1st Workshop
Applied Physics to PPB-based Environmental Biotechnology

Planet Earth sustains all its life forms by exploiting photosynthesis, a paramount biological process performed by plants, algae, and some bacteria. Photosynthesis is the sole biological process able to fix energy on Earth by harvesting sunlight. The planetary relevance of such metabolic process was clear to Giacomo Ciamician, one of the founders of modern photochemistry who delivered a visionary in 1912 speech [1] on the urgency of substituting fossil fuels with “The enormous quantity of energy that the Earth receives from the sun”, anticipating by more than one century what is now an almost universally shared opinion.
Two wars, the availability and handiness of fossil fuels and many skeptical world’s leaders have pushed the much-needed deadline for leaving the carbon based energy production further and further. Eventually, spurred by the vision of Ciamician, at the end of the last century the idea of artificial photosynthesis for producing sustainable energy kicked in the scientific community.
Twenty some years and many attempts later, awareness struck: complexity is the key to harness the energy from the Sun and no matter how hard you try, you cannot outdo the primary energy transducers fine-tuned by billions of years of evolution. A lesson learned the hard way, similarly to the way Pinocchio [2] learnt his after meeting the Fox and the Cat, agreed to reach the Field of Wonders in the City of Simple Simons and bury coins.
More recently, the possibility to use whole, metabolically active photosynthetic organisms in technological applications is gaining momentum in the scientific community. Results appear interesting and promising. In other words, if you can’t beat photosynthetic organisms, join them!
The recent attempts made in our laboratory to harness light and exploit it [3-7] are a useful little roadmap to employ photosynthetic bacteria in environmental applications.

In photosynthetic organisms, including purple photosynthetic bacteria, photon energy is captured by pigments in the light-harvesting antenna complexes and transferred to the photochemical reaction centers where electron transfer converts the excitation energy into energy of energy of the charge separated states. These excitation energy and electron transfer processes occur on ultrafast timescales – from femtoseconds to picoseconds. The efficiency of energy and electron transfer is crucial for the overall efficiency of photosynthesis and depends on the protein matrix and the external environment. Ultrafast optical spectroscopy techniques are essential to study the efficiency, dynamics, the intermediate steps and kinetic bottlenecks of the energy and electron transfer processes in photosynthetic systems. These techniques use short laser pulses to excite the sample and measure the time-dependent changes in the absorption and emission spectra. During the past two decades, two-dimensional electronic spectroscopy (2DES) has emerged as a powerful technique to study energy and electron transfer in photosynthetic systems. It provides a detailed map of the energy transfer pathways and can reveal the dynamics of energy transfer on femtosecond to picosecond timescales. 2DES has been used to study a variety of photosynthetic systems, from isolated pigments, antenna and reaction center complexes to living cells of photosynthetic bacteria. By measuring the interactions between different pigments in the photosynthetic system, this technique can reveal the mechanisms of energy transfer and provide insight into the factors that affect the efficiency of photosynthesis.

In photosynthetic organisms, including purple photosynthetic bacteria, photon energy is captured by pigments in the light-harvesting antenna complexes and transferred to the photochemical reaction centers where electron transfer converts the excitation energy into energy of energy of the charge separated states. These excitation energy and electron transfer processes occur on ultrafast timescales – from femtoseconds to picoseconds. The efficiency of energy and electron transfer is crucial for the overall efficiency of photosynthesis and depends on the protein matrix and the external environment. Ultrafast optical spectroscopy techniques are essential to study the efficiency, dynamics, the intermediate steps and kinetic bottlenecks of the energy and electron transfer processes in photosynthetic systems. These techniques use short laser pulses to excite the sample and measure the time-dependent changes in the absorption and emission spectra. During the past two decades, two-dimensional electronic spectroscopy (2DES) has emerged as a powerful technique to study energy and electron transfer in photosynthetic systems. It provides a detailed map of the energy transfer pathways and can reveal the dynamics of energy transfer on femtosecond to picosecond timescales. 2DES has been used to study a variety of photosynthetic systems, from isolated pigments, antenna and reaction center complexes to living cells of photosynthetic bacteria. By measuring the interactions between different pigments in the photosynthetic system, this technique can reveal the mechanisms of energy transfer and provide insight into the factors that affect the efficiency of photosynthesis.

Bacterium Sphingomonas glacialis AAP5 isolated from the alpine lake Gossenköllesee contains genes for anoxygenic phototrophy as well as proton-pumping xanthorhodopsin. However, these genes are not expressed in standard laboratory conditions. In order to find under which conditions the organisms expresses its light harvesting apparatus we conducted a larger investigation employing RNA sequen-cing, RTqPCR, metabolic assays and biochemical and biophysical investigation of its photosynthetic complexes. We found out that our strain readily express xanthorhodopsin when illuminated at tempera-tures below 14°C. In contrast bacteriochlorophyll-containing reaction centers are expressed between 4 and 23°C in the dark. Thus, cells grown at low temperature under natural light-dark cycle produced both photosystems (Fig. 1). The photosynthetic complexes consist of the type-2 reaction center surrounded by circular light-harvesting complex 1. The purified xanthorhodopsin contains carotenoid nostoxanthin serving as an auxiliary antenna and performs the standard photocycle. The xantho-rhodopsin-producing cells reduced upon illumination their respiration by 70%. This documents that the harvested light energy was utilized in the metabolism, which can represent a large benefit under carbon-limiting conditions.
The presence of two different photosystems may represent a metabolic advantage in alpine lakes where photoheterotrophic organisms face large changes in irradiance, limited organic substrates and low temperature.

Purple phototrophic bacteria (PPB) have gained importance in the biological treatment of wastewater and other streams due to their ability to recover C, N, and P through photoheterotrophic metabolism. Their light-harvesting complexes LHC2 and LHC1 transform the near-infrared (NIR) light into chemical energy in the reaction centers, which are composed of bacteriochlorophylls (BChl) and different carotenoids. However, their phototrophy is negatively affected in the presence of oxygen due to the oxidation of BChl, losing their photosynthetic machinery and causing displacement by aerobic bacteria in non-axenic conditions.

This work aims to evaluate the effect of oxygen on the photosynthetic capacity of a mixed culture of PPB (bleaching) and the recovery of these after subjecting them to anaerobic conditions (purpling). The evaluation is performed by directly analyzing the biochemical system response and through AFM to visualize the LHC structure loss and regain depending on dissolved oxygen (DO) concentrations.

We deal with the exciton transfer between light-harvesting complex 1(LH1) and reaction centres (RC) dimer. We show that the RC dimer does not act as a photo trap. The backward exciton rate constant from the dimer to the LH1 antenna complex depends on the number of chlorophyll molecules which create the LH1 ring complex. The electron transfer in PPB reaction centres is directed by the vibration modes into the needed site where the electron is localized. In the case of the PPB RC, the low-vibration modes produce such an effect.

The light-harvesting complexes incorporate pigment-protein systems that absorb light through pigments such as (Bacterio)chlorophylls, carotenoids, and bilins. Ultrafast spectroscopy has helped us learn more about energy transfer in photosynthesis and many antenna proteins have been studied. Most of these studies have excited the lowest-absorbing states of pigments, such as the Qy states of (B)Chl or the S2 state of carotenoids. The upper excited states, like the Soret or Qx bands of (B)Chl, also contribute to light harvesting. However, we have limited knowledge about how excess energy excitation affects the process. Here we would like to address the relaxation dynamics of upper excited state and the impact of excess energy dissipation on the system. The LH2 antenna complex of Rhodoblastus acidophilus has been chosen as a model system.
The study presents data comparison of the excited state dynamics of pigments in LH2 after excitation of either the Soret or B800 band of BChl a, and the S2 state of the carotenoid (rhodopin glucoside). There are weak signals in the spectral region of carotenoid absorption bands after BChl a excitation. These confirms that there is no energy transfer from the BChl a Soret band to the S2 state of rhodopin glucoside in LH2. The possible explanation is an unfavorable mutual orientation of the transition dipoles that are nearly orthogonal. The rich signal in the 450–650 nm spectral region after BChl a excitation is likely due to an electrochromic shift of the carotenoid S2 state resulting from a change in local electric field upon BChl a excitation.
The Soret band of BChl a decays to the Qy state within 200 fs and excess energy is stored in vibrational modes of the Qy state. The carotenoid nearby responds to this energy dissipation, indicating the cooling of the BChl a Qy state. We also found a sub-nanosecond triplet–triplet energy transfer between BChl a and carotenoid rhodopin glucoside, faster than singlet–triplet intersystem crossing. These findings have been summarized in a new model of energy flow in LH2 complex (see Figure).

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Purple phototrophic bacteria are a group of photosynthetic bacteria that use light energy to drive metabolic processes, making them promising candidates for sustainable biotechnological applications. They are metabolically diverse microorganisms with the ability to grow either photo- and chemo-heteroreophically.
Mechanistic models have been developed for PPB growth for further operation and control design. To overcome costly implementation of anaerobic implementation of PPB reactors, a mechanistic model for raceway reactors as an alternative has been developed. A metabolic switch among photo-, aerobic chemo- and anaerobic chemo- heterotrophic growth has been taken into account in the model. The idea is to consider photoheterotrophic biomass grown in aerobic and anaerobic chemoheterotrophic conversions and vice versa. Dependent PPB metabolic division of labor has been experimentally supported. The presentation aims to open up the discussion on the importance of a deeper level of mechanistical understanding of the associated biophysical and bioenergetics mechanisms influencing the metabolic pathways in PPB.

The Bose lab studies microbial metabolisms and their influence on biogeochemical cycling using a transdisciplinary approach. We apply the knowledge we gain to generate new ways of addressing issues such climate change, sustainability, and the circular economy. My lab’s recent work has focused on the ability of microbes to use solid-phase conductive materials as electron donors. The ability to use electrons from minerals and other solid-phase conductive materials (in essence using electricity) is called “Extracellular Electron Uptake” or (EEU). EEU fundamentally changes our perception of the ecological role of microbes (including phototrophs) in nature because it suggests that abundant elements (such as iron) can serve as electron sources for microbial productivity and survival. In addition, our work suggests that EEU might be a fundamental process that underlies microbial energy conservation ranging from terrestrial and aquatic ecosystems to the human body. The Bose lab has pioneered technologies to study EEU, driven fundamental knowledge in the field, and laid a solid foundation of tools and approaches to answer key questions about EEU in nature. We have applied this knowledge to produce carbon-negative bioplastics and carbon-neutral biofuels. Currently, we are pursuing fundamental and applied research on phototrophic EEU, and determining its role in microbial ecology in marine and freshwater ecosystems. We are leveraging this knowledge to develop a variety of climate technologies for the circular economy.

Fine tuning the metabolism in Purple Phototrophic Bacteria: the use of planktonic electrochemistry

Material surfaces can promote or inhibit microbial adhesion, depending on material and microbial surface characteristics. In my presentation I will present our recent experiments on different technological and medical relevant surfaces [1-5]. The surface roughness was controlled by atomic force microscope and profilometer. The zeta potential and contact angles were measured correspondingly by the tensiometer and kinetic analyzer. The rate of adhered bacteria on different type of surfaces was determined with spectrophotometer and scanning electron microscopy. The factors which impact the microbial adhesion to material surfaces will be discussed. At the end, the outlook will be given.

Purple phototrophic bacteria (PPB) are known due to their metabolic versatility. Thus, they could be exploited for the production of added value products such as H2 and PHB, through the electron interchange with an electrode (Manchon et al., 2023; Vasiliadou et al., 2018a).
Regarding PHB production, microbial electrosynthesis (MES) has been reported to be a suitable strategy under photoautotrophic conditions (Ranaivoarisoa et al., 2019). In this work, a mixed PPB culture enriched from brewery wastewater was grown under photoheterotrophic conditions with two different electrode configurations as electron donor: i) conventional graphite rod and ii) moving bed electrode (vitreous carbon). Both bioreactors were polarized at -0,8 V vs. Ag/AgCl to evaluate i) PHB production and ii) bioelectrochemical performance. In both bioelectrochemical reactors there was a significant increase in PHB production (figure 1) when PPB were grown in presence of an electrode as electron donor. Electrode-free and open circuit configurations were used as control.
Overall, the polarized reactors outperformed the non-polarized ones for PHB production with a similar carbon source consumption. In addition, the highest PHB production was achieved in presence of vitreous carbon as electron donor. Even though a low current consumption was shown for the graphite rod, the polarization did have an effect in electrofermentation (Virdis et al., 2022) as a redox potential controller in comparison with electrode-free control.

Climate change, water scarcity and soil degradation threaten global food security and may deplete natural resources by 2050. Pig farming generates a significant amount of manure, around 1.3-1.8 billion tons annually in the European Union, containing high nitrogen concentrations that contribute to pollution and greenhouse gas emissions. Current animal-based protein production for human consumption is unsustainable and inefficient, requiring a shift towards more sustainable protein production methods [1]. This study proposes photo-microbial electrosynthesis as a versatile solution for both manure treatment and sustainable protein production.
Purple phototrophic bacteria (PPB) are highly versatile microorganisms that have recently been proposed as a promising alternative source of protein. They perform anoxygenic photosynthesis using a wide range of soluble electron donors, and some can even accept electrons from solid phases such as minerals and graphite electrodes. The mechanism used for electron uptake from a cathode electrode is direct extracellular electron transfer (DEET), which is compatible with carbon fixation via the Calvin-Benson cycle. In electro-autotrophic conditions, PPB can achieve up to 60% protein yield, making them a potential alternative source of protein with an amino acid profile well suited for human consumption. [2,3,4].
The VALPIG4FOOD project proposes a novel photo-electro-biorefinery to treat pig manure and convert it into microbial protein and biomethane (Fig. 1). This project aims to increase the sustainability of pig manure treatment by avoiding the dissipation of nutrients into the soil and atmosphere and reducing carbon emissions to zero. The proposed biorefinery recovers the ammonium from hydrolyzed pig manure and the biogas generated in the anaerobic digestion to feed a photo-microbial electrosynthesis (photo-MES) system to upgrade biogas to biomethane and produce PPB protein for human food consumption [5,6].

Demand on petroleum derived materials constitute a bottleneck in transitioning from fossil fuel-based energy economy to more sustainable methods of energy production [1]. A major product of crude oil processing is commodity plastics [2]. Poly-hydroxybutyrate (PHB) is a compound synthesized by microbial species, with properties being similar to the aforementioned plastics [3]. PHB can be produced in abundance via purple non-sulfur bacteria (PNSB) [4]. The aim of this study is to investigate the effect of various stress conditions in the form of elevated sodium (Na+) concentration and pH levels on hydrogen and PHB production via photofermentation using uptake hydrogenase deficient Rhodobacter capsulatus YO3. To this purpose, two experimental designs were set up (Fig.1). In Set 1, effect of increasing Na+ concentration was investigated, using two different substrate types, acetate and sucrose. As a result of Na+ increase, elevated pH was observed to be a significant stress in acetate-containing reactors. Maximum PHB accumulation was 57.7 ± 5.2% and 3.3 ± 0.4% of cell dry weight (cdw) for acetate and sucrose, respectively. Maximum hydrogen productivities were 0.58 ± 0.03 mmol/L·h and 0.73 ± 0.06 mmol/L·h, respectively for acetate and sucrose. As pH contributed significantly to PHB accumulation, Set 2 was set up using acetate as the carbon source with three different pH levels, namely 7.0, 7.7 and 8.5, that were kept constant throughout the operation. In the 1st phase of reactor operation, Na+ concentration was kept at 1750 mgNa+/L, followed by around 3500 mgNa+/L. Maximum PHB accumulation was 57.5 ± 0.2% of cdw at 8.5 pH and 33.6 ± 2.0% of cdw at 7.7 pH for low and high Na+ conditions. Maximum hydrogen productivity was observed as 0.42 ± 0.01 mmol/L·h at pH 7.0.
It was concluded that sole Na+ or sole pH do not contribute significantly to PHB accumulation, rather elevated pH and Na+ concentration should be applied simultaneously to enhance accumulation of PHB. It was also observed that sucrose-containing reactors produced minimal amounts of PHB with higher H2 productivity, indicating that the system should be operated under more stressful conditions to favor the production of PHB over H2.