Mohammad A A Al-Najjar

Modular Spectral Imaging System for Discrimination of Pigments in Cells and Microbial Communities

ABSTRACT Here we describe a spectral imaging system for minimally invasive identification, localization, and relative quantification of pigments in cells and microbial communities. The modularity of the system allows pigment detection on spatial scales ranging from the single-cell level to regions whose areas are several tens of square centimeters. For pigment identification in vivo absorption and/or autofluorescence spectra are used as the analytical signals. Along with the hardware, which is easy to transport and simple to assemble and allows rapid measurement, we describe newly developed software that allows highly sensitive and pigment-specific analyses of the hyperspectral data. We also propose and describe a number of applications of the system for microbial ecology, including identification of pigments in living cells and high-spatial-resolution imaging of pigments and the associated phototrophic groups in complex microbial communities, such as photosynthetic endolithic biofilms, microbial mats, and intertidal sediments. This system provides new possibilities for studying the role of spatial organization of microorganisms in the ecological functioning of complex benthic microbial communities or for noninvasively monitoring changes in the spatial organization and/or composition of a microbial community in response to changing environmental factors.

Light utilization efficiency in photosynthetic microbial mats

Based on combined microsensor measurements of irradiance, temperature and O(2) , we compared light energy budgets in photosynthetic microbial mats, with a special focus on the efficiency of light energy conservation by photosynthesis. The euphotic zones in the three studied mats differed in their phototrophic community structure, pigment concentrations and thickness. In all mats, < 1% of the absorbed light energy was conserved via photosynthesis at high incident irradiance, while the rest was dissipated as heat. Under light-limiting conditions, the photosynthetic efficiency reached a maximum, which varied among the studied mats between 4.5% and 16.2% and was significantly lower than the theoretical maximum of 27.7%. The maximum efficiency correlated linearly with the light attenuation coefficient and photopigment concentration in the euphotic zone. Higher photosynthetic efficiency was found in mats with a thinner and more densely populated euphotic zone. Microbial mats exhibit a lower photosynthetic efficiency compared with ecosystems with a more open canopy-like organization of photosynthetic elements, where light propagation is not hindered to the same extent by photosynthetically inactive components; such components contributed about 40-80% to light absorption in the investigated microbial mats, which is in a similar range as in oceanic planktonic systems.

Conversion and conservation of light energy in a photosynthetic microbial mat ecosystem

Here we present, to the best of our knowledge, the first balanced light energy budget for a benthic microbial mat ecosystem, and show how the budget and the spatial distribution of the local photosynthetic efficiencies within the euphotic zone depend on the absorbed irradiance (Jabs). Our approach uses microscale measurements of the rates of heat dissipation, gross photosynthesis and light absorption in the system, and a model describing light propagation and conversion in a scattering–absorbing medium. The energy budget was dominated by heat dissipation on the expense of photosynthesis: in light-limiting conditions, 95.5% of the absorbed light energy dissipated as heat and 4.5% was channeled into photosynthesis. This energy disproportionation changed in favor of heat dissipation at increasing irradiance, with >99% of the absorbed light energy being dissipated as heat and <1% used by photosynthesis at Jabs>700 μmol photon m−2 s−1 (>150 J m−2 s−1). Maximum photosynthetic efficiencies varied with depth in the euphotic zone between 0.014−0.047 O2 per photon. Owing to steep light gradients, photosynthetic efficiencies varied differently with increasing irradiances at different depths in the euphotic zone; for example, at Jabs>700 μmol photon m−2 s−1, they reached around 10% of the maximum values at depths 0−0.3 mm and progressively increased toward 100% below 0.3 mm. This study provides the base for addressing, in much more detail, the photobiology of densely populated photosynthetic systems with intense absorption and scattering. Furthermore, our analysis has promising applications in other areas of photosynthesis research, such as plant biology and biotechnology.

Community Structure and Activity of a Highly Dynamic and Nutrient-Limited Hypersaline Microbial Mat in Um Alhool Sabkha, Qatar

The Um Alhool area in Qatar is a dynamic evaporative ecosystem that receives seawater from below as it is surrounded by sand dunes. We investigated the chemical composition, the microbial activity and biodiversity of the four main layers (L1–L4) in the photosynthetic mats. Chlorophyll a (Chl a) concentration and distribution (measured by HPLC and hyperspectral imaging, respectively), the phycocyanin distribution (scanned with hyperspectral imaging), oxygenic photosynthesis (determined by microsensor), and the abundance of photosynthetic microorganisms (from 16S and 18S rRNA sequencing) decreased with depth in the euphotic layer (L1). Incident irradiance exponentially attenuated in the same zone reaching 1% at 1.7-mm depth. Proteobacteria dominated all layers of the mat (24%–42% of the identified bacteria). Anoxygenic photosynthetic bacteria (dominated by Chloroflexus) were most abundant in the third red layer of the mat (L3), evidenced by the spectral signature of Bacteriochlorophyll as well as by sequencing. The deep, black layer (L4) was dominated by sulfate reducing bacteria belonging to the Deltaproteobacteria, which were responsible for high sulfate reduction rates (measured using 35S tracer). Members of Halobacteria were the dominant Archaea in all layers of the mat (92%–97%), whereas Nematodes were the main Eukaryotes (up to 87%). Primary productivity rates of Um Alhool mat were similar to those of other hypersaline microbial mats. However, sulfate reduction rates were relatively low, indicating that oxygenic respiration contributes more to organic material degradation than sulfate reduction, because of bioturbation. Although Um Alhool hypersaline mat is a nutrient-limited ecosystem, it is interestingly dynamic and phylogenetically highly diverse. All its components work in a highly efficient and synchronized way to compensate for the lack of nutrient supply provided during regular inundation periods.

Rapid Reactivation of Cyanobacterial Photosynthesis and Migration upon Rehydration of Desiccated Marine Microbial Mats

Desiccated cyanobacterial mats are the dominant biological feature in the Earth’s arid zones. While the response of desiccated cyanobacteria to rehydration is well-documented for terrestrial systems, information about the response in marine systems is lacking. We used high temporal resolution hyperspectral imaging, liquid chromatography, pulse-amplitude fluorometry, oxygen microsensors, and confocal laser microscopy to study this response in a desiccated microbial mat from Exmouth Gulf, Australia. During the initial 15 min after rehydration chlorophyll a concentrations increased 2–5 fold and cyanobacterial photosynthesis was re-established. Although the mechanism behind this rapid increase of chlorophyll a remains unknown, we hypothesize that it involves resynthesis from a precursor stored in desiccated cyanobacteria. The subsequent phase (15 min–48 h) involved migration of the reactivated cyanobacteria toward the mat surface, which led, together with a gradual increase in chlorophyll a, to a further increase in photosynthesis. We conclude that the response involving an increase in chlorophyll a and recovery of photosynthetic activity within minutes after rehydration is common for cyanobacteria from desiccated mats of both terrestrial and marine origin. However, the response of upward migration and its triggering factor appear to be mat-specific and likely linked to other factors.

Hyper-spectral imaging of biofilm growth dynamics

Spectrally resolved imaging was applied to study the growth dynamics of phototrophic biofilms comprizing a mixture of one cyanobacterial and one diatom species. Linear spectral unmixing was combined with liquid chromatography to quantitatively discriminate the areal biomass densities of the two populations. The grown biofilms exhibited highly heterogeneous distribution with patches of 1–2mm in size, although the conditions provided for growth, including substrate roughness, illumination and flow of the overlying water, were homogeneous. The biomass was initially dominated by cyanobacteria, which exhibited an exponential-like growth phase during days 2–7. Their population declined during days 9–17, which coincided with the growth phase of the diatom population. By allowing non-invasive and real-time measurements and data evaluation, the spectral imaging approach constitutes a useful tool for microbial ecologists.