of 44
Bacterial chemolithoautotrophy via manganese oxidation
Hang Yu
1
,
Jared R. Leadbetter
1,2
1
Division of Geological & Planetary Sciences, California Institute of Technology, Pasadena, CA,
USA 91125
2
Division of Engineering & Applied Science, California Institute of Technology, Pasadena, CA,
USA 91125
Abstract
Manganese is among Earth’s most abundant elements. Its oxidation had long been theorized
1
, yet
undemonstrated
2
4
, to fuel chemolithoautotrophic microbial growth. Here, an enrichment culture
exhibiting Mn(II)-oxidation-dependent, exponential growth was refined to a two species co-
culture. Oxidation required viable bacteria at permissive temperatures, resulting in the generation
of small Mn oxide nodules to which the cells associated. The majority member of the culture,
Candidatus
Manganitrophus noduliformans’, affiliates within phylum
Nitrospirae
(
Nitrospirota
)
but is distantly related to known
Nitrospira
and
Leptospirillum
species. The minority member has
been isolated, but does not oxidise Mn(II) alone. Stable isotope probing revealed Mn(II)-
oxidation-dependent,
13
CO
2
-fixation into cellular biomass. Transcriptomics reveals candidate
pathways for coupling extracellular manganese oxidation to aerobic energy conservation and to
autotrophic CO
2
-fixation. These findings expand the known diversity of inorganic metabolisms
supporting life, while completing a biogeochemical energy cycle for manganese
5
,
6
, one that may
interface with other major global elemental cycles.
Beijerinck and Winogradsky discovered biological redox reactions involving C, N, S, and Fe
over a century ago while pioneering methods for cultivating the responsible microbiota. This
led to the concept of chemolithoautotrophy
7
,
8
. The known breadth of inorganic electron
accepting and donating reactions in biology has continued to expand
9
13
. For example, the
anaerobic respiratory reduction of Mn(IV) oxides to Mn(II) by diverse microbes is now
understood to be widespread and of broad biogeochemical importance
5
,
6
,
14
,
15
. Over the past
century, a multitude of studies and reviews have focused on the details of Mn(II) oxidations
catalysed by diverse heterotrophs
2
4
; however the physiological roles of those activities
generally remains unclear. Despite experimental hints that the oxidation might be coupled to
energy conservation in some organisms
1
,
16
19
, whether Mn(II) oxidation drives the growth
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Corresponding author:
Jared R. Leadbetter, jleadbetter@caltech.edu.
Author contributions
H.Y. and J.R.L. together applied for funding, designed and conducted the experiments, performed data
analyses, prepared the figures, and wrote the manuscript.
Supplementary Information
is available in the online version of the paper.
Completing interests
The authors declare no competing financial interests.
NASA Public Access
Author manuscript
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Published in final edited form as:
Nature
. 2020 July ; 583(7816): 453–458. doi:10.1038/s41586-020-2468-5.
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of any chemolithotrophs has remained an open question [Mn(II) + ½ O
2
+ H
2
O
Mn(IV)O
2(s)
+ 2H
+
; ΔG°
= −68 kJ · (mol Mn)
−1
] (Supplementary Note 1).
Cultivation of manganese oxidisers
That unappreciated microbes from the environment might oxidise Mn(II) for energy was re-
examined. A glass jar was coated with a slurry of Mn(II)CO
3
and allowed to dry before
being filled with municipal tap water and left to incubate at room temperature. After several
months, the cream-coloured carbonate coating was oxidised to a dark Mn oxide. Serial
transfer of the material into a defined medium led to the establishment of a stable,
in vitro
culture. Unless otherwise noted: except for trace amounts of vitamins, this medium was free
of alternative organic and inorganic electron donors (e.g. nitrate was used instead of
ammonia as N source to preclude the growth of nitrifiers).
To distinguish between abiological and biological oxidation, flasks of sterile, defined,
Mn(II) media were either inoculated with a subculture of the promising enrichment, or left
uninoculated, and incubated under oxic conditions. Because Mn oxides have been suggested
to contribute to chemical autooxidation of Mn(II)
20
, other replicate flasks were inoculated
with a steam-sterilised sub-culture with oxide products. Even after a year, oxidation did not
occur in uninoculated flasks or those containing the steam-sterilised inocula, as predicted by
the known chemical stability of MnCO
3
under these conditions
21
,
22
. However, within 4
weeks, the flasks inoculated with “viable material” had generated dark, adherent Mn oxides
(Fig. 1a). Oxidation required O
2
and occurred up through 42 °C, occurring optimally
between 34 °C to 40 °C (Extended Data Fig. 1a), consistent with catalysis being enzymatic.
Mn(II) oxidation activity was also sensitive to exposure to antibiotics or to overnight
pasteurisation at 50 °C (Extended Data Fig. 1b). Phosphate was inhibitory above 0.3 mM.
When amended with MnCO
3
, the pH of unbuffered media ranged between 5.7 and 6.3.
While pH buffer was not required, Mn(II) oxidation was faster in media buffered with 5 mM
MOPS at its pKa (7.1 at 37 °C). In buffered cultures, the final pH ranged between 6.5 and
6.8. With or without buffer, increases in culture pH during or after oxidation (that might
potentially lead to chemical oxidation of unreacted MnCO
3
) were not observed. No growth
could be detected in the MOPS-buffered basal medium without addition of MnCO
3
(Extended Data Fig. 2a).
An rRNA-gene iTag community analysis of the initial enrichment culture revealed ~70
different species representing 11 bacterial phyla (Supplementary Table 1). The responsible
microbes did not generate oxide-forming colonies on MnCO
3
agar media, but successive
rounds of serial-dilution-to-extinction in MnCO
3
liquid media refined the community to a 2
species co-culture (Supplementary Table 1). Species A belongs to the phylum
Nitrospirae
(
Nitrospirota
), whereas Species B is a betaproteobacterium, occurring at cell ratio of ca. 7:1
(Supplementary Table 1; Extended Data Fig. 3a). Attempts to isolate Species A have thus far
failed. Species B can be isolated from disrupted oxides as single colonies on succinate or
other heterotrophic media (Supplementary Note 2), but does not oxidise MnCO
3
alone.
Either Species A is solely responsible for Mn(II) oxidation (Extended Data Fig. 3b), or the
activity is consortial (Extended Data Fig. 3c–e). Several betaproteobacteria have proven
recalcitrant to elimination from multi-species cultures: some were seemingly
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unimportant
9
,
23
, whereas others engaged in metabolite cross-feeding
24
, and at least one
example central to a consortium
25
.
DNA harvested from both the co-culture and from a pure culture of Species B lead to near
complete genome sequences for both species (Extended Data Fig. 3f), facilitating later
experiments and analyses (below).
Manganese oxide nodules
Mn(II) oxidation yielded morphologically conspicuous, Mn oxide nodules ~ 20–500 μm in
diameter (Fig. 1; Extended Data Fig. 4). These formed in both static and shaken liquid
media, often adhering to the glass and to each other, as well as in media solidified with
agarose (Fig. 1b). The surfaces were dark brown but often reflective, and typically
invaginated around deeper depressions having a rough, dark orange surface (Fig. 1c,d).
Attenuated total reflectance FTIR analysis revealed that Mn oxide nodules, hypochlorite-
bleached to remove cellular and other organic carbon, are poorly-ordered and similar to
birnessite.
Epifluorescence microscopic examination of nucleic acid-stained Mn oxide nodules
localized the majority of the exposed biomass to the invaginations (Fig 1d,e), with few cells
observed attached to the substrate or found planktonic. In agarose solidified media, these
could be well separated from the carbonate substrate, dissolving it from a distance (Fig. 1b).
The latter is partially explained by the solubility products of the MnCO
3
precipitate; under
the incubation conditions, these can be expected to include free Mn
2+
ion, manganese
bicarbonate, and soluble MnCO
3
22
. The mean concentration of dissolved Mn in
uninoculated and active MnCO
3
cultures was 0.214 mM (s.d. = 0.107, n=3) and 0.119 (s.d. =
0.081, n=3), respectively, before falling to 0.010 mM (s.d. = 0.009, n=3), after oxidations.
Soluble Mn(II) chloride does not appear to be utilized; instead, it appears to inhibit MnCO
3
oxidation when amended to active co-cultures at concentrations >2.0 mM (Extended Data
Fig. 1c). No evidence for motility was observed: the oxides did not accumulate as a band
across the interface between counter opposing gradients of Mn(II) and O
2
in agarose-
solidified media (Fig. 1b), as is commonly observed for microaerophilic iron-oxidising
bacteria
26
. It is not yet understood whether the tight association of the cells with the
oxidation product is circumstantial or is more intrinsic to the process due to some role of
adsorptive, conductive, catalytic, and/or other properties of Mn oxides.
Additional biomass was revealed upon chemical dissolution of Mn oxide nodules, and was
examined using fluorescence
in situ
hybridisation (FISH) with specific rRNA-targeted
probes (Supplementary Note 3). Consistent with the iTag analysis, Species A cells were
more abundant (Extended Data Fig. 3a). No stereotypic patterns of association with Species
B were observed (Fig. 1g; Extended Data Fig. 4a–e). Cells of both were often pleomorphic.
Species A cells were typically crescents, 1.07 μm by 0.40 μm (s.d. = 0.17 by 0.08, n=50);
Species B were typically rods, 1.22 μm by 0.56 μm (s.d. = 0.20 by 0.09, n=50) in co-culture,
but at high cell densities in pure cultures, cells elongate and form flocs.
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Growth rates and yields
If truly chemolithotrophic, one or both species in the co-culture should exhibit a)
exponential increases in the rate of Mn(II) oxidation during and in parallel to b) Mn(II)-
dependent exponential growth. Indeed, after inoculation, the rates of Mn(II) oxidation in the
basal media increased exponentially, initially doubling every 6.2 days (s.d. = 1.1; n = 4
replicate cultures; Fig 2a; Extended Data Fig. 2e–g; Supplementary Note 4) before
decelerating to 10.8 days (s.d. = 0.1). Concurrent with Mn(II) oxidation, Species A exhibited
sustained exponential growth, roughly matched by the less numerous and perhaps
commensal Species B (T
d
= 6.1 and 7.7 day, respectively; Fig. 2b; Extended Data Fig. 2b;
Supplementary Note 5). The co-culture oxidised Mn(II) at a combined rate of 3.4 to 9.0 ·
10
−15
mol Mn(II) · cell
−1
· hr
−1
. A linear relationship between Mn(II) oxidised by the co-
culture and the cell yields was observed (Fig. 2c; Supplementary Note 6): 6.4 · 10
11
cells of
Species A, 1.0 × 10
11
cells of Species B, for a combined yield of 7.4 · 10
11
cells · (mol
Mn(II) oxidised)
−1
. The total amount of DNA extracted from samples also increased
exponentially with time and oxidation of Mn(II) (Extended Data Fig. 2c,d), yielding 3.1 ·
10
6
ng DNA · (mol Mn(II) oxidised)
−1
. Based on either of two estimates (both using the
known dry weight of a single cell of
E. coli
27
), the growth yield in the co-culture is
estimated to be ~100–200 mg dry biomass · (mol Mn(II) oxidised)
−1
. Such growth yields
and normalised substrate oxidation rates are comparable with those observed for
chemolithotrophic microbial nitrite oxidation (Extended Data Fig. 3g), a metabolism
predicted to yield a similar free energy
12
.
Phylogenetic analyses
Species A affiliates remotely with the genera
Nitrospira
,
Leptospirillum
, and other members
of the phylum
Nitrospirae
(
Nitrospirota
), yet shares <84% 16S rRNA identity with any
cultivated organism, and <87% identity to all but ~50 16S rRNA gene sequences from not-
yet-cultivated organisms (Fig. 3a; Extended Data Fig. 5a,c; Supplementary Table 2). The
genome of Species A does not encode recognisable genes for the chemolithotrophic
oxidation of ammonia, hydroxylamine, nitrite, or reduced sulfur compounds. Several closely
related sequences have been recovered from drinking and karst-impacted groundwater,
marine sites, and a subsurface oxic-anoxic transition zone (Fig. 3a; Extended Data Fig. 5a).
Together, these cluster with ‘
Candidatus
Troglogloea absoloni’
28
, an uncultivated cave
organism of unknown physiology. Few genome datasets are available for comparison.
Species A shares <88% 16S rRNA gene identity to the best reference genomes (from
metagenome-assembled genomes from groundwater
29
,
30
), and is the only genome currently
available in the
Candidatus
Class ‘Trogloglia’ (Fig. 3a; Extended Data Fig. 5b,c).
Species B affiliates with heterotrophs from the betaproteobacterial genus
Ramlibacter
(Extended Data Fig. 6a,b). It exhibited H
2
+ O
2
dependent growth; encodes genes for
hydrogenases; encodes both Sox and DsrMKJOP gene clusters for the oxidation of reduced
sulfur; encodes a Calvin-Benson-Bassham Cycle; and may capable of anaerobic respirations
such as denitrification and dissimilatory metal reduction. The potential for facultative
lithotrophy had not been previously reported for members of this genus.
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Transcriptomics of Mn dependent growth
The transcriptomes of the co-culture (in particular Species A) were examined, during
different stages of Mn(II) oxidation in replicate cultures (n=7; Extended Data Fig. 3h).
Although both species encode genes for swimming and twitching motility, as well as for
chemotaxis, they were not expressed (Supplementary Table 3), matching the observations in
agarose solidified media (above). In contrast, genes for different compatibles solutes (e.g.
hydroxyectoine, trehalose, and betaines) were expressed by both species and, consistent with
this, co-cultures oxidised Mn when grown at a range of brackish salinities, up through nearly
40% of that of seawater.
Candidate genes that might underly Mn(II) chemolithotrophy have been identified. Species
A transcribed 4 gene clusters encoding outer membrane complexes that evoke comparisons
with lithotrophic iron oxidisers and respiratory metal reducers. By analogy, these might play
a role in extracellular electron transfer (EET) by ferrying Mn(II)-derived electrons to
periplasmic carriers (candidates for which were also expressed; Supplementary Table 4),
leaving the resultant, insoluble oxide outside the cell (Fig. 3b). In iron-oxidising microbes,
an outer membrane c-type cytochrome (Cyc2 or Cyt572) is often employed as the initial
oxidant and carrier for the Fe(II)-derived electron
31
,
32
. Species A expressed a Cyc2 homolog
with the predicted heme-binding site and outer membrane beta-barrel structure (Fig. 3b;
Supplementary Table 4). In iron-oxidising anoxygenic phototrophs
33
,
34
and in several
neutrophilic iron-oxidising chemolithotrophs
35
, an alternative mechanism involves a porin-
cytochrome c protein complex (PCC)
36
. Species A expresses genes for three recognisable
PCCs: a porin-dodecaheme cytochrome c with no homologs in the databases (PCC_1); and
two distinct porin-decaheme cytochrome c modules (PCC_2 and PCC_3; Fig. 3b).
Curiously, during growth in the Mn(II) oxidising co-culture, Species B expresses an
MtrABC-like PCC and other multiheme cytochromes c with greater resemblance to the
complexes involved in anaerobic reduction of metals by
Shewanella
sp.
36
(Supplementary
Table 5).
After the transfer of Mn(II)-derived electrons from outside of the cell into the periplasm,
their flow through respiratory complexes in the cytoplasmic membrane is central to
understanding this novel mode of energy conservation. On average, the two Mn(II)-derived
electrons are generally considered to be of high potential [Mn(II)/Mn(IV), E° ́=+466 mV;
Supplementary Note 1]. However, the energetics of each of the two sequential one-electron
transfers can be impacted by inorganic and organic binding ligands
22
,
37
, leading to a degree
of uncertainty here. Of the respiratory complexes, canonical respiratory Complex I is
unlikely to be employed for energy conservation, leaving canonical or alternative Complex
III, Complex IV, or cytochrome bd oxidases as possible candidates for generating a proton
motive force during Mn(II) chemolithotrophy.
The genome of Species A contains a host gene clusters for terminal oxidases (TO) that could
link the quinone pool to O
2
, many of which are strongly transcribed (Supplementary Table
4). Although a role in the process can’t be ruled out, the single Complex IV (cbb3-type
cytochrome c oxidase) of Species A is not as well expressed (24th percentile) as four
unconventional terminal oxidase complexes containing cytochrome bd-like oxidase (Fig. 3b;
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Supplementary Table 4; Extended Data Figs. 7 and 8; Supplementary Note 7). The most
highly expressed of the latter is “TO_1” (99th percentile; Fig. 3b), a complex generally
similar to those observed in ammonia- and nitrite-oxidising
Nitrospirae
38
,
39
, but without the
hypothesized candidate catalytic and iron-sulfur subunits, NxrAB. The second highest
expressed terminal oxidase (TO_2; 83rd percentile) also misses these and is highly unusual
amongst cultivated organisms, having extra heme c domains, cytochrome c, and two MrpD-
like ion-pumping subunits (Fig. 3b; Supplementary Table 4) that might, hypothetically,
facilitate electron transfer while generating a motive force. In both TO_1 and TO_2, a
membrane attached di-heme cytochrome b may connect EET and periplasmic electron
carriers to these membrane complexes (Fig. 3b; Supplementary Note 7). Whether either of
these will have a high affinity for O
2
, as canonical bd-oxidases do, is not known. Future
studies are required to examine the function of these unusual terminal oxidases, their roles in
energy conservation, and how any energetic challenge of the initial oxidation of Mn(II) to
Mn(III) is offset or met by the organism.
Mn(II)-oxidation-dependent CO
2
-fixation
With the demonstration that Mn(II) oxidation drives lithotrophic energy metabolism and
growth, that the co-culture might generate biomass via autotrophic CO
2
-fixation with
Mn(II)-derived electrons was examined. For this, the co-culture was grown with labelled
13
C-MnCO
3
(and
15
N-nitrate, to aid in tracking the synthesis of new biomass), with the
isotopic compositions of cells visualized microscopically by species-specific FISH coupled
to nanometre-scale secondary ion mass spectrometry (nanoSIMS) (Fig. 4). The results are
consistent with the co-culture being autotrophic. Both Species A and Species B were
confirmed to incorporate significant amounts of
13
C and
15
N isotopes (Fig. 4;
Supplementary Note 9). Species A showed a higher enrichment for both isotopes, as
compared to Species B (Extended Data Fig. 9a), suggesting that it is the main if not sole
driver of Mn(II)-dependent CO
2
-fixation and lithoautotrophic growth in the co-culture,
especially when taken together with its greater abundance (Extended Data Fig. 3a). While it
can’t be ruled out that some degree of anabolic mixotrophy might occur via the uptake of
trace contaminating organic carbon, it is likely that Species A has an inoperable oxidative
TCA cycle: it does not encode a recognisable homodimeric 2-oxoglutarate dehydrogenase
(OGDH) complex (Fig. 3b; Supplementary Note 10), a hallmark deficiency observed in
many autotrophs that are unable to mineralize organic carbon (including N- and Fe-oxidising
Nitrospirae
species, which grow autotrophically using the rTCA pathway
38
,
40
,
41
).
During Mn(II)-dependent growth in the co-culture, Species A expresses genes for a
complete Reverse Tricarboxylic Acid Cycle (rTCA; Fig. 3b; Supplementary Table 4;
Supplementary Note 10). Curiously, although its role in Mn oxidation is speculative, Species
B expresses genes for the Calvin-Benson-Bassham Cycle (Supplementary Table 5).
For Species A to fix CO
2
via the rTCA pathway, low potential electron carriers [e.g.
NAD(P)
+
and ferredoxin]
42
need to be reduced with high potential, Mn(II)-derived electrons
(see discussion above), possibly via a reverse electron transport chain, at the very least
involving Complex I (Fig. 3b). Three distinct Complex I gene clusters can be found in
Species A, two of which (Complex I_1&2; Fig. 3b; Supplementary Table 4) hypothetically
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might dissipate energy when operated in reverse and generate NAD(P)H (E° ́=−320 mV)
43
from reduced quinones with a more positive reduction potential. Similarly, a remarkably
unusual Complex I (Complex I_3; Fig. 3b; Supplementary Table 4) might be used to reduce
ferredoxin (E° ́=−398 mV)
43
. This complex encodes five ion-pumping subunits (NuoN, 2
NuoM, and 2 MrpD-like subunits, but no NuoL), whereas the canonical Complex I only
encodes three, NuoLMN
44
. Recently, rare and unusual variants of Complex I with four ion-
pumping subunits (NuoLMMN) have been identified and postulated to couple the inward
flow of 5 protons to drive the endergonic reduction of ferredoxin from a quinone
45
,
46
. In
Species A, Complex I_3 hypothetically may use electrons from the quinone pool to drive the
reduction of ferredoxin via the inward flow of 6 protons or ions (Fig. 3b). If the entry
point(s) of Mn(II) derived electrons into the electron transport chain have reduction
potentials more positive than the quinone pool, then additional respiratory complexes (Fig.
3b) would have to be involved to accomplish productive reverse electron flow
(Supplementary Note 7).
Discussion
Whether chemolithoautotrophic manganese-oxidising microbes exist had been an open
question for over a century. This study establishes their existence and provides insights into
the details and dynamics of the process at cellular, physiological, genomic, transcriptomic,
and isotopic levels. Manganese chemolithoautotrophy extends the known physiologies in the
phylum
Nitrospirae
that leverage meagre differences in redox potentials between inorganic
electron donors and acceptors
9
,
12
,
47
49
. Based on its physiology, phylogeny, genomics, and
other characteristics, the epithet ‘
Ca.
Manganitrophus noduliformans’ is proposed for
Species A (Supplementary Taxonomic Proposal).
The potential impact of Mn(II) oxidation coupled to seemingly slow exponential growth of
the co-culture (Extended Data Fig. 3g) has current and past environmental implications.
Starting with a single cell each of Species A and B, unrestricted chemolithotrophic growth at
the observed cell doubling times and oxidation rates would be sufficient to generate Mn
oxides equalling global Mn reserves within 2 years (Supplementary Note 11). Based on
phylogenetic inferences, close relatives of Species A reside in many subsurface and karst
environments (Fig. 3a; Extended Data Fig. 5a), including at oxic-anoxic transition zones
(OATZ) such as Hanford sediments
50
. At such interfaces, Mn could be cycled between
aerobic Mn(II)-oxidising chemolithoautotrophs and anaerobic, Mn oxide respiring
(reducing) chemotrophs
5
,
6
,
14
,
15
, thereby stimulating significant electron flows through the
element over even brief geological time scales. This has implications for the interconnected
biogeochemical cycles of C, N, S, Fe, H, and O, with which a newfound, complete Mn
energy cycle likely interacts.
Methods
No statistical methods were used to predetermine sample size, and the investigators were not
blinded to allocation during experiments and outcome assessment.
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Media and culture enrichment, refinement, and maintenance.
A fortuitous enrichment culture for Mn(II) lithotrophs was established over the Summer and
Fall of 2015. A dense slurry of freshly precipitated MnCO
3
(see below) was distributed onto
the internal surface of wide mouth glass jar and the coat was allowed to dry. The jar was
filled completely with unsterilised Pasadena municipal drinking water (typically a blend
from surface and aquifer sources) collected and allowed to stand for approximately 10
weeks, open and without agitation in an unoccupied room maintained at ca. 21°C (GPS
34°08’12.1”N 118°07’38.3”W). Additional freshly precipitated MnCO
3
was added as a
dense suspension after the cream-coloured coating had blackened, and the jar was covered
with a loose fitting lid and allowed to incubate for several more months, after which a small
amount (ca. 5% v/v) of the dark product was used to inoculate flasks of MnCO
3
suspended
in municipal tap water. After this, a separate line of cultures was initiated in a defined,
deionized water medium (below) incubated in the laboratory at 37 °C.
The basal medium used for routine growth and maintenance of this line and in related
experiments was adapted and modified from prior formulations
51
,
52
. The medium contained,
per liter deionized H
2
O: NaCl, 1 g; MgCl
2
·6H
2
O, 400 mg; CaCl
2
·2H
2
O, 1 g; KCl, 5 g;
Na
2
SO
4
, 142 mg; FeCl
3
·6H
2
O, 2 mg; H
3
BO
3,
30 μg; MnCl
2
·4H
2
O, 100 μg; CoCl
2
·6H
2
O,
190 μg; NiCl
2
·6H
2
O, 24 μg; CuCl
2
·2H
2
O, 2 μg; ZnCl
2
, 68 μg; Na
2
SeO
3
, 4 μg; Na
2
MoO
4
, 31
μg; riboflavin, 100 μg; biotin, 30 μg; thiamine HCl, 100 μg; L-ascorbic acid, 100 μg; d-Ca-
pantothenate, 100 μg; folic acid, 100 μg; nicotinic acid, 100 μg; niacinamide, 100 μg; 4-
aminobenzoic acid, 100 μg; pyridoxine HCl, 100 μg; lipoic acid, 100 μg; NAD, 100 μg;
thiamine pyrophosphate, 100 μg; cyanocobalamin, 10 μg. As P source, a solution of
potassium phosphate (pH 7.2) was added to a final concentration of 0.15 mM. As N source,
NaNO
3
was added to a final concentration of 1 mM; alternatively, 1 mM NH
4
Cl or other N
sources were employed as noted. MOPS at its pKa was added as a buffer to a final
concentration of 5 mM, when noted. MnCO
3
, or alternative, heat-stable substrate for growth
was added to the basal medium prior to steam sterilisation; alternatively, heat-unstable
substrate for growth was added using filter sterilised stock solutions after media had
adequately cooled.
Freshly precipitated MnCO
3
was employed, for the initial enrichment culture, for routine
Mn(II)-dependent cultures in small volumes; or for the serial dilution-to-extinction
resolution of complex cultures; or for stock and starter cultures. To prepare this, 25 g MnCl
2
(Sigma-Aldrich #221279) was dissolved into 100 ml deionized water, yielding 125 ml of a
1.59 M MnCl
2
solution. Over the course of several minutes, this solution was slowly poured
into 3 liters of 0.33 M NaHCO
3
while vigorously stirring. After the cessation of stirring after
ca. 1 hour, the resultant precipitate was allowed to settle by gravity. Thereafter, the overlying
reaction fluid was decanted and discarded. The precipitate was resuspended in 3 liters of
deionized water, and the stirring, settling, decanting, and resuspension steps were repeated at
least 10 times. After the final wash, the precipitate was resuspended in deionized water to a
final volume of 100 ml, stored in a clean glass bottle, and refrigerated at 4°C in the dark.
Initially, the precipitate appeared white to light pink, but aged to a light tan within ca. 24 h.
Thereafter, the material remained stable for months to years. Alternatively, a hydrated
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MnCO
3
substrate (Sigma-Aldrich #63539) was employed for larger culture volumes, and/or
for reproducible mass balances, as noted.
Cells were typically cultured in 10 ml of medium in 18-mm diameter culture tubes, or in 100
ml of medium in 250-ml Erlenmeyer flasks at 37 °C, with 25 to 200 mM MnCO
3
, as noted.
To prevent dehydration of cultures over the long periods of incubation, cut strips of Parafilm
(Heathrow Scientific, Vernon Hills, IL, USA) were used to seal the bottom edge of the 18-
mm plastic test tube enclosures. Cultures were incubated stationary without agitation, or
with shaking at 200 rpm, as noted.
To refine the number of species in the complex manganese-oxidising enrichment, 5
successive rounds of serial tenfold dilution-to-extinction series were performed using 9 ml
of MnCO
3
nitrate basal media in 18-mm culture tubes, incubated at 32 °C. Culture tubes in
each dilution series were scored as positive for the presence of lithotrophic manganese
oxidisers when, after 2–12 weeks of incubation, the small and easily dispersed particles of
the MnCO
3
substrate (light cream in colour) were converted to larger Mn oxide nodules or a
single continuous Mn oxide coating (dark brownish-black in colour) that typically tightly
adhered to the bottom of the glass culture tubes. Mn oxides from the final dilution tube
showing such oxidation were used as the inocula for the next serial-dilution-to-extinction
series.
To examine whether Mn(II) oxidation in the cultures was biological in nature, an active co-
culture with Mn oxides was used to inoculate 18-mm culture tubes containing the basal
media with 50 mM MnCO
3
. Cultures were amended with either of two antibiotics,
kanamycin (30 μg/ml) or vancomycin (20 μg/ml), or pasteurised overnight at 50 °C before
incubation at 32 °C. To examine the impact of incubation temperature on oxidation, cultures
were incubated without agitation in different incubators set at a diversity of temperatures;
incubation temperatures were regularly and independently confirmed with >2 thermometers.
After 2 weeks, 2 ml mixture from the cultures were sampled and stored at −80 °C for later
ICP-MS analysis (below). Reported values were corrected for Mn oxides carried over in the
inoculum, as ascertained by the lowest amount determined in the 50 °C pasteurisation
experiments.
To examine the growth of the culture in the absence of Mn(II) substrate, a stationary co-
culture (confirmed separately to be viable) was used to inoculate 4 replicates of 250-ml
Erlenmeyer flasks containing 120 ml of the basal media without MnCO
3
. The flasks were
incubated at 37 °C with shaking at 200 rpm. After inoculation and after 10 and 21 days, 20
ml mixture from the cultures were sampled and centrifuged at 5250 × g for 10 min; the
pellet was stored at −80 °C. DNA was extracted from the thawed pellets using the DNeasy
PowerSoil kit (Qiagen, Valencia, CA, USA) following the manufacturer’s instructions, with
the bead beating option using FastPrep FP120 (Thermo Electron Corporation, Milford, MA,
USA) at setting 5.5 for 45 s instead of the 10 min vortex step. DNA concentration was
quantified using Qubit dsDNA High Sensitivity Assay Kit (Thermo Fisher Scientific,
Waltham, MA, USA).
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To examine whether MnCl
2
can be oxidised by, or otherwise impacts growth: an active co-
culture, which had oxidized 38% of the 35 mM MnCO
3
initially provided (measured using
the ICP-MS method below), was used to inoculate (10% v/v) 18-mm culture tubes
containing 10 ml of the basal media with 0.5–20 mM MnCl
2
instead of MnCO
3
. After
inoculation, and after days 5 and 10, 0.5 ml of the oxides and culture fluid mixture was
sampled and stored at −80 °C for later ICP-MS analysis (below). Reported values were
corrected for Mn oxides carried over in the inoculum, as ascertained by the lowest amount
determined in the 0.5 mM MnCl
2
culture.
For attempts to observe single colony formation by Mn(II) lithotrophs on agar media, the
basal medium was adjusted to contain 200 mM MnCO
3
and 1.5% washed agar (BD Difco),
and distributed into petri dishes after steam sterilisation.
Genomics predicts that each species in the co-culture may be able to produce compatible
solutes (e.g. trehalose and hydroxyectoine, by Species A), and thus may be able to grow
under a range of salinities. To examine the impact of increased salinity on the Mn(II)-
oxidising lithotrophs, the basal medium was amended with NaCl to achieve final salt
concentrations of 2 ppt, 2.8 ppt, 3.8 ppt, 4.6 ppt, 5.5 ppt, 9 ppt, 16 ppt, 23 ppt, 30 ppt, and 37
ppt (equivalent to 6%, 8%, 11%, 13%, 16%, 26%, 45%, 65%, 85%, and 105% of the salinity
of seawater, respectively). After inoculation, oxidation in the tubes was monitored visually
over time.
For isolation and maintenance of strains of “Species B” (
Ramlibacter lithotrophicus
) from
the co-culture (Supplementary Note 2), plates of agar-solidified (1.5% agar w/v, BD Life
Sciences) basal media were employed except that sodium succinate (10 mM, final conc.) or
tryptone (0.5% w/v, BD Life Sciences) were used in place of MnCO
3.
Viable cells of this
bacterium could rarely be retrieved from the co-culture as planktonic cells overlying Mn
oxide nodules. For clonal isolation, 200 μl of a dense suspension of the dark Mn oxides
produced by the co-culture were spotted onto the surface of succinate agar medium and
allowed to dry, after which the Mn oxides were vigorously and heavily streaked over the
agar surface and monitored for the development of colonies thereafter. After 3–5 days of
incubation, colonies of Species B appeared small, leathery, and adherent to the agar surface;
transfer of cells to new plates or liquid media was facilitated by the use of a sterile syringe
needle for the removal of an entire single colony from the agar surface. In liquid, newly
isolated strains of Species B can be grown with tryptone (0.5% w/v) or acetate (10 mM, final
conc.) (Extended Data Fig. 6c,d), and can form fabric-like biofilms blanketing at the bottom
of culture tubes. Transfer of such material proved challenging, as the fabric like biomass
typically adhered to the insides of both plastic and glass pipette surfaces, leading to the rapid
selection for strain variants that do not form flocs.
For the examination of anaerobic growth of Species B, 10 ml of basal medium is dispensed
into 18-mm glass “Balch” tubes (Bellco, Vineland, NJ, USA) and stoppered with 1 cm butyl
rubber stoppers under an N
2
headspace. Autotrophic growth of the isolate using H
2
+ O
2
+
CO
2
was examined similarly, except that an air headspace, periodically spiked with 1 ml of
H
2
+ CO
2
(80%/20%, v/v), was used. Colonies of Species B did not develop on standard
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