PLFA distribution
The PCA of PLFAs indicated that the oxic, suboxic and anoxic groundwaters had
distinct bacterial communities, with the anoxic groundwater additionally
differentiated into two distinct bacterial communities (Fig. 4). Of the
environmental variables tested, the variation partitioning showed that
NH4+, O2 and Fet concentration explained 22.0, 19.7
and 13.4 % of the PLFA variations, respectively (Fig. 6), and
differentiated between those three bacterial communities. Variation partitioning
analyses revealed, along those environmental variables, clusters of covarying
PLFAs that may originate from the same functional group of organisms or
closely affiliated organisms that react similarly to certain environmental
conditions. While the ladderanes are unequivocally attributed to anammox
bacteria (Sinninghe Damsté et al., 2002, 2005), the other PLFAs are not
exclusive to a phylogenetic or functional microbial group, which complicates
their use in understanding the role of microbes in environments. The t-value
biplots of variation partitioning analyses evidenced the PLFAs that
significantly correlated with the environmental variables O2 (Fig. 6a),
Fet (Fig. 6b) and NH4+ (Fig. 6c), and provided better
insights into the functional diversity of active microorganisms in the
subdivided groundwaters. Additional supports of the bacterial community
structure, assessed by the PLFA patterns, were found in the 16S rRNA-based
results. Although a large fraction of the microbial community remains poorly
classified and thus precludes the knowledge of the metabolic capacities, high
sequence similarity to genera known to be involved in iron oxidation or
reduction, sulfate reduction, anammox, and nitrite oxidation allowed an
estimation of the fraction of the microbial population potentially involved
in these processes. By combining the PLFA-based and sequencing-based
approaches, we aimed, here, to compensate for biases introduced by PCR as
well as for the limited phylogenetic resolution of PLFA-based analysis. This
combined approach resulted in highly supported evidence of some key
microbial players and associated biogeochemical processes in physico-chemical
distinct aquifer assemblages of the aquifer transect.
PLFA cluster in oxic to suboxic groundwater (wells 5.1, 4.1, 3.1
and 3.2)
A cluster of the covarying 20:4, 20:5, 22:5 and 22:6 PLFAs has to our
knowledge heretofore never been observed in groundwater. Associations of
those PLFAs have been commonly found in eukaryotes such as microalgae
(Volkman et al., 1989; Shinmen et al., 1989; Kennedy et al., 1993; Olsson,
1999; Qi et al., 2004) and protozoans (White, 1988), etc. Protozoa act as
detritivores and are expected to be key predators in the microbial loop
feeding on different subsets of the bacterial communities and other protozoa
(Brad et al., 2008; Akob and Küsel, 2011). Consistently, sessile and
free-swimming suspension-feeding flagellates (e.g. Spumella sp.),
mobile naked amoebae, and ciliates could be detected in this aquifer, with a
cultivable protist abundance of up to 8000 cells L-1 (Risse-Buhl et
al., 2013). The 18S rRNA gene sequences
also revealed high relative fractions of Spumella-like
Stramenopiles, and sequences affiliated with fungi and metazoan grazers.
DNA-based pyro-tag sequencing of fungal internal transcribed spacer sequences
revealed a fungi community structure dominated by Ascomycota and
Basidiomycota (Nawaz et al., 2016), with the majority of the observed fungal
groups being involved in ectomycorrhizal symbioses. In general, the abundance
of micro-eukaryotes in pristine groundwater is estimated to be low, because
they are limited in nutrients and space and are unable to cope with oxygen
limitations (Akob and Küsel, 2011). Consistently, they are commonly found
in higher concentrations in OM-rich contaminated groundwaters (Ludvigsen et
al., 1997). In pristine aquifers, the origin of those eukaryotic organisms is
difficult to determine as they may be autochthonous, allochthonous or both.
In the studied sites, the close relation of eukaryotic PLFA biomarkers with
O2 concentrations (Fig. 6a) suggests their association with recharging
groundwater within larger conduits prone to faster water flow. Freshly
introduced surface OC and O2 could fuel the heterotrophic bacterial
growth in groundwater. This may subsequently stimulate protists that
selectively graze on the prokaryotic biomass and result in the observed
relationship between the eukaryotic PLFAs and the O2 concentration. It
is possible to speculate that some surface microorganisms would also survive
the transport from surface to the aquifer (Dibbern et al., 2014), especially
if the transport is fast. In this case, high cy17:0-to-16:1ω7c ratios
(Table 3) may show physiological stress due to change of the environmental
conditions within the Gram-negative communities (Balkwill et al., 1998).
The 16:1ω11c and particularly the 11Me16:0 are major components of
Nitrospira moscoviensis (Lipski et al., 2001) cell
membrane, an obligatory
chemo-lithoautotrophic nitrite-oxidizing bacterium (Ehrich et al., 1995). In
the oxic groundwater, the occurrence of 16S rRNA gene sequence reads that are
closely related to Nitrospira moscoviensis (Herrmann et al., 2015)
further supports the potential of 11Me16:0 as a biomarker for
Nitrospira moscoviensis and confirms previous assumptions about an
important role of nitrite oxidizers within the autotrophic community of the
lower aquifers (Herrmann et al., 2015). The correlation of 11Me16:0 and
16:1ω11c with O2 (Fig. 6a) indicated the occurrence of active
nitrification in oxic zones of the aquifers, in agreement with observation of
experiments (Satoh et al., 2003). Nitrospira use the reverse
tricarboxylic acid cycle as the pathway for CO2 fixation (Lücker et
al., 2010), which leads to small 13C fractionation (2–6 ‰)
between biomass and CO2 (van der Meer et al., 1998). The
13C-enrichment of 11Me16:0 and 16:1ω11c relative to the other
PLFAs (up to 18 ‰ in well H4.1) thus supports a major
Nitrospira contribution to those PLFAs found in oxic groundwaters
(Fig. 7).
PLFA cluster in anoxic, more Fe<inline-formula><mml:math id="M502" display="inline"><mml:msub><mml:mi/><mml:mi mathvariant="normal">t</mml:mi></mml:msub></mml:math></inline-formula>-rich groundwater (wells
H4.2 and H4.3)
In groundwater, the concentration of dissolved iron is often inversely related
to oxygen, as O2 in water will chemically oxidize iron that will
precipitate as insoluble iron-hydroxides at neutral pH. In the wells
H4.2 and H4.3, the increase of the PLFAs 10Me12:0, 12:0, 17:1, 18:1ω7c and
18:1ω9c with concentrations of Fet, Fe2+ and
HCO3- (Figs. 5 and 6b) and the DNA- and RNA-based analyses (Fig. 9)
suggested degradation of OM by anaerobic iron-reducing bacteria (IRB).
Because many IRB are highly versatile, i.e. they can use different metal
substrates as electron acceptors coupled to the oxidation of the OM (Coleman
et al., 1993; Lovley et al., 1993; Holmes et al., 2004), specific PLFAs
linked to the reduction of iron in anoxic environments are poorly described.
The two most studied genera of IRB are Geobacter and
Shewanella, which contain most of those PLFAs (Coleman et al., 1993;
Lovley et al., 1993; Hedrick et al., 2009). However, none of these PLFAs are
specific to a certain genus or species. The 17:1 is generally related to
anaerobic SRB (Dowling et al., 1986) as Desulfobulbus (Parkes and
Calder, 1985; Macalady et al., 2000) but also occurs in dissimilatory IRB as
Shewanella (Coleman et al., 1993). The ability of some sulfate
reducers to reduce iron rather than sulfate has long been recognized in
groundwater (Coleman et al., 1993).
The 18:1ω9c is common and abundant in fungi (Frostegård and
Bååth, 1996; Kaiser et al., 2010), but may also occur in micro-algae
(Arts et al., 2001) and Gram-negative bacteria (Kandeler, 2007). The
18:1ω9c, 18:2ω6,9 and 18:3ω6 are typically used as
fungi biomarkers in soil (Frostegård and Bååth, 1996; Ruzicka et
al., 2000; Bååth and Anderson, 2003) and more particularly for
saprotrophs (Etingoff, 2014). The correlations between 18:1ω9c,
18:2ω6,9 and 18:3ω6 suggested a major fungal origin of those
PLFAs in the studied groundwaters. In soil, fungi are well known for their
role in accelerating weathering and solubilisation of iron-containing
minerals by excreting organic acids including phenolic compounds,
siderophores and protons (Arrieta and Grez, 1971; Landeweert et al., 2001).
By forming dense hyphae tunnelling in soils and shallow rocks, fungi mediate
and facilitate iron transport in plants and increase iron availability in the
environment (van Schöll et al., 2008). Therefore, several studies have
linked enhanced rates of iron cycling to the presence of fungal biomass
(Gadd, 2010). Moreover, in a recent study, it is been shown that rhizoplanes
are important root channels for preferential vertical transport from soil to
seepage area of soil colloids including microbes (Dibbern et al., 2014).
Limitation of ferric iron may restrain the growth and activity of IRB in the
subsurface (O'Neil et al., 2008). In the groundwater of wells H4.2 and H4.3,
the close relation of 18:1ω9c and 18:2ω6,9 with
Fet concentration (Fig. 6b) suggested that fungal biomass may, by
mediating and facilitating the transport of different types of organic and/or
inorganic particles and colloids, play a key
role in iron bioavailability and thus sustain IRB growth and activity.
PLFA cluster in anoxic, more NH<inline-formula><mml:math id="M519" display="inline"><mml:mrow><mml:msubsup><mml:mi/><mml:mn mathvariant="normal">4</mml:mn><mml:mo>+</mml:mo></mml:msubsup></mml:mrow></mml:math></inline-formula>-rich groundwater (wells
H5.2, H5.3 and H3.2)
To our knowledge, this is the first time
phospholipid [3]-ladderane and [5]-ladderane, which attest the presence of
viable or recently degraded anammox bacteria (Jaeschke et al., 2009), have
been identified in groundwater. The occurrence of anammox bacteria in those
groundwaters is consistent with the DNA- and RNA-based analyses (Fig. 9) and
coincided with higher concentrations of ammonium (Fig. 2). The difference
between DIC and ladderane δ13C values of 55 ‰ was within
the range previously reported for anammox in the Black Sea (Schouten et al.,
2004), further suggesting that autotrophic carbon fixation pathways within
the diverse group of anaerobic ammonium-oxidizing bacteria are similar. In
the sub-oxic (well H3.2) and anoxic groundwaters (well H5.2 and H5.3), the
increasing concentration of ladderane lipids derived from anammox bacteria
with decreasing O2 concentration (Fig. 6a) agrees well with the reported
high sensitivity of the anammox process to O2 (Kalvelage et al., 2011).
Denitrification and anammox are the dominant nitrogen loss pathways in
aquatic ecosystems (Burgin and Hamilton, 2008; Koeve and Kähler, 2010).
The occurrence of lipids derived from anammox bacteria in those groundwaters
indicates that the anammox process may be critically important in the
nitrogen loss from this part of the aquifer assemblage.
High amounts of 10Me16:0 are typically found in SRB (Dowling et al., 1986;
Vainshtein et al., 1992; Kohring et al., 1994) but also occur in anammox
bacteria (Sinninghe Damsté et al., 2002). Anammox bacteria strongly
fractionate against 13C, producing ladderane lipids which are
13C-depleted by 47 ‰ compared to the inorganic carbon source
(Schouten et al., 2004). Relative to ladderanes, SRB-derived lipids are
expected to be 13C-enriched as cultured SRB under heterotrophic and
autotrophic growth fractionated against 13C by up 27 ‰ (Londry
et al., 2004). Therefore, the 13C-enrichment of 10Me16:0 (up to
19 ‰) relative to the ladderanes supported major SRB contribution to
the 10Me16:0 found in these groundwaters. The i13:0, i15:0 and i17:1 are
typically, as with 10Me16:0, associated with SRB (Edlund et al., 1985; Kohring et
al., 1994). In those groundwaters, similar δ13C values, in the
-44 to -56 ‰ range, also supported a common SRB origin of those
PLFAs.
Variation partitioning analyses showed that the concentrations of
[3]-ladderane, [5]-ladderane, 10Me16:0 and i17:1 correlated with NH4+
concentration (Fig. 6c). Many studies in other aquatic environments showed
that the relative importance of the anammox process is directly related to
the availability of NH4+ (Dalsgaard and Thamdrup, 2002; Kuypers et
al., 2003). Commonly, the breakdown of OM via ammonification or dissimilatory
nitrate reduction to ammonia is presumed to be the major source of
NH4+ for anammox (Kartal et al., 2007). However, the recent discovery
of comammox organisms capable of complete nitrification underlines the
complexity of the nitrogen cycle and the variability of ammonium sources for
anammox (van Kessel et al., 2015). The availability of OM is known as an
additional important factor influencing the anammox process. Higher anammox
activity has been observed in OM-poor environments and interpreted as a
decrease in competition for NO2- by heterotrophic denitrifiers (Hu et
al., 2011). Consistently, high anammox activity was observed in redox zones
associated with sulfate reduction or sulfur oxidation (Mills et al., 2006;
Canfield et al., 2010; Prokopenko et al., 2013; Wenk et al., 2013). In the
groundwater of the wells H5.2 and H5.3, the occurrence of anammox bacteria
and SRB thus supported low groundwater–surface interactions which likely
threatened the availability of generically favourable electron acceptors and
labile OM.