BGBiogeosciencesBGBiogeosciences1726-4189Copernicus PublicationsGöttingen, Germany10.5194/bg-15-1415-2018Revisiting chlorophyll extraction methods in biological soil crusts –
methodology for determination of chlorophyll a and chlorophyll a+b as
compared to previous methodsRevisiting chlorophyll extraction methods in biological soil crustsCaesarJennifercaesar@hs-nb.deTammAlexandraRuckteschlerNinaLeifkeAnna LenaWeberBettinab.weber@mpic.dehttps://orcid.org/0000-0002-5453-3967Multiphase Chemistry Department, Max Planck Institute for Chemistry,
Hahn-Meitner-Weg 1, 55128 Mainz, GermanyPresent address: Department of Agriculture & Food Sciences,
University of Applied Sciences, Brodaer Str. 2, 17033 Neubrandenburg,
GermanyThese authors contributed equally to this work.Jennifer Caesar (caesar@hs-nb.de) and Bettina Weber (b.weber@mpic.de)8March20181551415142419September201725September201713January201824January2018This work is licensed under the Creative Commons Attribution 4.0 International License. To view a copy of this licence, visit https://creativecommons.org/licenses/by/4.0/This article is available from https://bg.copernicus.org/articles/15/1415/2018/bg-15-1415-2018.htmlThe full text article is available as a PDF file from https://bg.copernicus.org/articles/15/1415/2018/bg-15-1415-2018.pdf
Chlorophyll concentrations of biological soil crust (biocrust) samples are
commonly determined to quantify the relevance of photosynthetically active
organisms within these surface soil communities. Whereas chlorophyll
extraction methods for freshwater algae and leaf tissues of vascular plants
are well established, there is still some uncertainty regarding the optimal
extraction method for biocrusts, where organism composition is highly
variable and samples comprise major amounts of soil. In this study we
analyzed the efficiency of two different chlorophyll extraction solvents, the
effect of grinding the soil samples prior to the extraction procedure, and
the impact of shaking as an intermediate step during extraction. The analyses
were conducted on four different types of biocrusts. Our results show that
for all biocrust types chlorophyll contents obtained with ethanol were
significantly lower than those obtained using dimethyl sulfoxide (DMSO) as a
solvent. Grinding of biocrust samples prior to analysis caused a highly
significant decrease in chlorophyll content for green algal lichen- and
cyanolichen-dominated biocrusts, and a tendency towards lower values for
moss- and algae-dominated biocrusts. Shaking of the samples after each
extraction step had a significant positive effect on the chlorophyll content
of green algal lichen- and cyanolichen-dominated biocrusts. Based on our
results we confirm a DMSO-based chlorophyll extraction method without
grinding pretreatment and suggest the addition of an intermediate shaking
step for complete chlorophyll extraction (see Supplement S6 for detailed
manual). Determination of a universal chlorophyll extraction method for
biocrusts is essential for the inter-comparability of publications conducted
across all continents.
Introduction
Chlorophyll (Chl) is a pigment commonly occurring in photosynthesizing
organisms. It facilitates organisms to utilize sunlight as an energy source
to build glucose and carbohydrates from CO2 and water. The composition
of photosynthetic pigments varies between organisms: cyanobacteria
possess only Chl a and the accessory antenna pigments, phycocyanin and
phycoerythrin; green algae and vascular plants comprise Chl a and
Chl b; brown algae contain Chl c instead of Chl b and Rhodophyta
contain only Chl a whilst Chl b is replaced by Chl d (Mohr and
Schopfer, 1995).
Biological soil crusts (biocrusts) are surface soil communities commonly
occurring in arid and semiarid regions throughout the world, as well as in
areas where the lack of water or other environmental conditions (e.g.,
disturbance) restrict the development of vascular plants (Garcia-Pichel et
al., 2003; Zaady et al., 2016). They grow within the uppermost millimeters of
the soil in close association with soil particles. It has been shown that
biocrusts play significant functional roles in desert ecosystems (Eldridge
and Greene, 1994; Evans and Belnap, 1999; Lan et al., 2011), as they
stabilize the soil surface and reduce erosion by wind and water (Zhao et al.,
2014; Belnap et al., 2014; Belnap and Büdel, 2016), they contribute to
soil fertility through carbon and nitrogen fixation (Elbert et al., 2012;
Sancho et al., 2016; Barger et al., 2016; Brankatschk et al., 2013), and they
positively affect water retention and distribution in drylands
(Rodriguez-Caballero et al., 2014; Chamizo et al., 2016). Biocrusts and their
organisms have also been shown to release gaseous nitrogen compounds, as
nitrous acid (Lenhart et al., 2015), nitric oxide, and nitrous oxide into the
atmosphere (Weber et al., 2015; Meusel et al., 2018). Biocrusts are composed
of photosynthesizing cyanobacteria, algae, lichens, and bryophytes plus
decomposers, i.e., fungi, bacteria and archaea (Maier et al., 2016), and
heterotrophic consumers, like protozoa, collembolans, and snails (Darby and
Neher, 2016; Bamforth, 2008). Thus, they form one of the smallest ecosystems
with photosynthetic carbon fixation being the main source of carbohydrates.
The chlorophyll content of these communities is therefore a good indicator
of the photosynthetic capacity and thus the capability of these systems to
acquire energy, jointly used by the community but also exchanged with the
surrounding environment. The capability to acquire energy under favorable
environmental conditions is in turn a relevant proxy indicating successional
stage, system stability, and its ability to recover from disturbance (Dojani
et al., 2011; Gomez et al., 2012; Weber et al., 2016). As cyanobacteria and
cyanolichens (photobiont: cyanobacteria) only comprise Chl a, high
Chl a/ Chl b ratios may indicate their dominance. Unfortunately, the
Chl a/ Chl b ratio in algae, chlorolichens (photobiont: green
algae), and bryophytes (i.e., liverworts and mosses) is variable, thus not
facilitating the proportional quantification of both groups; nevertheless, it
allows tentative estimates on the relevance of eukaryotic Chl b-comprising
partners (Thorne et al., 1977).
Suitable chlorophyll extraction procedures are essential to obtain reliable
results, and standardized methods are needed to allow comparison between
studies (Schagerl and Künzl, 2007). During the last decades, several
different methods have been described. The solvents most commonly used for
chlorophyll extraction have been ethanol, acetone, N, N-Dimethylformamide
(DMF), and dimethyl sulfoxide (DMSO) (Mackinney, 1941; Shoaf and Lium, 1976;
Moran and Porath, 1980; Barnes et al., 1992; Inskeep and Bloom, 1985). In a
recent publication, Castle et al. (2011) compared the efficiency of four
different solvents: acetone, ethanol, DMSO, and methanol for biological soil
crusts of three different successional stages. They found that ethanol and
DMSO extracted the greatest amount of Chl a using a double extraction
technique. At a similar time, Lan et al. (2011) also compared the Chl a
extraction efficiency using ethanol, acetone, DMF, and DMSO as solvents to
analyze algal-, lichen-, and moss-dominated biocrusts. They concluded ethanol
extractions expressed on an area basis to be most efficient and found DMSO
extractions of lichens to be unreliable. Apart from the extraction solvent,
preparatory steps and handling during extraction also varied between methods.
Both Castle et al. (2011) and Lan et al. (2011) ground the samples with a
mortar and pestle to facilitate the following extraction steps, and Castle et
al. (2011) also applied a two-step extraction and placed the samples on a
shaker after each extraction cycle.
In this study we investigate these methodological techniques, which have been
recently suggested for the extraction of Chl a from biocrusts. Our overall
analytical technique is based on the photometric method established by Ronen
and Galun (Ronen and Galun, 1984; Hiscox and Israelstam, 1978) and on
methodological adaptions made in the lab of O. L. Lange, personal
communication, 2000. We compare the efficiency of two extraction methods,
using the two solvents DMSO and ethanol, which have been rated as most
effective during the most recent studies by Lan et al. (2011) and Castle et
al. (2011), for green algae-dominated, green algal lichen-dominated,
moss-dominated, and cyanolichen-dominated biocrusts.
In this research we address the following three questions:
Which is the most potent chlorophyll extraction method for biocrusts? The
ethanol method by Castle et al. (2011) or the DMSO method by Ronen and Galun (1984)?
Is a disruption of the cells (grinding) necessary prior to the extraction
procedure and does it influence the chlorophyll yield?
Does shaking (20 min) after each extraction step influence the
chlorophyll yield?
The overall goal of this study is to determine an extraction method best
suited for chlorophyll determination of the analyzed samples, which we
consider as an important step towards a universal chlorophyll determination
method for different types of biocrusts.
Material and methodsSampling site
The biocrust samples for this study were collected in: the Mehlinger Heide nature reserve, located about 15 km north of Kaiserslautern, in Rhineland-Palatinate; and the Ruine Homburg nature reserve at
Gössenheim, in northern Bavaria.
The Mehlinger Heide is about 410 ha in size, being one of the biggest
heathlands in southern Germany. Until 1912, the area was completely covered
by forest. In the First and Second World War, the region was partly
deforested to build a military training ground (http://mehlinger-heide.de/).
After the Second World War, the area was used for military training by
French and American troops until 1992 and 1994, respectively. The
prolonged continuous disturbance by military use caused the formation of a
distinct flora and fauna typical of nutrient-depleted sandy soils, which are rarely found in central Europe. The vegetation is characterized by dwarf shrubs (heather), lichens (fruticose, foliose and crustose) and
bryophytes (mosses and liverworts). The Mehlinger Heide has been
under conservation since 2001.
The Ruine Homburg nature reserve is an
open anthropogenic landscape with bare rock and gravel spots covered by a
thin vegetation layer dominated by cryptogams including lichens, bryophytes,
and cyanobacteria. Its bedrock is Triassic shell limestone and its flora is
composed of a relic flora from after the last ice age with
sub-Mediterranean–continental and sub-Mediterranean–sub-Atlantic elements
(Lösch, 1980). The landscape has remained open owing to the presence of a
castle which was built nearby in 1080.
Sample collection
Four different biocrust types were collected:
Green algae-dominated biocrust with Klebsormidium sp. as the dominating organism.
Green algal lichen-dominated biocrust with Cladonia sp. as the dominating genus.
Moss-dominated biocrust with Hypnum sp. as the dominating organism.
Cyanolichen-dominated biocrust with Peltigera rufescens as the dominating lichen.
All four biocrusts types were collected in 2014 (January and May) and 2016
(June and September) in the Mehlinger Heide. Some samples of the
cyanolichen-dominated biocrusts were collected in May 2014 in
Aschfeld/Gössenheim. Within each experiment, only samples of the same
sampling batch were used. During sampling, special care was taken to avoid
variability between replicate samples within each type. For sampling, a
metal ring of 14 mm diameter (surface size: 153.9 mm2) and
3.5 cm in height was used. The metal ring was pressed 3 cm deep into the
soil, a trowel was pushed underneath, both were pulled out again, and the
biocrust sample within the ring was transferred into a plastic zip lock bag.
For each biocrust type five replicates were collected (total number of samples:
n=20). The samples were transported back to the institute, where they
were air dried and stored at room temperature at low light intensities for
less than four weeks until the chlorophyll extraction experiments were
conducted.
Chlorophyll extractionPretreatment of the biocrust samples
Prior to chlorophyll extraction, all biocrust samples were dried in a drying
oven at 60 ∘C for at least 24 h until constant weight was
reached and the dry weight was determined. On the day before the chlorophyll
extraction the samples were slightly sprinkled with distilled water to
activate the biocrust organisms, which is known to facilitate the subsequent
chlorophyll extraction. The photosynthetic apparatus undergoes disassembly
during desiccation, so rehydration is required to repair the photosynthetic
apparatus (Harel et al., 2004). During the entire chlorophyll extraction
procedure, the samples were kept in the dark or at minimum light to prevent the
degradation of chlorophyll (Molnár et al., 2013; Hosikian et al.,
2010).
Chlorophyll extraction with dimethyl sulfoxide (DMSO)
Chlorophyll extraction with DMSO as solvent was conducted based on the method
described by Ronen and Galun (Hiscox and Israelstam, 1978; Ronen and Galun,
1984) and on methodological adaptions made in the lab of O. L. Lange,
personal communication, 2000.
The soil crust samples of the four types (n=5) were placed in 15 mL
screw-cap vials without prior grinding. A spatula tip of MgCO3 or
CaCO3 was added to avoid acidification and the associated chlorophyll
degradation (Weber et al., 2013; Rapsch and Ascaso, 1985). Following this, 6 mL of DMSO
(ROTIDRY®≥ 99.5 %, ≤ 200 ppm H2O, Carl Roth GmbH + Co. KG, Karlsruhe, Germany)
were
added to the samples. A vial with only DMSO and
MgCO3/ CaCO3 (no sample added) was used as a blank. All vials were placed on a
water bath at 65 ∘C for 90 min. The caps of the vials were half
tightened to allow extension of the liquid during heating but avoid
evaporation loss. After the first extraction cycle the supernatant was poured
into a separate vial, and another 6 mL of DMSO were added to the samples for
a second extraction cycle. After a second extraction in the water bath for
90 min, the supernatants of both extractions were pooled and centrifuged
(Megafuge 16R, Thermo Fisher Scientific, Dreieich, Germany) for 10 min at
3000 × g and 15 ∘C before photometric determination.
The absorption was measured with a spectrophotometer (Lambda 25 UV/VIS,
PerkinElmer, Rodgau, Germany) at 648, 665, and 700 nm. If absorption values
at 665 nm were above 0.8, the sample was diluted 1:1 with DMSO and the
equation was adjusted accordingly, changing the dilution factor from 1 to 2.
The Chl a+b concentrations were calculated according to O. L. Lange,
personal communication, 2000.
Total Chl a+b amount in sample:
Chla+bµg=A665-A700×8.02+A648-A700×20.2×DF×S
Chl a+b amount based on surface area:
Chla+bmg×m-2=Chla+bµg/AR×1000
Chl a+b amount based on dry weight:
Chla+bµg×g-1=Chla+bµg/DW,
where
Ax=absorbanceatcertainwavelength,DF=dilutionfactor,S=amountofsolvent[mL],AR=aream2,theareainthiscaseisalways153.9mm2,astheradiusofthesamplingringis7mmAR=π×r2,DW=dryweight[g].
The Chl a concentration of biocrusts dominated by cyanobacterial lichens
was calculated according to Arnon et al. (1949).
Total Chla amount in sample:
Chlaµg=A665-A700×12.19×DF×S
Chl a amount based on surface area:
Chlamg×m-2=Chlaµg/AR×1000
Chl a amount based on dry weight:
Chlaµg×g-1=Chlaµg/DW
Reproducibility of chlorophyll extraction with dimethyl sulfoxide
(DMSO)
In order to analyze the reproducibility of chlorophyll extraction with DMSO,
∼ 90 g of green algae-dominated biocrust were oven-dried (see
Sect. 2.3.1), homogenized with a mortar and pestle and evenly distributed
into eight screw-cap vials. The subsequent chlorophyll extraction followed
the described DMSO extraction procedure (see Sect. 2.3.2). The analyzed
chlorophyll content refers to the initial sample weight.
Chlorophyll extraction with ethanol
The Chl a double extraction with ethanol was carried out according to the
protocol of Castle et al. (2011). The soil crust samples (four types, n=5) were ground with mortar and pestle until they were homogenous and placed
in screw-cap vials, a spatula tip of MgCO3 and 6 mL of ethanol (≥ 99.8 %, Sigma-Aldrich, Steinheim, Germany) were added and the caps
were half-tightened. A vial with ethanol and a spatula tip of MgCO3 was
used as blank. All samples were heated in a water bath at 80 ∘C
until they started to boil. Once they had started to boil they were kept in
the water bath for 5 min. After boiling, the vials were cooled down for
10 min and then placed on a horizontal shaker for 20 min. Subsequently, the
samples were centrifuged at 3000× g and 15 ∘C for 10 min.
The supernatant was poured into separate vials and another 6 mL of ethanol
were
added to the samples for a second extraction cycle conducted in the same way
as the first.
After the second cycle, the supernatants of both extractions were combined
and the absorbance was measured spectrophotometrically at 665, 649, and
750 nm. The absorption at 750 nm was measured in addition to eliminate a
potential effect of the intrinsic color of the samples. The Chl a+b content
was determined according to the formula of Ritchie (2006).
Total Chl amount in sample:
Chlaµg=13.5275×A665-A750-5.201×A649-A750×DF×SChlbµg=22.4327×A649-A750-7.0741×A665-A750×DF×S
The total Chl a+b content is the sum of the results obtained from
formula (7) and (8).
Analogous to the DMSO method, the total chlorophyll content can be calculated
per area by dividing the total amount of Chl a+b by the area (AR) (see also
Sect. 2.3.2).
Chl amount based on surface area:
Chla+bmg×m-2=Chla+b/AR×1000
The Chl a concentration of biocrusts dominated by cyanobacterial lichens
was calculated according to the formula by Ritchie (2006).
Total Chl a amount in sample:
Chlaµg=11.9035×A665-A750×DF×S
Chl a amount based on surface area:
Chlamg×m-2=Chlaµg/AR×1000
Effect of prior grinding and additional shaking on chlorophyll
extraction efficiency
Additional experiments were performed to evaluate the effect of two
methodological steps, i.e., grinding prior to extraction and shaking after
each extraction cycle of the samples, as described in the protocol by Castle
et al. (2011). These experiments were only carried out with the DMSO method
described above. To analyze the effect of grinding, the dry samples were
ground to homogeneity with a mortar and pestle prior to the extraction
procedure. Based on a reviewer suggestion, a second set of samples was ground
in a wet state with some of the solvent (DMSO) added prior to grinding. In a
separate approach, the effect of shaking the samples after extraction was
analyzed. To achieve this, the samples were placed on a horizontal shaker for
20 min after each extraction cycle. Both treatments were applied to the four
biocrust types (n=5, except in the shaker experiment: green algal- and green algal
lichen-dominated biocrust: n=4).
Statistical evaluation
All data were analyzed with the statistical and analytical software
OriginPro (Version 8.6; OriginLab Corporation, Northampton, MA, USA). Before
statistical analyses all data were tested for normality and variance
homogeneity. To determine statistical differences between normally
distributed samples, where homogeneity of variance was given, unpaired t tests
were performed. For samples, where normal distribution could not be reached,
a Mann–Whitney U test was performed. Normally distributed samples without
homogeneity of variance were analyzed using the Welch test.
For some samples the relative standard deviation (RSD) was calculated to
reveal the extent of variability in relation to the average value of the
population, using the following formula:
RSD=σ/μ×100%,
where is σ= standard deviation and μ= average value.
The RSD indicates the precision of the data and shows if the data is tightly
clustered around the mean.
Chlorophyll a+b/chlorophyll a content (mg m-2) of green algae-
(a), cyanolichen- (b), green algal lichen- (c), and
moss-dominated biocrusts (d) using the DMSO and ethanol extraction
method. To prove statistical differences between both extraction methods, the
Mann–Whitney U test was performed for moss-dominated biocrusts (data not
normally distributed), for the other three biocrust types a Welch test was
performed (data without homogeneity of variance) (*p≤0.05;
**p≤0.01; ***p≤0.001).
ResultsComparison of chlorophyll extraction methods: DMSO versus
ethanol
The two different chlorophyll extraction methods for DMSO and ethanol showed
significant differences in chlorophyll yield for all four biocrust types
(Fig. 1). Using the DMSO method, significantly higher chlorophyll contents
were obtained compared to the ethanol method. Whereas Chl a+b/ Chl a contents
determined with the DMSO extraction method ranged between mean values of
∼ 550 and 900 mg m-2 (i.e., 598 mg m-2 for green
algae-dominated, 547 mg m-2 for cyanolichen-dominated, 772 mg m-2
for green algal lichen-dominated, and 903 mg m-2 for moss-dominated
biocrusts), values obtained by the ethanol extraction method were between
∼ 110 and 370 mg m-2 (i.e., 182 mg m-2 for green
algae-dominated, 111 mg m-2 for cyanolichen-dominated, 125 mg m-2
for green algal lichen-dominated, and 367 mg mg-2 for moss-dominated
biocrusts; Supplement Table S1). Thus, the mean values obtained by the
ethanol extraction procedure were ∼ 70, 80, 84, and 59 %
lower for green algae-, cyanolichen-, green algal lichen-, and moss-dominated
biocrusts than those obtained by the DMSO method. However, for all biocrust
types, the results showed higher standard deviations for the DMSO as compared
to the ethanol extraction method, and all types had higher relative standard
deviations (Table S2).
Chlorophyll a+b/chlorophyll a content (mg m-2) depending on preparatory
grinding in a dry state (GDRY) as compared to control extractions
without grinding (NG). Investigation of green algae- (a),
cyanolichen-
(b), green algal lichen- (c), and moss-dominated biocrusts
(d) using the DMSO extraction method. To prove statistical
differences, an unpaired t test was performed for all four biocrust types
(*p≤0.05; **p≤0.01; ***p≤0.001).
To evaluate reproducibility of the chlorophyll extraction procedure by means
of the DMSO method, eight replicate samples of homogeneous biocrust material
were analyzed. Here, a RSD of 12.1 % was obtained, certifying a good
reproducibility of the DMSO extraction method (Table S3).
Chlorophyll a+b/chlorophyll a content (mg m-2) depending on
preparatory grinding in a wet state with DMSO added (GDMSO) as
compared to control extractions without grinding (NG). Investigation of green
algae- (a), cyanolichen- (b), green algal lichen- (c),
and moss-dominated biocrusts (d) using the DMSO extraction method.
To prove statistical differences, an unpaired t test was performed for all
four biocrust types (*p≤0.05; **p≤0.01; ***p≤0.001).
Pretreatment of the samples: grinding versus non-grinding
As the ethanol extraction method contains an additional grinding step during
sample pretreatment, the impact of this step was tested for the DMSO
extraction method, which had gained higher chlorophyll extraction yields
(Fig. 1). The results reveal that preparatory grinding caused a decrease in
Chl a+b/ Chl a contents, resulting in significantly lower values for cyanolichen- and
green algal lichen- and a similar tendency in green algae- and moss-dominated
biocrusts (Fig. 2). In fact, this preparatory step caused mean Chl a+b/ Chl a
yields to be ∼ 36 and 51 % lower in cyanolichen-, and green
algal lichen-dominated biocrusts (Table S4).
In an additional experiment the samples were ground in a wet state with
solvent (DMSO) added prior to grinding (Fig. 3). Also under these conditions
grinding had a negative effect and caused Chl a+b/ Chl a contents of ground
samples to be significantly lower for cyanolichen-, green algal lichen-, and
moss-dominated biocrusts. Solely for green algae-dominated biocrusts no
significant reduction was observed.
Intermediate step during chlorophyll extraction: shaking versus
non-shaking
The effect of shaking after each extraction cycle was evaluated for all four
crust types. As shown in Fig. 4, shaking caused increased chlorophyll yields
in green algal lichen- and cyanolichen-dominated biocrusts and a trend in the
same direction was observed for green algae-dominated crusts.
Chl a+b/ Chl a
values were ∼ 73 and 42 % higher in cyanolichen- and green
algal lichen-dominated biocrusts, respectively (Table S5).
Chlorophyll a+b/chlorophyll a content (mg m-2) depending on intermediate
shaking (S) after each extraction cycle as compared to control extractions
without shaking (NS). Investigation of green algae- (a),
cyanolichen-
(b), green algal lichen- (c), and moss-dominated biocrusts
(d) using the DMSO extraction method. To prove statistical
differences between shaking (S) and non-shaking (NS) a Welch test was performed for
cyanolichen-dominated biocrusts (data without homogeneity of variance). For all
other biocrust types an unpaired t test was carried out. (*p≤0.05; **p≤0.01;
***p≤0.001).
Discussion
Whilst biocrusts represent an important component of the landscape in arid
and semi-arid environments and are a natural and most effective force in
land stabilization and recovery (Campbell, 1979; Belnap et al., 2003;
Weber et al., 2016), no universal method has yet been established to
determine the chlorophyll content of the photosynthesizers within these
microbial communities. In this study we evaluated the usefulness of
different preparatory steps and methods proposed in recent publications to
determine the most suitable chlorophyll extraction technique for biocrust
samples. A perfect extraction procedure should deliver rapid and
reproducible results and must be simple to execute. Furthermore, the
solvent utilized should bring all pigments into solution, resolve pigments to
extremely low levels of detection, be hazard-free, and cause no chemical
changes to the pigments (Jeffrey, 1981).
Our investigations illustrate that DMSO extracted Chl a and Chl a+b
pigments to a significantly larger extent than ethanol. Significant differences
were observed for all types of biocrusts (Fig. 1). Grinding of samples prior to the extraction procedure, which was
analyzed for both dry samples (Fig. 2) and samples that had solvent added (Fig. 3), had a significant
negative effect on the extracted amounts of chlorophyll, whereas shaking of
samples after each extraction cycle caused significantly increased
chlorophyll contents for two of four biocrust types (Fig. 4).
Hiscox and Israelstam (1978) previously stated that DMSO is applicable for a
wide range of plant types with variable leaf tissues, Lan et al. (2011)
furthermore measured maximum extraction efficiency when using DMSO as the
solvent for biocrust analysis. When employing the DMSO method, the
duration of the chlorophyll extraction procedure is less critical; after
extraction the samples can even be stored in the fridge overnight and
spectrometrically analyzed the next day (Barnes et al., 1992). During
chlorophyll extraction with DMSO, CaCO3 is added to prevent
acidification and minimize the phaeophytinization of Chl a+b (Barnes et
al., 1992), which otherwise happens easily, as chlorophyll is sensitive to
extreme light exposure, pH, and temperature (Molnár et al., 2013;
Hosikian et al., 2010). In studies, where acidification has not been
prevented, unnaturally high absorption values were observed for lichen
samples around 665 nm (Lan et al., 2011), where pheophytin also absorbs
(Ritchie, 2008), therefore suggesting the phaeophytinization of Chl a by
lichen acids. Solvents containing methanol, ethanol or 1-propanol are also
known to easily degrade Chl a and Chl a+b, as isomerization and
allomerization of chlorophyll molecules occurs very easily under acidic
conditions (Hynninen, 1977), which need to be avoided by the addition of
magnesium carbonate (Ritchie, 2008). The extraction efficiency of DMSO was
higher, but also showed larger variability between replicates compared to the
ethanol extraction method (Table S1). As thorough chlorophyll extraction has
been reported for DMSO as solvent in previous studies (Barnes et al., 1992;
Castle et al., 2011) and a good reproducibility of the results has been shown
for biocrusts in this study (Table S3) and for leaf fragments by Tait and
Hik (2003), we believe that this variability reflects the actual variability
in chlorophyll contents between samples. In contrast to the extraction
efficiency, ethanol has the advantage of being non-toxic (Lan et al., 2011),
whereas DMSO has a potential to carry dissolved substances into the body
through the skin (Horita and Weber, 1964; Sulzberger et al., 1967).
Grinding of dry biocrust samples before extraction had a particularly
negative effect on green algal lichen- and cyanolichen-dominated biocrusts,
which seem to be particularly sensitive to cell damage, leading to
chlorophyll degradation. This again, may be caused by lichen acids; however,
this negative effect also persisted when grinding was conducted in a wet
state with DMSO added prior to grinding. In addition, also moss-dominated biocrusts
seemed to suffer from this treatment. The biocrust samples in
the current experiments were ground by hand with a mortar and pestle and no
high temperatures were reached. In other experiments, cooling with ice may be
necessary to avoid overheating, as chlorophyll is easily degraded by heat
(Braumann and Grimme, 1979). If the cells are disrupted within less than 20 s,
very rapidly and efficiently by a motor driven pestle, cooling with ice may
also not be necessary (Schagerl and Künzl, 2007). Loss of extraction
efficiency due to the increasing interference of humus, polysaccharides, and
clay also cannot be excluded (Lan et al., 2011). In contrast to our results,
in other laboratories grinding was observed to improve chlorophyll extraction
efficiency (J. Belnap, personal communication, 2017). This could be caused by
minor differences in extraction procedures or different sample properties,
which will be analyzed in future experiments.
Shaking of biocrust samples after each extraction cycle improved extraction
efficiency, as mean extraction quantities were significantly higher for
cyanolichen- and chlorolichen-dominated biocrusts, and showed the same
tendency for green algal-dominated biocrusts. Solely for moss-dominated
biocrusts shaking of samples had no effect. The effectiveness of shaking has
also been shown for other substances, such as in the extraction of
polyphenols from basil leaves and of toxic elements from artificial saliva
(Zlotek et al., 2016; Arain et al., 2013).
Error sources, which may cause an increased variability of the chlorophyll
contents, are differences in sample size and composition. Whilst variation
in sample size seems negligible, as defined sampling rings of fixed area and
height were used, variation in sample growth could not be completely
excluded, although during sampling special care was taken to minimize this
effect.
Conclusions
In conclusion, the determination of chlorophyll content using DMSO as a
solvent ensures a simple, rapid, and stable extraction. A cell-disrupting
pretreatment, like sample grinding or homogenization, is not required for a
complete chlorophyll extraction and can even cause chlorophyll loss.
Moreover, this turned out to be a time consuming step, especially when large
sample numbers were processed. In contrast, shaking between two extraction
cycles turned out to improve extraction efficiency. An advantage of
chlorophyll extraction with DMSO is that the samples are stable for 6–10 days
after incubation and can be stored at 4 to 8 ∘C without
degradation of the pigments (Ronen and Galun, 1984; Barnes et al., 1992),
while in other solvents significant amounts of chlorophyll are lost during
storage (Hiscox and Israelstam, 1978). Thus, based on our experiments, we
developed a DMSO-based chlorophyll extraction method optimized for green
algae-, lichen-, and moss-dominated biocrusts (Supplement S6).
Mean values and standard deviations of all data are listed
in the supplement. Raw data can be obtained from the corresponding authors
upon request.
The supplement related to this article is available online at: https://doi.org/10.5194/bg-15-1415-2018-supplement.
BW, JC, AT, and NR designed the experiments. AT, ALL, and NR collected
soil samples and carried out the laboratory work. JC and BW prepared the
manuscript with contributions from all co-authors.
The authors declare that they have no conflict of
interest.
This article is part of the special issue “Biological soil
crusts and their role in biogeochemical processes and cycling”. It is a
result of the BIOCRUST3 conference, Moab, USA, 26 to 30 September 2016.
Acknowledgements
This work was supported by the Max Planck Society. Bettina Weber would like
to thank Paul Crutzen for the award of a Nobel Laureate Fellowship
(2013–2015), and was financed by the German Research Foundation (DFG-FOR
1525: INUIT; WE2393/2). Alexandra Tamm and Nina Ruckteschler were supported
by the Max Planck Graduate Center (MPGC). We would like to thank Ulrich Pöschl for his overall support and the provision of lab space, as well as Jens Weber
and Heike Pfaff for their help during lab work. For field work at the study
sites, permissions were obtained from the Untere Naturschutzbehörde
Kaiserslautern and the Regierung Unterfranken (in the framework of the SCIN
project). The article processing charges for
this open-access publication were covered by the Max Planck
Society. Edited by: Kees Jan van Groenigen
Reviewed by: Jayne Belnap and Kees Jan van Groenigen
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