Near infrared radiation-driven oxygenic photosynthesis contributes substantially to primary production in biofilms harboring chlorophyll f-containing cyanobacteria

Reviewer #1 (Public Review):

Summary:

Mosshammer et al. studied the oxygenic photosynthetic productivity of beachrock samples containing cyanobacteria with different pigment compositions. The use of longer wavelength absorbing chlorophylls in some cyanobacteria (chlorophylls d and f) allows their photosystems to use light further in the red than canonical chlorophyll a photosystems. As such, their distribution in visible light-shaded environments, such as the beachrock studied by Mosshammer et al., allows them to perform oxygenic photosynthesis using wavelengths not capable of driving photosynthesis in most cyanobacteria, algae, or plants.

By adapting measuring systems they have previously used to study these types of beachrock samples, the authors attempt to mimic a more natural light penetration through the beachrock in order to measure oxygen production. By doing so with different wavelengths and intensities, the authors are able to show that far-red light-driven oxygen production is potentially capable of driving high levels of gross primary production.

Strengths:

The manuscript builds on previous measurement techniques used by the authors while focussing on illumination from the top of a sample rather than the specific microbial layers themselves. This provides a more environmentally realistic understanding of the beachrock community, as well as far-red light-driven photosynthesis.

The manuscript benefits from using previously defined methods to further characterize complex environmental samples.

Weaknesses:

The manuscript suffers from a lack of discussion and interpretation of the findings, and as such is more of a report.

Using the envionmental beachrock samples has inherent complications, from the variation in rock morphology, to the microbial community composition of different samples as well as within a single sample. It would benefit the authors to discuss these technical difficulties in more detail, as the light penetration through the beachrock is likely greatly limiting measurements of chlorophyll f and/or chlorophyll d-driven photosynthesis in the beachrock.

This can be seen in the different luminescence measurements (Figure 2 and supplements), that the different samples have clear differences in far-red light-driven oxygen production. While the BLACK sample produces oxygen with 740nm LED filtered with a NIR-75N filter, neither of the other two samples produce measureable oxygen under this condition. Conversely, this sample results in the lowest level of gross photosynthesis when measuring dissolved oxygen. A more detailed discussion of the variation between and within samples and measurements would benefit the overall results of the manuscript.

The PINK beachrock sample has the highest level of chlorophyll d per chlorophyll a. As FaRLiP cyanobacteria only incorporate 1 chlorophyll d per photosystem II, and none in photosytem I, is there a (relatively) high composition of Acaryochloris species in the PINK sample? If normalized to the reflectance minima can more distinct populations be identified?

For Figure 1, multiple points should be clarified. The first is that the HPLC methods are estimates of concentrations, as the extinction coefficients are not correct for the solvent solution for which the pigments elute, and are likely to be differently incorrect for each pigment. This results in quantitatively incorrect data, but qualitative comparisons between samples likely remain valid. Secondly, the pigment concentrations can also be misleading. Within the cyanobacterial cells, photosystem I harbors approximately 3 times as many chlorophylls as photosystem II. While the community numbers and photosystem stoichiometry are not necessarily relevant to the current study, the red shift in absorbance between photosystem II and photosystem I is of importance for the measurements performed. How cyanobacterial cells with differing concentrations of photosystems will absorb the red tail of the far-red LEDs, as well as impact the light penetration would be a useful discussion point.

The different samples used are from varying beachrock zonations but have the same chlorophyll f per chlorophyll a concentrations. A discussion of why this might be would be useful.

For the luminescence measurements (Figure 2 and supplements), no oxygen production is seen in the BROWN or PINK beachrock samples when the 740nm LED is filtered with a NIR-75N filter. This is likely due to multiple factors (low initial intensity compounded by penetration depth, community composition, etc.) but should be discussed. While the authors say that Chrooccidiopsis species dominate the samples, variation of absorbance between different chlorophyll f containing cyanobacteria has also been measured (see Tros et al. 2021, Chem), and the extent to which even chlorophyll f species extend into the far-red varies. Discussions about these implications would help with their characterization of the luminescence data. While the authors discuss that based on their respiration measurements the oxygen may be being consumed, resulting in an inability to measure it (lines 147-150), other explanations are clearly viable.

For the luminescence measurements, no oxygen production is discernable in the endolithic region when excited with visible light, which is at a much stronger intensity than the near-infrared light used. However, both Acaryochloris and chlorophyll f cyanobacteria are capable of driving photosynthesis with visible light. As the intensities used are much brighter than for the NIR measurements, presumably generated oxygen would be higher than what could be immediately consumed by respiration. It is important that the authors address this.

A highlighted point by the authors is the >20% of photosynthesis driven by NIR in the beachrock at comparable irradiation. However, this statement is deceiving for multiple reasons.
(1) The irradiation is likely not comparable for what is reaching the cells. This is not a problem per se as illumination from above is the point, but does skew the interpretation.
(2) The >20% value comes from the maximum amount of gross photosynthesis driven by NIR at ~1400 umol photons m-2s-1, whereas at other comparable illuminations the value is much, much lower (<1%). A likely interpretation of such data is that while the chlorophyll f endolithic layer is capable of producing a relatively large amount of oxygen, it is likely far less productive under most illuminations, though not zero.

The authors have the difficult task of weaving in results from laboratory, uniculture or isolated photosystem measurements with their environmental-based results. This is especially clear in lines 172-183. While the authors are correct that measurements of trapping times in chlorophyll f containing photosystems have been measured and are slower in chlorophyll f photosystem II and photosystem I relative to all chlorophyll a photosystems, the quantum yield for trapping remains high in chlorophyll f photosystem I (Tros et al. 2021, Chem). The quantum yield of trapping for chlorophyll f photosystem II is much lower for chlorophyll f than chlorophyll a complex, though improved by the attachment of phycobilisomes. However, these are intrinsic physical properties of the complexes that are not modulated in response to the environments. This could be interpreted that at low photon flux densities as measured in these experiments, the endolithic near infrared-driven oxygen production could be limited by an overall lower quantum efficiency of trapping the captured light and thus minimizing photosynthetic productivity relative to a theoretical level based on the efficiency of the chlorophyll a photosystem II. How the variations in intensity and spectral composition impact the cyanobacterial community likely involves many other factors and has not been addressed (though see Nurnberg et al. 2018, Science and Viola et al. 2022 eLife for further discussions).

Reviewer #2 (Public Review):

The authors investigate the role of near-infrared photosynthesis in primary production across three beachrock communities. This work is particularly pertinent as more cyanobacteria with far-red light acclimation capacities are discovered, underscoring the need to assess their contributions to primary production. However, the manuscript is currently very difficult to follow due to unclear correlations between the text and figures and the samples analyzed in the different experiments.. Additional explanations would also enhance clarity. For example, it would be beneficial for the authors to better define the three communities, as distinctions are not apparent. Another example is the pigment analysis, where the extinction coefficients for pigments vary in different solvents. Quantification by chromatography should use calibration curves for all pigments, not just Chl a, as is currently done. Pigments can be easily purified from cyanobacteria for this purpose.

Reviewer #3 (Public Review):

Summary:

On islands in the pacific, beachrock occurs near high tide level, composed of calcareous material. The surface of the beach rock is colonised by cyanobacteria and some eukaryotic algae. On Heron Island on the Southern Great Barrier Reef, beach rock occurs on the north and south side of the island in continuous slabs, which slope gently upwards toward the island. Thus the upper beach rock is only inundated at extreme high tides. On the south side, the major photosynthetic organism is a cyanobacterium Chroococcidiopsis, which forms tough smooth mats over all the beach rock. This cyanobacterium belongs to a newly discovered class called FaRLiP photosynthesisers, which carry out conventional photosynthesis under visible radiation using chlorophyll a (Chl a) but which deactivate most of the Chl a under near infra -red radiation (NIR) and produce chlorophyll f and chlorophyll d which can absorb NIR (700 - 760 nm). These NIR Chl molecules are repositioned in the reaction centres. In addition, an NIR-activated allophycocyanin (a phycobiliprotein) is synthesised and placed in the reaction centres. These FaRLiP cyanobacteria can carry out photosynthesis and primary production when placed under NIR. Here it is shown that in the mats of Chroococcidiopsis on the beach rock the upper layers carry out conventional photosynthesis while the lower layers carry out FaRLiP photosynthesis. It is shown that the FaRLiP-activated lower layers can produce up to 20% of the total photosynthetic primary production.

Strengths:

The authors have researched sections of beachrock obtained from the beach rock on Heron Island. The Beach Rock on Heron Island occurs on both sides of the Island lying in a semi-horizontal position slightly sloping upwards toward the Island. At normal high tide, only the upper parts are not submerged. Black crusts occur in the uppermost parts of the beachrock. Brown crusts occur in the intermediate sites and pink crusts occur at the lowest part of the beachrock.

The crusts are made up largely of cyanobacteria and the major component is a cyanobacterium of one species, tentatively identified by shape, pigmentation, and partial DNA analysis as Chroococcidiopsis.

In this investigation sections of the beach rock from different levels have been analysed using three techniques:

(1) Hyperspectral analysis to determine the layout of pigmented cells and their spectra.

(2) Bioluminescence to determine the spectra of the cells in the sections.

(3) Oxygen analysis, using luminescence lifetime imaging on special films closely applied to vertical sections of the beachrock.

(4) Oxygen production from the surface of three-dimensional blocks of beach rock, illuminated with white light or Near Infra Red (NIR) radiation, from above.

In addition, pigmentation has been analysed by High Performance Liquid Chromatography (HPLC).

These techniques allow the following conclusions:

(1) Scytonemin is a main screening compound for UV irradiation.

(2) Carotenoids also play a part in screening from UV and probably visible radiation.

(3) The cyanobacteria occur near the rock surface and contain Chl a plus some Chl f and a small amount of Chl d.

(4) HPLC pigment analysis confirms the presence of Chl a plus Chl f and a small amount of Chl d.

(5) The deeper layer with FaRLiP cyanobacteria produces oxygen under both visible light and NIR irradiation, with different P vs I curves.

(6) Using the oxygen chamber to measure oxygen exchange above the beach rock surface, it was shown that high respiration meant that only with the brown samples was significant oxygen released to the water column at lower light levels, i.e. respiration accounted for most of the primary production of oxygen except at the highest visible light intensities. And with NIR much lower levels of oxygen production only breaking compensation significantly in the brown samples.

(7) FaRLiP primary production was significant in the deeper layer.

The major new conclusion from these studies is that FaRLiP photosynthesis is a significant factor in this biofilm, and possibly other biofilms. Visible light is mostly absorbed in the upper layers and NIR reaching the lower layers induces FaRLiP photosynthesis and primary production, which can be up to 20% of the total primary production of the film.

Weaknesses:

The techniques are sufficient to justify the conclusions, especially the new result that the FaRLiP photosynthesis deeper in the films is surprisingly active with relatively high primary productivity. This is an important conclusion but it must be realised that there is some way to go to polish up the results and gain more quantitative results.

Firstly the beachrock is a heterogeneous material. So cutting a section leaves a non-homogeneous surface where various sand grains are removed, cut, or not removed. This means that when applying a luminescence film, the results are dependent on the uniformity of the surface or rather the lack of conformity. This needs to be taken into consideration in future studies.

Furthermore, previous papers have revealed that pits in the beach rock are important sites for FaRLiP cyanobacteria and the paper needs to make clear that these pits were avoided here.

Secondly, while Chroococcidiopsis is the major alga/cyanobacterium present, other algae/cyanobacteria are present and their presence needs to be factored into the results. In this regard we need more microscopic images of the surface and cross-sections of the beachrock, to reveal the nature of the bacterial and algal organisms.

Thirdly, it is not clear from this paper how far the identification of Chroococcidiopsis is firm. Presumably preliminary DNA analyses have been carried out on tell-tale genes (rRNA?). At some stage, a complete genome will be needed. Mention should be made about what has been done and what is contemplated.

Fourthly, the acclimation to FaRLiP is time-dependent. How long does it take in these beach rock sections? And has sufficient notice been taken of this time-dependent process?

Fifthly, FaRLiP is a sophisticated system as shown by Mascoli et al, 2022. It is activated in NIR by red-shifted allophycocyanin. It is also dependent on the allocation of Chl f and Chl d to special positions in the reaction centre. All this may take some time and be light-dependent. This may explain the curious increase in the slopes of light vs productivity of Fig 4 (Pink and Black) for NIR light.

The fifth point needs to be taken into account in any rewrite of the paper. The authors assume that the upwardly sloping P vs I curve is explained as follows:
"This can be explained by the light attenuation due to scattering and absorption in the compacted beachrock biofilm, which prevented saturation of NIR-driven photosynthesis in the endolithic layer even at levels of incident light similar to solar irradiation on mid-day exposed beachrock."

Activation of the FaRLiP system also needs to be considered.