Optimization of the in-situ growth conditions based on a novel photo-microcalorimeter for the sustainable cultivation of photosynthetic microorganisms

Despite the great potential of photosynthetic microbes in the production of renewable fuels, value-adding chemicals, and water treatment, etc., commercial utilization of them is significantly hindered by the lack of techniques to accurately monitor the thermodynamic and kinetic characteristics of the in-situ growth of microbes under controlled light illumination for optimal cultivation. Herein, we demonstrated that a newly developed highly sensitive photo-microcalorimetric system successfully captured the impacts of the light wavelength and strength on the thermodynamic and kinetic parameters of the in-situ growth of Rhodopseudomonas palustris, a representative photosynthetic microorganism. To our best knowledge, this is the first time that highly precise microcalorimetry is employed to monitor exam the in-situ growth of photosynthetic microorganisms under controllable photo illumination. We envision this technique can help for the optimization of the growth conditions of photosynthetic microorganisms for scale-up applications. Abbreviation P: heat output


Introduction
Photosynthetic bacteria are strains of distinct microorganisms that can use their light-absorbing pigments to convert solar energy into chemical energy. For example, in the most important natural processes, photosynthesis, cyanobacteria, and microalgae can use carbon dioxide and water to produce sugar with the help of sunlight. Traditionally, photosynthetic microorganisms have been cultivated for food and feed supplements (Linder, 2019). Photosynthetic bacteria are well-acknowledged as an important source of alternative energy based on their application in the mass production of biofuel (Tiwari et al., 2018) and hydrogen (Lu et al., 2016). Photosynthetic microorganisms have been utilized in the areas of environmental bioremediation (Idi et al., 2015), wastewater treatment (Lu et al., 2019;Talaiekhozani and Rezania, 2017), desalination (Amezaga et al., 2014;Kokabian and Gude, 2015), production of valueadding chemicals (Milano et al., 2019;Zhou et al., 2015), bioenergy (Liu and Choi, 2017;Sekar and Ramasamy, 2015), agriculture (De Godos et al., 2010;Wei et al., 2016) as well as pharmaceutical industry (Jeffryes et al., 2015;Sakaki et al., 2005).
It is believed that photosynthetic microorganisms will have an important impact on the future of human beings (Mazard et al., 2016). The key to the successful commercial application of photosynthetic bacteria is the optimization of the cultivation process of the organisms (Fernandes et al., 2015). Since light illumination is the source of energy for photosynthetic bacteria (Benemann et al., 1973;Donia et al., 2011), it is necessary to investigate the detailed influence of different illumination conditions on the growth of the photosynthetic microorganisms.
Due to the lack of techniques for online measurements, the research on the thermodynamic and kinetic of the growth of photosynthetic microorganisms under light illumination is still limited. To our best knowledge, there is no literature reporting the in-situ simultaneous quantification of the thermodynamics and kinetics during the cultivation process of these bacteria. Studies on the effects of illumination conditions on the growth of photosynthetic bacteria have mainly relied on intermittent sampling rather than dynamic monitoring due to the absence of tools to precisely regulate light wavelengths and intensities. Carlozzi et al. (2006) demonstrated that different wavelengths had distinct effects on the growth of photosynthetic bacteria during the alternating dark-light cycles. Benemann et al. (1973) showed that extremely high and low light intensities were unfavorable for the growth of photosynthetic bacteria by measuring the optical density (OD) of bacterial cultures at various time points during growth. Wakayama et al. (2000) reported high hydrogen yields (22 L/m 2 ·d) from photosynthetic bacteria grown under 30-min dark-light cycles by OD measured. Xu et al. (1995) used calorimetry to show that the growth kinetics of photosynthetic bacteria followed the Malthusian equation. However, the rate constant (k) was calculated based only on the effect of temperature without considering synergistic photothermal effects. Therefore, it is desired to carry out in-situ studies that could reveal the association between thermodynamics and kinetics in photosynthetic bacteria.
Photocalorimetry is an in-situ, real-time, non-invasive, direct, and precise method for measuring thermodynamic parameters during biological and photochemical reactions. The principle of photocalorimetry was first described by Magee et al. (1939), who measured the efficiency of photosynthesis by Chlorella vulgaris as thermal changes per unit time. Since then, researchers have invested a great amount of effort in improving the experimental precision and sensitivity of this technique (Adamson et al., 1978;Dhuna et al., 2007;Dhuna et al., 2008;Gordon et al., 1981;Mukhanov and Kemp, 2009;Olmsted Iii, 1979;Schaarschmidt and Lamprecht, 1973;Seybold et al., 1969;Teixeira and Wadsö, 1990). In a novel photo-microcalorimetric system developed by our group (Fan and Huang, 2011;Wang et al., 2011), a monochromatic light source of a specific wavelength (532 nm) was introduced into the RD496-CK 2000 microcalorimeter through an optical fiber. This design allows for the accurate detection of changes in thermal power and energy without additional requirement on the solvent, spectral, or electrical property of the system under investigation. In addition, the device can precisely regulate the microenvironment of the reaction system by maintaining a temperature of 10 −4°C to 10 −5°C . Using this device, we have reported results of studies on the in-situ growth of nanomaterials as well as photocatalytic studies (Guo et al., 2012;Huang et al., 2015).
In the present study, we used the photo-microcalorimetric system developed by our group to monitor the in-situ growth processes of a representative photosynthetic bacteria. The thermodynamic characteristics and patterns of bacterial growth under various conditions were extracted from the thermograms collected on this unique photo-microcalorimeter. The results provide a theoretical foundation for a better understanding of the in-situ growth mechanisms, as well as the optimized growth conditions of the photosynthetic microorganism.

Materials and Methods
The photosynthetic microorganism used in this study was Rhodopseudomonas (R.) palustris, CICC 2312, which was purchased from the China Center of Industrial Culture Collection (Beijing, China). The strains were facultative bacteria grown in CM0847 culture media (15.0 g peptone, 5.0 g soya peptone, 5.0 g NaCl, 13.0 g agar, and 1.0 L distilled water), which was adjusted to pH 7.3 ± 0.2 and sterilized at 121°C for 30 min prior to use.
Experiments were conducted using a DHG Series Heating and Drying Oven, pH meter, electronic balance, autoclave (TOMY, Tokyo, Japan), UV-Vis spectrophotometer (model needed), ultraclean bench, PGX-270B programmable multiwavelength illumination incubator (Ningbo Southeast Instrument Co. Ltd., Ningbo, China), incandescent lamp, and light-emitting diodes. A novel photoreaction-microcalorimetric system was constructed by combining an RD 496-CK 2000 microcalorimeter (Mianyang Zhongwu Thermal Instrument Co. Ltd., Sichuan, China), a 532 nm light source and optical fiber (Nanjing Chunhui Science and Technology Industrial Co. Ltd., Nanjing, China), and a solar power light intensity meter (TES1333, TES Electrical Electronic Corp., Taipei) (Fig. 1a). A detailed description of this novel instrument can be found in our previous report .

Experimental methods
Determination of culture concentrations Thawed bacteria were cultured and passaged for three generations. The bacteria were cultured with CM0847 culture media at pH 7.3 for 4 days at 30°C and did not require strict deoxygenation. The resulting culture was used as the bacterial stock solution. Cell counts for photosynthetic bacteria were determined by manual counting with a Petroff-Hausser counting chamber. The concentration standard curve was prepared by plotting OD values of bacterial culture against the number of bacterial cells on blood agar plates (Fig. 1b). The OD value was obtained by measuring at 660 nm on the UV spectrometer. Perform zero adjustments with culture medium and read 5 times for each measurement.
Effect of light source on growth of R. palustris R. palustris was cultured under blue light (400−520 nm), green light (520−570 nm), red light (620−700 nm), and white light (incandescent light composed of mixed light spectrums) at an intensity of 4 W/m 2 for 84 h, respectively. Aliquots of samples were collected every 12 h for OD measurements at 660 nm on the UV-Vis spectrometer.
Effect of light intensity on growth of R. palustris R. palustris was cultured under the optimal light source, as determined in the experiment as described above, at 1, 2, 4, 6, and 8 W/m 2 for 84 h. Aliquots of samples were collected every 12 h for OD measurements at 660 nm.

Determination of thermodynamic parameters during growth of R. palustris
The scheme of the photoreaction-microcalorimetric system for the microcalorimetry experiments is shown in Fig. 1a. In a typical test, blank media and R. palustris liquid culture (1.5 mL) were added to the reference cell and sample cell, respectively. The OD value of the bacterial culture was measured prior to each microcalorimetric measurement, and the bacterial count was adjusted to approximately 1.96 × 10 8 cells/mL based on a standard curve (Fig. 1b). The temperature of the system was set to 30°C, and the 532 nm light source was turned on after the baseline was stabilized. The in-situ growth of R. palustris was monitored at intensities of 2, 4, and 6 W/m 2 for a duration of 290,000 s.

Results
Effect of wavelength and light intensity on R. palustris growth Using a single-factor experimental design, the OD of bacterial cultures grown at four different wavelengths was plotted over time (Fig. 2a). It is evident that the growth rate of the photosynthetic microorganism is affected by the wavelength, and it follows the order of green light > blue light > white light ≅ red light. Therefore, green light (520−570 nm) is the optimal wavelength range for the growth of R. palustris. Cultures were then grown at green light, and the OD of bacterial cultures grown under five different light intensities were plotted over time (Fig. 2b). The results suggested that the fastest growth rate of R. palustris was achieved after 12 h of culture under a light intensity of 4 W/m 2 , followed by 6 W/m 2 , and were lowest under a light intensity of 2 W/m 2 · OD of the bacterial cultures at the end of the experiment was highest at a light intensity of 4 W/m 2 (0.665), followed by 6 W/m 2 (0.649) and lowest at 2 W/m 2 (0.182).
Construction of R. palustris thermograms using the novel photoreaction-microcalorimetric system The power-time (p-t) curve of the in-situ growth of R. palustris in the photoreaction-microcalorimetric system, set at 30°C under 523 nm light at an intensity of 2 W/m 2 , is shown in Fig. 3a and Tab. 1. As indicated by the p-t curve, the growth of the photosynthetic bacteria included four phases: The lag phase (I), log phase (II), stationary phase (III), and decline phase (IV). The average k of R. palustris from five replicate experiments was 1.191 × 10 −4 ± 7.071 × 10 −8 min −1 with a correlation coefficient R > 0.9975, indicating that the results were highly reproducible and correlated.

Thermograms of R. palustris growth
The thermograms of R. palustris cultured in the photoreactionmicrocalorimetric system under 532 nm illumination at light intensities of 2 W/m 2 (A-E), 4 W/m 2 (A'-E'), and 6 W/m 2 (A"-E") during the lag (AB), log (BC), stationary (CD), and decline (DE) phases are given in Fig. 4. Upon baseline stabilization at t = 2000 s and during the entire lag phase (AB), bacterial growth was slow, and heat release was limited (1.048 J). At t = 5791 s (B), heat release accelerated and peaked at P max = 48.19 μW. This was followed by slow heat release throughout the stationary phase of stable growth (CD). Heat production declined substantially from t = 20,868 s until the end of the experiment.
Energy changes in R. palustris during different growth phases Heat production by R. palustris as a result of growth and metabolism was significantly different under the illumination of green light with three different light intensities (P < 0.001). During the lag phase, heat production was highest at 2 W/m 2 , followed by 4 W/m 2 , and lowest at 6 W/m 2 . During the log, stationary, and decline phases, heat production was highest at 4 W/m 2 , followed by 6 W/m 2 , and lowest at 2 W/m 2 (Fig. 5a, Tab. 2).
Thermodynamic parameters of R. palustris during the log phase The growth rate, maximum heat production, peak time, and generation time of R. palustris are summarized in Fig. 5 and Tab. 3. Growth rate constant k (s −1 ) was greatest under a light intensity of 2 W/m 2 , followed by 4 W/m 2 , and lowest at 6 W/m 2 . The maximum heat production was greatest under a light intensity of 4 W/m 2 , followed by 6 W/m 2 , and lowest at 2 W/m 2 . The peak time was greatest under a light intensity of 6 W/m 2 , followed by 2 W/m 2 , and shortest at 4 W/m 2 . Generation time was longest under a light intensity of 2 W/m 2 , followed by 4 W/m 2 , and shortest at 6 W/m 2 . Although the growth rate at 2 W/m 2 was greater than at 4 or 6 W/m 2 , the maximum heat production at 2 W/m 2 was delayed by 10,283 s compared to 4 W/m 2 .
Under a light intensity of 2 W/m 2 , the decline phase was the longest growth phase, followed by the lag phase, the   stationary phase, and the log phase. Under a light intensity of 4 W/m 2 , the decline phase was the longest growth phase, followed by the log phase, the stationary phase, and the lag phase. Under a light intensity of 6 W/m 2 , the lag phase was the longest growth phase, followed by the decline phase, the log phase, and the stationary phase. The lag phase was longest under a light intensity of 2 W/m 2 , followed by 6 W/m 2 , and shortest under 4 W/m 2 . The log phase was longest under a light intensity of 4 W/m 2 , followed by 6 W/m 2 , and shortest under 2 W/m 2 . R. palustris cultured under a light intensity of 4 W/m 2 started the earliest, spent the longest amount of time in the log phase, and had the highest growth efficiency. Heat release by R. palustris intensities during the lag phase was highest under a light intensity of 4 W/m 2 , followed by 6 W/m 2 , and was lowest under a light intensity of 2 W/m 2 .

Effect of wavelength and light intensity on R. palustris growth and metabolism
It is important to identify the optimal wavelength and intensity of the light to achieve the desired bacterial growth. In the present study, single-factor experiments assessing different wavelengths showed that growth of R. palustris was highest under green light (520−570 nm), followed by blue light, white light, and red light (Fig. 2a). The optimal range of light intensities for the growth of R. palustris was 2-6 W/m 2 , and growth performance was not enhanced by green light with higher intensities. The cultures reached their maximum OD (1.56) 24 h after inoculation under a light intensity of 4 W/m 2 . The growth of the bacteria was reduced under light intensities >6 W/m 2 , suggesting that there is a certain threshold value of the light intensity above FIGURE 5. Heat production changes in R. palustris during different growth phases when cultured under 532 nm light at various intensities. (a) Change in heat production of R. palustris at light intensities of 2, 4, and 6 W/m 2 . Changes in heat production and growth phase duration at light intensities of (b) 2 W/m 2 , (c) 4 W/m 2 , and (d) 6 W/m 2 . *P < 0.05; **P < 0.01; ***P < 0.001. which the growth of R. palustris was not significantly promoted (Fig. 2b). In fact, high-intensity light may even reduce bacterial growth due to overexcitation of photosystem I by excess energy absorbed by photosynthetic organs, also known as the reports of (Ichii et al., 2003;Luque and Clark, 2010;Melis, 2012;Zhang et al., 2016). In the present study, the light saturation effect was apparent when the light intensity was increased to 8 W/m 2 . However, the OD values of R. palustris cultured under various light intensities were not significantly different during the four growth phases, indicating that the effect of illumination on bacterial growth is complex. These results will contribute to the development of a theoretical explanation of the mechanisms underlying reproductive and metabolic processes in bacteria.
Although the single-factor experiments showed that green light with intensities between 2 and 6 W/m 2 was optimal for R. palustris growth, it did not provide information on in-situ growth under a single wavelength at specific light intensities. With the addition of experiments that screen a wide range of factors, integration of photocalorimetry allows for the examination of subtle energy changes during bacterial growth. In the present study, thermograms and relevant parameters of R. palustris were determined using a highly precise and sensitive photoreaction-microcalorimetric system. Because the bacterial cells in the log phase are viable and consistent in size, they are often used in research or as "seeds" in large-scale production (Pommerville, 2012). Metabolic and enzymatic activities of R. palustris are also high and stable during the log phase. In the context of production, the fermentation efficiency of bacteria is often increased by prolonging the log phase. Both cell number and heat production increase exponentially during the log phase, as shown by Eq. (1).
where P t is heat output at time t in the log phase, P 0 is heat output at t = 0, k is the growth rate constant, and t is the duration of the experiment (Huang et al., 1998). Since the log phase of each p-t curve was strongly correlated with Eq.
(1), a series of P t and t values from the log phase were assessed to find the best fit for Eq. (1), and k and growth kinetics equations were then established. Generation time, t G , was determined based on equation t G = ln 2/k, and the maximum heat production P max and peak time t max (corresponding to maximum heat production) were obtained from the p-t curve. In the present study, R. palustris had the fastest growth rate but smallest P max when cultured under a light intensity of 2 W/m 2 , indicating that this amount of light was insufficient to activate photosynthesis and that bacteria were unable to obtain enough energy (Idi et al., 2015;Ormerod et al., 1961). Despite the rapid growth of R. palustris during the log phase, when generation time was the shortest, and growth rate was the highest, energy release was not at its maximum. Therefore, a light intensity of 2 W/m 2 is not optimal for the growth of R. palustris.
In the lag phase, bacteria accumulate energy in order to undergo rapid growth and proliferation during the log phase once the energy level reaches a threshold level. This could explain why R. palustris cultured under a light intensity of 2 W/m 2 had the highest growth rate but the shortest generation time. Additionally, although the growth rate of R. palustris was reduced to 7.652 × 10 −5 s −1 and the generation time was extended to 151.01 min under a light intensity of 4 W/m 2 , maximum heat output (48.19 μW) was much higher at 2 W/m 2 . These results are consistent with the fact that the log phase started earlier under light intensities of 2 and 6 W/m 2 in the single-factor experiment.
Interestingly, it was found that growth rate, generation time, and peak time of R. palustris during the log phase, and heat release during the stationary and decline phases, were lower under a light intensity of 6 W/m 2 compared to 4 W/m 2 (Fig. 5a). These results suggested that 6 W/m 2 was also not the optimal light intensity for R. palustris growth. Overall, our data demonstrated that light intensities above or below 4 W/m 2 are not optimal for R. palustris growth. The growth of R. palustris was dependent on a combination of optimal conditions and was not simply proportional to light intensity. This conclusion is in good agreement with established knowledge of photosynthesis bacteria, proving that the information collected on the novel photo-calorimeter reported in the present study. This system can precisely collect the in-situ, real-time, and uninterrupted thermodynamic parameters of the growth process of the photosynthesis bacteria, therefore providing accurate information needed to control the growth of the photosynthesis microbes. To our best knowledge, the application of photocalorimetry for this purpose has not been reported.
R. palustris growth characteristcs Metabolic processes are influenced by changes in the environment, and this can lead to significant changes in the p-t curve of the bacteria (Moulisová et al., 2009). Photocalorimetry can reveal details of bacterial growth and metabolism that are undetectable by other methods. Therefore, this novel method is extremely useful for elucidating the growth mechanisms of bacteria, as it provides information on the thermodynamics, kinetic patterns, and characteristics at different stages of growth. The result also was similar to Sardaro et al. (2013), who presented a calorimetric investigation of R. sphaeroides life cycle by two different experimental procedures based on the use of a static ampoule and a flow cell, respectively (Sardaro et al., 2013). Although photo-microcalorimeter technology provides accurate in-situ thermodynamic information about the growth process of bacteria, during the growth process, each process of bacterial growth does not completely match with the increase of light intensity. Maybe it is related to the photosynthetic system, even because the dimension of the light-harvesting apparatus and with the physical dimension of the cells is possible.

Conclusions
Although photosynthetic microorganisms are suggested to have great applications in the green production of alternative energy and value-adding products as well as for bioremediation of the environment, the industrial cultivation of these important microbes has received limited success partially due to the lack of a precise online measurement tool which can monitor the in-situ growth of them. In the present study, we have demonstrated the integration of light source with microcalorimeter provided the unique opportunity to investigate the effect of light illumination conditions on the in-situ growth of the photosynthetic microorganisms. The results clearly proved that the photo-microcalorimeter designed in our project can conveniently monitor the processes of bacterial growth and metabolism under light with a desired wavelength and intensity and generate the desired thermodynamic and kinetic parameters of the in-situ growth of the photosynthetic microbes. This novel technique can detect tiny differences in the in-situ bacterial growth under different growth conditions that cannot be detected by conventional analytical methods. Thus, it is envisioned that the photo-microcalorimeter will become a powerful tool to help the optimization of the industrial cultivation of photosynthetic microorganisms.
Availability of Data and Materials: The datasets used or analysed during the current study are available from the corresponding author on reasonable request.