vapor cave.
                                              150                                                                         We also noted that the greater the light intensity at
                                                                                                                        the Vapor cave study site, the higher the nitrogenase
                                              100
                                                                                                                        activity. This coincides with the results of Dodds
                                                                                                                        (1989), even to the point that the times the highest
                                                                                                                        nitrogenase activities were recorded coincide with the
                                               50
                                                                                                           light        maximum PAR, temperature and relative humidity
                                                                                                           dark         values.
                                                0                                                                         Ethylene nighttime production by Scytonema
                                                         8       10        12               14        16
                                                                           Time (h)
                                                                                                                        julianum was between 65.1 and 120.6 nmol of
                                                                                                                        C2 H4 m-2 s-1. These values were lower than those
Fig. 6. Nitrogenase activity by Scytonema julianum (light and dark) in Vapor
                                                                                                                        obtained in the daytime, which occurred with all
cave.
                                                                                                                        the heterocysted cyanobacteria. Although these
                                                                                                                        prokaryotes are considered totally photoautotroph
                                                                                                                        organisms, some may grow in the darkness at a slower
                                                                        VAPOR cave
                                                                                                                        rate (Stewart, 1973; Jones, 1992).
                                                70                                                                        Temperature inside the Vapor cave changed in
                                                60                                                                      accordance with the daytime or nighttime nitrogenase
    PAR (µEm-2s-1)




                                                50
                                                                                                                        activity. When temperatures were lower, nitrogenase
                                                40
                                                30
                                                                                                                        activity dropped, which is clearly demonstrated in the
                                                20                                                                      daytime when ethylene production dropped sharply as
                                                10                                                                      temperatures lowered, and again increased at higher
                                                    0
                                                                                                                        temperatures. The exact opposite took place with
                                                                                                                        nighttime nitrogen fixation as nitrogenase activity
                                                120
                                                                                                                        dropped at higher temperatures, and increased with
                                                                                                                        lower temperatures.
         Relative humidity (%)




                                                100
                                                    80
                                                                                                                          Nitrogenase activity by Scytonema julianum was
                                                    60
                                                                                                                        roughly 30 times higher than that of Scytonema
                                                                                                                        mirabile, which also grew in a similar environment
                                                    40
                                                                                                                        (Asencio & Aboal, 2010), due to the characteristics of
                                                    20
                                                                                                                        each site. The Andragulla cave was 2.0 m deep, 2.0
                                                     0
                                                                                                                        m high and 6.0 m wide. Its lack of depth meant that
                                                                                                                        the microclimate was very similar to that experienced
                                                    32
                                                                                                                        externally, which explained its extreme PAR (0.5-
                                                    31                                                                  582.7 μE.m-2.s-1), temperature (1.5-20.3 ºC) and
                         Temperature (ºC)




                                                    30                                                                  relative humidity (24.0-79.9 %) winter values. The
                                                    29                                                                  Vapor cave, however, was very deep so it was isolated
                                                    28
                                                                                                                        from external influences. Therefore, its PAR values
                                                    27
                                                                                                                        remained constant and it had very high temperature
                                                    26
                                                    25
                                                                                                                        and relative humidity values.
                                                             8    10      12           14        16                18     The first ever daytime and nighttime nitrogen
                                                                            Time (h)                                    fixation values by Scytonema julianum recorded in the
                                                                                                                        Vapor cave differed considerably, but coincided with
Fig. 7. PAR (Photosynthetically active radiation), temperature and relative                                             Calothrix in tropical environments (Jones, 1992). This
humidity from sunrise to sunset in Vapor cave from a cyanobacterial mat of                                              was likely due to the energy reserves stored during
Scytonema julianum.                                                                                                     photosynthesis being exhausted and used in the dark
                                                                       DISCUSSION                                       phase, which occurred with Nostoc in Californian
  Nitrogenase activity by Scytonema julianum recorded                                                                   streams (Horne & Carmiggelt, 1974). This fact
in the Vapor cave over a 24-h period ranged from                                                                        suggests that Scytonema julianum, like other tropical
129.9 to 215.7 nmol of C2 H4 m-2 s-1. These values are                                                                  terrestrial cyanobacteria (Jones, 1981, Saino &
very close to those obtained for the Calothrix genus:                                                                   Hattori, 1978), preferred high-levels of sunlight which
284 nmol of C2 H4 m-2 s-1 in tropical environments                                                                      could substantially contribute to the overall nitrogen
(Jones, 1992), where similar relative humidity and                                                                      cycle in N-poor environments, such as cave entrances
temperature conditions to those in the Vapor cave                                                                       and other lighted areas.
were registered.
  If we compare the aforementioned values with                                                                                         ACKNOWLEDGEMENTS
the mean 28.2 nmol value of C2 H4 m-2 s-1 recorded                                                                        We sincerely thank J.J. Ruíz de Almirón for his help
in Scytonema sp. crusts from semi-arid areas of                                                                         in the field, H. Warburton for his assistance with the

                                                                          International Journal of Speleology, 40 (1), 17-21. Tampa, FL (USA). January 2011
In situ acetylene reduction activity of Scytonema julianum in Vapor cave (Spain)                            21


English version of the text and Dr. D.E. Northup and                    Hill C.A., 1987 – Geology of Carlsbad Cavern and other
an anonymous reviewer for their comments on the                           caves in the Guadalupe Mountains, New Mexico and
manuscript.                                                               Texas: Socorro, N.M. New Mexico Bureau of Mines and
                                                                          Mineral Resources Bulletin, 117: 150.
                      REFERENCES                                        Hoffmann L., 1989 – Algae of terrestrial habitats.
Aboal M., Asencio A.D. & Prefasi M., 1994 – Studies on                    Botanical Review, 55: 77-105.
  cave cyanophytes from southeastern Spain: Scytonema                   Hoffmann L., 1992 – Variability in the crystal morphology
  julianum Richter. Archiv für Hydrobiology/ Algological                  of calcified terrestrial      Scytonema       populations
  Studies, 75: 31-36.                                                     (Cyanobacteria,     Cyanophyceae).       Geomicrobiology
Antonopoulou S., Oikonomou A., Karantonis H.C.,                           Journal, 10: 59-64.
  Fragopoulou E. & Pantazidou A., 2002 – Isolation and                  Horne A.J. & Carmiggelt C.J.W., 1974 – Algal nitrogen
  structural elucidation of biologically active phospholipids             fixation in Californian streams: seasonal cycles.
  from Scytonema julianum (Cyanobacteria). Biochemical                    Freshwater Biology, 5, 461-470.
  Journal, 367: 287-293.                                                Houssman D.C., Powers H.H., Collins A.D. & Belnap
Antonopoulou S., Karantonis H.C., Nomikos T.,                             J., 2006 – Carbon and nitrogen fixation differ between
  Oikonomou A., Fragopoulou E. & Pantazidou A.,                           successional stages of biological soil crusts in the
  2005 – Bioactive polar lipids from Chroococcidiopsis                    Colorado Plateau and Chihuahuan Desert. Journal of
  sp. (Cyanobacteria). Comparative Biochemistry and                       Arid Environments, 66: 620-634.
  Physiology-B Biochemistry and Molecular Biology,                      Jeffries D.L., Klopatek J.M., Link S.O. & Bolton H.Jr.,
  142: 269-282.                                                           1992 – Acetylene reduction by cryptogamic crusts from
Asencio A.D. & Aboal M., 2010 – In situ nitrogen fixation                 a blackbrush community as related to resaturation and
  by cyanobacteria at the Andragulla cave (Spain).                        dehydration. Soil Biology and Biochemistry, 24: 1101-
  Journal of Cave and Karst Studies, 72(3): in press.                     1105.
Bellezza S., Albertano P., De Philippis R. & Paradossi                  Jones K., 1981 – Diurnal acetylene reduction by mats of
  G., 2006 – Exopolysaccharides of two cyanobacterial                     blue-green algae in sub-tropical grassland: use of short-
  strains from Roman hypogea. Geomicrobiology Journal,                    term and long-term assays. New Phytology, 88: 73-78.
  23: 301-310.                                                          Jones K., 1992 – Diurnal nitrogen fixation in tropical
Bothe H., 1982 – Nitrogen fixation. In: Carr, N.G. &                      marine cyanobacteria: a comparison between adjacent
  Whiton, B.A. (Eds), The Biology of Cyanobacteria.                       communities of non-heterocystous Lyngbya sp. and
  University of California Press, Berkeley, 87-104.                       heterocystous Calothrix sp. British Phycological
Couté A. & Bury E., 1988 – Ultrastructure d’une                           Journal, 27:107-118.
  cyanophycée aérienne calcifiée cavernicole: Scytonema                 Ruíz de Almirón J.J., 1998 – La sima del Vapor.
  julianum     (Frank)    Richter (Hormogonophycideae,                    Subterránea, 9: 36-43.
  Nostocales, Scytonemataceae). Hydrobiologia, 160:                     Saino T. & Hattori A., 1978 – Dial variation in nitrogen
  219-239.                                                                fixation by a marine blue-green alga, Trichodesmium
Dodds W.K., 1989 – Microscale vertical profiles of N2                     thiebautii. Deep Sea Research, 25: 1259-1263.
  fixation, photosynthesis, O2, chlorophyll a and light in a            Stewart W.D.P., 1973 – Nitrogen fixation. In Carr, N.G.
  cyanobacterial assemblage. Applied and Environmental                    & Whiton, B.A. (Eds), The Biology of Blue-Green Algae.
  Microbiology, 55: 882-886.                                              Blackwell Scientific Publications, Oxford: 260-278.
Friedman E.I., 1973 – Scanning electron microscopy of                   Zielke M., Solheim B., Spjelkavik S. & Olsen R. A., 2005
  three lime encrusting aerophytic blue-green algae.                      – Nitrogen fixation in the high Arctic: role of vegetation
  Journal of Phycology, 9: 17.                                            and environmental conditions. Arctic, Antarctic and
Griffiths M.S.H., Gallon J.R. & Chaplin A. E., 1987 – The                 Alpine Research, 37: 372-378.
  diurnal pattern of dinitrogen fixation by cyanobacteria
  in situ. New Phytologist, 107: p. 649-657.




                            International Journal of Speleology, 40 (1), 17-21. Tampa, FL (USA). January 2011

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Vapor

  • 1. International Journal of Speleology 40 (1) 17-21 Tampa, FL (USA) January 2011 Available online at www.ijs.speleo.it & scholarcommons.usf.edu/ijs/ International Journal of Speleology Official Journal of Union Internationale de Spéléologie In situ acetylene reduction activity of Scytonema julianum in Vapor cave (Spain) Antonia Dolores Asencio1 and Marina Aboal2 Abstract: Asencio A.D., Aboal M. 2011. In situ acetylene reduction activity of Scytonema julianum in Vapor cave (Spain). International Journal of Speleology, 40 (1), 17-21. Tampa, FL (USA). ISSN 0392-6672. DOI: 10.5038/1827-806X.40.1.3 Nitrogen fixation was measured in situ for the first time by acetylene reduction for a greyish mat composed of Scytonema julianum in cave- like environments. Mat-specific rates (129.9-215.7 nmol C2 H4 m-2 s-1 for daytime fixation and 65.1-120.6 nmol C2 H4 m-2 s-1 for nighttime fixation) recorded in the Vapor cave differed considerably due to the energy reserves stored during photosynthesis being exhausted and used in the dark phase. The most influential environmental parameter for nitrogen fixation in the Vapor cave is temperature in the daytime and nighttime fixations. Nitrogen fixation by cyanobacteria may contribute considerably to the overall nitrogen cycle in harsh environments such as caves. Nitrogenase activity in Scytonema julianum was roughly 30 times higher than that of Scytonema mirabile, which also grew in cave environments, which is due to the characteristics of each site. The entrance of Vapour cave (Spain) faces SE, measures 0.75 x 0.6 m and opens to shafts of a total depth of 80 m. Its dimensions and environmental conditions (relative humidity up to 100%; maximum temperature, 43ºC) imply that it is isolated from external influences, and that the microclimate differs substantially from that experienced externally. Nitrogen fixation, photon flux density, relative humidity and temperature in the Vapor cave were taken hourly over a 24-hour period in winter. Keywords: caves, cyanobacteria, nitrogen fixation, Scytonema julianum, SE Spain Received 14 August 2010; Revised 28 September 2010; Accepted 12 October 2010 INTRODUCTION branched, heterocysted species characterized by the Nitrogen is a nutrient that limits the growth presence of calcium carbonate crystals on sheaths. of microorganisms in all kinds of environments, Most authors consider this species as typical of especially N-poor ones, like caves where nutrients are lit walls close to cave openings (Hoffmann, 1989), provided fundamentally thanks to the visits of certain whereas others have detected it in poorly lit conditions animals that use caves as shelters (Hill, 1987). (Aboal et al., 1994). Cyanobacteria are the most abundant phototrophic Scytonema julianum has been studied from microorganisms to grow in caves. Most of them have morphological (Aboal et al., 1994; Couté & Bury, 1988; structures called heterocysts in which the enzyme Friedman, 1973; Hoffmann, 1992) and biochemical responsible for nitrogen fixation, nitrogenase, is (Bellezza et al., 2006; Antonopoulou et al., 2002, found to protect the enzyme from oxygen since this 2005) viewpoints, but nothing is known about its gas irreversibly inhibits this enzyme (Bothe, 1982). atmospheric nitrogen fixation capacity. The capacity to fix atmospheric nitrogen allows There are very few previous studies on in situ nitrogen cyanobacteria to play an important role in extreme fixation by cyanobacteria in caves (Griffiths et al., environments (Houssman et al., 2006; Zielke et al., 1987; Asencio & Aboal, 2010), despite its importance 2005). owing to the fact that certain environmental factors, Scytonema julianum (Meneghini ex Franck) Richter which cannot be reproduced in the laboratory, is one of the most abundant cyanobacteria in caves intervene in this process. This research work focuses (Hoffmann, 1989). It is an aerophytic, filamentous, on investigating the in situ nitrogen fixation capacity of a typical cave species, Scytonema julianum, in special 1 División Botánica. Departamento de Biología Aplicada. environmental conditions (maximum temperature, Facultad de Ciencias Experimentales. Universidad Miguel 43ºC and relative humidity up to 100%) where it Hernández. Campus de Elche. Avenida Universidad, s/n. grows. 03202 Elche, Alicante, Spain. STUDY AREA e-mail: aasencio@umh.es (corresponding author) The Vapor cave (Fig. 1) is located in Alhama (Murcia 2 División Botánica. Departamento de Biología Vegetal. Region, SE Spain), a municipal area crossed by the Facultad de Biología. Universidad de Murcia. Campus de Alhama Fault. This Fault is considered one of the most Espinardo. 30100 Murcia, Spain. active in the Iberian Peninsula, and is responsible for e-mail: maboal@um.es heating ground waters in this region.
  • 2. 18 Antonia Dolores Asencio and Marina Aboal Fig. 1. The Vapor cave entrance in Murcia (SE Spain). This enclave is located on the Castillo Hill at an altitude of 295 m (UTM 30SXG389912) and calcite is the dominant mineral (95%). At the bottom of this hill we find a warm water spring, used by different Fig. 3. Wall of Vapor cave showing the Scytonema julianum mat. civilizations for centuries. The phreatic level of the region has dropped by more than 100 m over time, and currently the Alhama warm-water spring does not actually flow; but when approaching the Vapor cave a constant steam flow can be observed (Fig. 2). Fig. 2. Panoramic view of Vapor cave with the steam flowing (arrow) from the entrance. The entrance to this cave, facing SE, is oval-shaped and the axes measure 0.6 x 0.75 m. It opens to form a diaclase with the strata of conglomerated Triassic limestone edges. Then a small 3.5 m deep vertical Fig. 4. Drawing showing the different passages and shafts in the Vapor shaft is found in which only plant organisms have been cave. registered. The initial 0.5 m are covered by bryophytes giving it a greenish tone whereas the following 3 m are two descents commence with an uneven level at made up of cyanobacteria that create a greyish hue approximately -20 m, which places the cavity on a (Fig. 3). final uneven level of -80 m. The cave continues over approximately 50 m along a The relative humidity inside the cavity is 100%. sharp downward sloping passage, which runs unlevel Temperatures vary and increase with depth, ranging at -30 m in a SE-NW direction until it reaches the between 27ºC and 43ºC. The carbon dioxide and top of the Agobio shaft (Fig. 4). This shaft is split in oxygen concentrations inside the cave are 1.8% for CO2 two sections: one measuring 18.30 m that reaches the and 18.5% for O2 as opposed to 0.03% and 20.93%, Chopard shelf and the other is the bottom measuring respectively, recorded in the atmosphere. Given these 13.70 m. The total shaft length comes to 32 m where environmental conditions, the visitors only can access the uneven level lies at -61.41 m. From this point, the cave for short periods of time. International Journal of Speleology, 40 (1), 17-21. Tampa, FL (USA). January 2011
  • 3. In situ acetylene reduction activity of Scytonema julianum in Vapor cave (Spain) 19 MATERIALS AND METHODS Material was collected aseptically from the wall near the Vapor cave opening where Scytonema julianum grows. We were careful not to remain inside too long to avoid any ill effects to our organisms because of the high temperature and high relative humidity of this cave (Ruíz de Almirón, 1998). Non-hydrated samples were incubated in situ from sunrise to sunset in triplicate inside transparent and opaque sterile vials to reproduce the light and dark conditions, respectively. Then 10% of the gaseous phase of these vials was replaced with acetylene by placing syringes through the rubber vial stoppers. Gaseous samples were collected on an hourly basis over a 24-hour period in winter between sunrise and sunset. The following values were recorded hourly throughout the incubation period: photon flux density (PAR - photosynthetically active radiation), air temperature and relative humidity. A LI-1400 datalogger model (LICOR) with a LI-192 sensor and a Delta Ohm HD 8501 H thermohygrometre were used. Electrodes were placed on the rock surface. The concentrations of CO2 and O2 in the cave air were measured with a hand pump (Gastec Corporation, Japan) and detector tubes (measurement range: CO2, 300-5,000 ppm; O2, 3-24%). Nitrogen fixation was quantified by acetylene-ethylene reduction and was subsequently analyzed in a Shimadzu GC 14 A gas chromatograph. RESULTS Nitrogen fixation by Scytonema julianum (Fig. 5), a species growing in the Vapor cave forming a greyish covering, was measured for the first time. During the daytime, nitrogenase activity ranged between 129.9 and 215.7 nmol of C2 H4 m-2 s-1 (Fig. 6). It reached its peak value after a series of high values followed by low values. Thus, the graphic representation of nitrogenase activity is a zigzag pattern. The highest values were recorded at 11:00h, 13:00h and 15:00h at 201.2, 215.7 and 213.0 nmol of C2 H4 m-2 s-1, respectively, while the lowest values were observed at 08:00h, 10:00h, 12:00h, 14:00h and 17:00h at 125.1, 129.9, 157.8 and 130.4 nmol of C2 H4 m-2 s-1, respectively. Fig. 5. Light micrographs [scale bar: 10 µm] of Scytonema julianum. The PAR values in the Vapor cave ranged between 5.2 and 57.7 μE.m-2.s-1 (Fig. 7). From 08:00h to 10:00h, 27.5, 28.1, 29.1 and 27.4ºC noted at 08:00h, 10:00h, these values increased from 7.5 to 18.9 μE.m-2.s-1. 12:00h, 14:00h and 18:00h, respectively. Then at this time, a substantial increase took place at Nighttime nitrogen fixation varied from 65.1 to 11:00h reaching 48.6 μE.m-2.s-1. Finally, these values 120.6 nmol of C2 H4 m-2 s-1 (Fig. 6) and nitrogenase continued rising until a peak value of 57.7 μE.m-2.s-1 activity was graphically depicted as a zigzagging line. was noted at 13:00h, to drop later. At 08:00h, a value of 79.2 nmol of C2 H4 m-2 s-1 was The minimum value of relative humidity (Fig. 7) noted, which rose to 120.6 nmol of C2 H4 m-2 s-1 at was 72% recorded at 08:00h. Relative humidity 10:00h. After this time, it dropped to 78.3 nmol of C2 progressively increased to a maximum value of 100% H4 m-2 s-1 at 11:00h. Nitrogenase activity started rising at 11:00h, and remained so until 17:00h when it again to reach 94.4 nmol of C2 H4 m-2 s-1 at 12:00h and dropped slightly to 99.2% at 18:00h. then dropped to 75.8 C2 H4 m-2 s-1 at 13:00h. Finally, Temperatures in the Vapor cave ranged between at 14:00h it rose to 97.3 C2 H4 m-2 s-1 at 15:00h, from 27.2ºC, recorded at 08:00h, to 31.0ºC noted at 13:00h which time nitrogenase activity descended to reach a (Fig. 7). The highest temperatures were 28.9, 31.0 minimum value of 65.1 to 120.6 nmol of C2 H4 m-2 s-1 and 29.5ºC recorded at 11:00h, 13:00h and 15:00h, at 17:00h. respectively, while the lowest temperatures were 27.2, International Journal of Speleology, 40 (1), 17-21. Tampa, FL (USA). January 2011
  • 4. 20 Antonia Dolores Asencio and Marina Aboal 250 the U.S.A. (Jeffries et al., 1992), we notice a vast difference, despite previously rehydrating samples, which confirms the importance of a high, constant 200 Nitrogenase activity (nmol C 2H4 m -2s-1) relative humidity in the nitrogen fixation process, which also occurs in the Vapor cave. 150 We also noted that the greater the light intensity at the Vapor cave study site, the higher the nitrogenase 100 activity. This coincides with the results of Dodds (1989), even to the point that the times the highest nitrogenase activities were recorded coincide with the 50 light maximum PAR, temperature and relative humidity dark values. 0 Ethylene nighttime production by Scytonema 8 10 12 14 16 Time (h) julianum was between 65.1 and 120.6 nmol of C2 H4 m-2 s-1. These values were lower than those Fig. 6. Nitrogenase activity by Scytonema julianum (light and dark) in Vapor obtained in the daytime, which occurred with all cave. the heterocysted cyanobacteria. Although these prokaryotes are considered totally photoautotroph organisms, some may grow in the darkness at a slower VAPOR cave rate (Stewart, 1973; Jones, 1992). 70 Temperature inside the Vapor cave changed in 60 accordance with the daytime or nighttime nitrogenase PAR (µEm-2s-1) 50 activity. When temperatures were lower, nitrogenase 40 30 activity dropped, which is clearly demonstrated in the 20 daytime when ethylene production dropped sharply as 10 temperatures lowered, and again increased at higher 0 temperatures. The exact opposite took place with nighttime nitrogen fixation as nitrogenase activity 120 dropped at higher temperatures, and increased with lower temperatures. Relative humidity (%) 100 80 Nitrogenase activity by Scytonema julianum was 60 roughly 30 times higher than that of Scytonema mirabile, which also grew in a similar environment 40 (Asencio & Aboal, 2010), due to the characteristics of 20 each site. The Andragulla cave was 2.0 m deep, 2.0 0 m high and 6.0 m wide. Its lack of depth meant that the microclimate was very similar to that experienced 32 externally, which explained its extreme PAR (0.5- 31 582.7 μE.m-2.s-1), temperature (1.5-20.3 ºC) and Temperature (ºC) 30 relative humidity (24.0-79.9 %) winter values. The 29 Vapor cave, however, was very deep so it was isolated 28 from external influences. Therefore, its PAR values 27 remained constant and it had very high temperature 26 25 and relative humidity values. 8 10 12 14 16 18 The first ever daytime and nighttime nitrogen Time (h) fixation values by Scytonema julianum recorded in the Vapor cave differed considerably, but coincided with Fig. 7. PAR (Photosynthetically active radiation), temperature and relative Calothrix in tropical environments (Jones, 1992). This humidity from sunrise to sunset in Vapor cave from a cyanobacterial mat of was likely due to the energy reserves stored during Scytonema julianum. photosynthesis being exhausted and used in the dark DISCUSSION phase, which occurred with Nostoc in Californian Nitrogenase activity by Scytonema julianum recorded streams (Horne & Carmiggelt, 1974). This fact in the Vapor cave over a 24-h period ranged from suggests that Scytonema julianum, like other tropical 129.9 to 215.7 nmol of C2 H4 m-2 s-1. These values are terrestrial cyanobacteria (Jones, 1981, Saino & very close to those obtained for the Calothrix genus: Hattori, 1978), preferred high-levels of sunlight which 284 nmol of C2 H4 m-2 s-1 in tropical environments could substantially contribute to the overall nitrogen (Jones, 1992), where similar relative humidity and cycle in N-poor environments, such as cave entrances temperature conditions to those in the Vapor cave and other lighted areas. were registered. If we compare the aforementioned values with ACKNOWLEDGEMENTS the mean 28.2 nmol value of C2 H4 m-2 s-1 recorded We sincerely thank J.J. Ruíz de Almirón for his help in Scytonema sp. crusts from semi-arid areas of in the field, H. Warburton for his assistance with the International Journal of Speleology, 40 (1), 17-21. Tampa, FL (USA). January 2011
  • 5. In situ acetylene reduction activity of Scytonema julianum in Vapor cave (Spain) 21 English version of the text and Dr. D.E. Northup and Hill C.A., 1987 – Geology of Carlsbad Cavern and other an anonymous reviewer for their comments on the caves in the Guadalupe Mountains, New Mexico and manuscript. Texas: Socorro, N.M. New Mexico Bureau of Mines and Mineral Resources Bulletin, 117: 150. REFERENCES Hoffmann L., 1989 – Algae of terrestrial habitats. Aboal M., Asencio A.D. & Prefasi M., 1994 – Studies on Botanical Review, 55: 77-105. cave cyanophytes from southeastern Spain: Scytonema Hoffmann L., 1992 – Variability in the crystal morphology julianum Richter. Archiv für Hydrobiology/ Algological of calcified terrestrial Scytonema populations Studies, 75: 31-36. (Cyanobacteria, Cyanophyceae). Geomicrobiology Antonopoulou S., Oikonomou A., Karantonis H.C., Journal, 10: 59-64. Fragopoulou E. & Pantazidou A., 2002 – Isolation and Horne A.J. & Carmiggelt C.J.W., 1974 – Algal nitrogen structural elucidation of biologically active phospholipids fixation in Californian streams: seasonal cycles. from Scytonema julianum (Cyanobacteria). Biochemical Freshwater Biology, 5, 461-470. Journal, 367: 287-293. Houssman D.C., Powers H.H., Collins A.D. & Belnap Antonopoulou S., Karantonis H.C., Nomikos T., J., 2006 – Carbon and nitrogen fixation differ between Oikonomou A., Fragopoulou E. & Pantazidou A., successional stages of biological soil crusts in the 2005 – Bioactive polar lipids from Chroococcidiopsis Colorado Plateau and Chihuahuan Desert. Journal of sp. (Cyanobacteria). Comparative Biochemistry and Arid Environments, 66: 620-634. Physiology-B Biochemistry and Molecular Biology, Jeffries D.L., Klopatek J.M., Link S.O. & Bolton H.Jr., 142: 269-282. 1992 – Acetylene reduction by cryptogamic crusts from Asencio A.D. & Aboal M., 2010 – In situ nitrogen fixation a blackbrush community as related to resaturation and by cyanobacteria at the Andragulla cave (Spain). dehydration. Soil Biology and Biochemistry, 24: 1101- Journal of Cave and Karst Studies, 72(3): in press. 1105. Bellezza S., Albertano P., De Philippis R. & Paradossi Jones K., 1981 – Diurnal acetylene reduction by mats of G., 2006 – Exopolysaccharides of two cyanobacterial blue-green algae in sub-tropical grassland: use of short- strains from Roman hypogea. Geomicrobiology Journal, term and long-term assays. New Phytology, 88: 73-78. 23: 301-310. Jones K., 1992 – Diurnal nitrogen fixation in tropical Bothe H., 1982 – Nitrogen fixation. In: Carr, N.G. & marine cyanobacteria: a comparison between adjacent Whiton, B.A. (Eds), The Biology of Cyanobacteria. communities of non-heterocystous Lyngbya sp. and University of California Press, Berkeley, 87-104. heterocystous Calothrix sp. British Phycological Couté A. & Bury E., 1988 – Ultrastructure d’une Journal, 27:107-118. cyanophycée aérienne calcifiée cavernicole: Scytonema Ruíz de Almirón J.J., 1998 – La sima del Vapor. julianum (Frank) Richter (Hormogonophycideae, Subterránea, 9: 36-43. Nostocales, Scytonemataceae). Hydrobiologia, 160: Saino T. & Hattori A., 1978 – Dial variation in nitrogen 219-239. fixation by a marine blue-green alga, Trichodesmium Dodds W.K., 1989 – Microscale vertical profiles of N2 thiebautii. Deep Sea Research, 25: 1259-1263. fixation, photosynthesis, O2, chlorophyll a and light in a Stewart W.D.P., 1973 – Nitrogen fixation. In Carr, N.G. cyanobacterial assemblage. Applied and Environmental & Whiton, B.A. (Eds), The Biology of Blue-Green Algae. Microbiology, 55: 882-886. Blackwell Scientific Publications, Oxford: 260-278. Friedman E.I., 1973 – Scanning electron microscopy of Zielke M., Solheim B., Spjelkavik S. & Olsen R. A., 2005 three lime encrusting aerophytic blue-green algae. – Nitrogen fixation in the high Arctic: role of vegetation Journal of Phycology, 9: 17. and environmental conditions. Arctic, Antarctic and Griffiths M.S.H., Gallon J.R. & Chaplin A. E., 1987 – The Alpine Research, 37: 372-378. diurnal pattern of dinitrogen fixation by cyanobacteria in situ. New Phytologist, 107: p. 649-657. International Journal of Speleology, 40 (1), 17-21. Tampa, FL (USA). January 2011