Feedback-controlled LED photobioreactor for photophysiological

studies of cyanobacteria.

JITENDRA SINGH

VERMA

INTRODUCTION

What is a Bioreactor ?• An apparatus for growing organisms

(yeast, bacteria, or animal cells) under controlled conditions.

• Used in industrial processes to produce pharmaceuticals, vaccines, or antibodies

• Also used to convert raw materials into useful byproducts such as in the bioconversion of corn into ethanol.

•Bioreactors supply a homogeneous (same throughout) environment by constantly stirring the contents.

•Bioreactors give the cells a controlled environment by ensuring the same temperature, pH, and oxygen levels.

Types of Bioreactors• Batch: Media and cells are added to the reactor and it is run

until a predetermined set point (i.e. time, concentration). The bioreactor has a constant volume (the initial volume).

• Fed-Batch: The bioreactor is a batch process in the beginning and after a certain point a feed input is introduced and the volume of the vessel increases.

• Continuous: The bioreactor starts with an initial volume and media is constantly introduced and product is constantly taken out. The inputs and outputs are at the same rate, so the volume always remains the same.

Why interest in photosynthetic microorganisms cultivation :

•Pivotal components of major ecosystems.

•Potential catalyst for sustainable production of biofuels.

The study of cyanobacteria is confounded by the properties of light (Photon flux density).

Pattern and degree of light attenuation is dependent on :▫Cell shape;▫Physiological status;▫Culture density;▫Light quality;▫Mixing properties;▫Vessel geometry.

Cultivation of photosynthetic organisms can be effectively done in turbidostats whereby relative amount of light governs the addition of fresh growth medium.

Operating modes for cultivation:

▫Batch

▫Chemostat

▫Luminostat

▫Turbididostat

Key feautures in the design of photobioreator :•Automatic adjustments of irradiance

properties;• Excellent reproducibility;• Minimization of light gradient effects;•Avoidance of over-illumination;•Rebalancing of the irradiance profile resulting

in the turbidostat control with superior responses;

•Rapid light mesurements ;•Online feedbak-control over phsiological &

photosynthetic parameters.

METHODS :1. Construction of the LED-based

photobioreactor utilizes :▫Bioflo 3000/310 fermenter platform ▫Custom made borosilicate glass vessel

and an enclosure containing built-in arrays of LEDs and light detectors

▫Immersible probes▫Sampling port was custom-modified to allow

clearance of the dead-volume after sampling by evacuation with the sparge gas.

Fig. 1 Schematic of the photobioreactor and LED light enclosure. (A) The cylindrical culture vessel is surrounded by a removable black anodized aluminum shell, the interior of which acts as a mounting-point for 32 illuminator chips and six quantum sensors. (B) The enclosure also serves as a heat sink for the LEDs and as a shield from ambient room lighting. (C) A cross-section of the photobioreactor, illustrating the equidistant arrangement of the 630 and 680 nm LED illuminator chips which provide a balanced illumination profile at surface of the culture vessel. The illuminators are also staggered longitudinally, in a similar fashion

•The aluminum enclosure has a black anodized coating to minimize reflection of stray light and acts as an efficient heat-transfer material, while the rubber seals and gaskets establish a barrier for stray light, isolating the interior from ambient room lighting.

•16 LED illuminator chips (60 LEDs on each chip) with a peak emission of 630nm, interpersed with another 16 LED chips with peak emission at 680nm.

1 LED 8% of Photons in sunlight (measured by PAR).

•630 nm for excitation of phycocyanin (cyanobacterial light harvesting equipment )

•680 nm for excitation of chlorophyll•Sensors facing the illuninators chips to

determine the incident radiance were also applied.

•Sensors for measuring light transmitted through the culture were also applied.

Irradiance measurements

• To determine the impact of a radial distribution of illuminators upon irradiance within the reactor, a correlation was established between the voltage produced by the incident light sensors hardmounted on the enclosure and the average light-intensity of a separate spherical quantum sensor .

• A custom software was developed to control the LED output and monitor optical changes in the reactor via an intermittent series of rapid light measurements for both wavelengths.

• Standard use of a polarographic dissolved O2 probe and pH sensor, the reactor head-plate was retrofitted to house an extra sensor port for an additional immersible probe (e.g., dissolved CO2, dissolved H2, redox potential).

Bacterial strains and growth conditions :• Strains : Cyanothece sp. ATCC 51142 and

Synechococcus sp. PCC 7002;

• Medium : modified ASP2 and A+.

• Controlled batch and continuous cultivations using the photobioreactor.

• Agitation speed 250rpm, 30°C, pH 7.5 &.% (2M NaOH and 2M HCl)

Analytical procedures :• Ash-free dry weight and chlorophyll content of

biomass were estimated.• O2 concentration in the off-gas was measured by MS

based gas analyzer.• Culture optical density were recorded at 730 nm

using a Thermo Spectronic Genesys 20 spectrophotometer and were used to calibrate the variable irradiance and obtain high-resolution optical data.

• Chlorophyll and phycocyanin were estimated upon baseline correction of whole-cell absorbance spectra using an exponential fit.

• Protein was quantified using BCA protein reagent.

Results and discussion.• Fig. 2 Growth of Synechococcus 7002 in LED PBR

batch cultures using different illumination strategies.

(A) Under fixed-incident irradiance (red), culture growth results in a gradual decrease of the transmitted light (pink). Using a fixed transmitted light setpoint (light green), the incident irradiance (dark green) is adjusted to increase as growth proceeds. Under these treatments, the depthaveraged light intensity in the reactor increased from 26 to 42 lmol m2 s1 in the fixed-transmitted batch, but dropped from 22 to 16 lmol m2 s1 in the fixed-incident batch.

(B) The effect of different irradiance setpoints upon batch growth rate. Auto-OD730 was obtained from calibrations with manual measurements (Supplementary Fig. S1), and is plotted on a logarithmic yaxis to emphasize changes in growth rate, as depicted by deviations from linearity. All batches were pre-grown in turbidostat mode for at least 24 h using 54 lmol m2 s1 fixed incident irradiance, and were controlled for OD730 = 0.010, which resulted in a dilution rate of 0.096 h1. All BioLume setpoints used a balanced ratio of 630:680 nm light. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig 3 (A )The feedback loop control can also be exploited to investigatethe effects of light quality. For example, an elevated ratio of 630:680 nm light leads to a higher growth rate in batch cultures of Cyanothece sp. 51142.

Fig.3(B) Changes in feedback-controlled incident irradiances over the course of an experiment also illustrate how the relative importance of phycocyanin and chlorophyll a may change over time. As expected, more 630 nm light was required to maintain the transmitted light setpoint when the ratio of 630:680 nm was high.

Fig. 3C At a lower 630:680 ratio for an equivalent transmitted setpoint, 680 nm light comprised an increasingly larger proportion of the total incident light as the experiment progressed suggesting that the abundance and/or light utilization efficiency of the chlorophyll antenna may be different from that of the phycobilisome antenna.

Chemostat operation

Fig. 4 (A) Light absorption dynamics of Synechococcus 7002 during acclimation from light-limited chemostat growth to carbon limitation (7.7 mM HCO3)

Fig 4 (B) Light absorption dynamics to nitrogof Synechococcus 7002 during acclimation from light-limited chemostat growth en limitation (0.9 mM NH4+).

Turbidostat Operation

Fig 5 (A). Dynamic response of Synechococcus 7002 turbidostat cultures during (A)down-shift from high to moderate irradiance, The start of each transition is indicated by a black arrow. Labeled irradiances represent set incident intensities for 630 and 680 nm. Cultures were constantly sparged with N2 gas containing 1.3% CO2 at 4.1 L/min and the targetOD730 for all conditions was 0.10. The media dilution rate was automatically varied using a feedback loop based on the transmitted light signal.

Fig 5 (B)Dynamic response of Synechococcus 7002 turbidostat cultures during (B) up-shift from moderate to high irradiance. The start of each transition is indicated by a black arrow. Labeled irradiances represent set incident intensities for 630 and 680 nm. Cultures were constantly sparged with N2 gas containing 1.3% CO2 at 4.1 L/min and the target OD730 for all conditions was 0.10. The media dilution rate was automatically varied using a feedback loop based on the transmitted light signal.

Photophysiological measurements

Fig. 6 A. In-situ measurement of photosynthetic rate by Cyanothece 51142 as a function of irradiance. Upon imposition of dark pre-treatment, a pre-programmed light routine cycled through a series of fixed-incident irradiances in 7 min increments. The first four settings employed an equal ratio of 630 and 680 nm light, whereas the final setting used a 630:680 ratio of 1.7, due to power limitations of the 680 nm LEDs. Throughout the experiment, media delivery and gas sparging were maintained.

Fig. 6 B. In-situ measurement of photosynthetic rate by Cyanothece 51142 as a function of irradiance.The steady-state concentrations of dO2 at each light-step were converted to net production rates by accounting for O2 removal as a first-order process empirically determined in the dark (Supplementary Fig. S3). Photosynthetic oxygen evolution was normalized to chlorophyll and plotted vs. incident irradiance. The data was fit to a hyperbolic tangent function (Jassby and Platt, 1976) (R2 = 0.99), yielding the following photosynthetic parameters: a = 1.96 ± 0.06, Pmax = 758 ± 24 lmol O2h1mg Chl1, and Ik = 386 lmol m2 s1.

Conclusion• Feedback-controlled LED photobioreactor improved the

robustness of cyanobacterial cultivation and achieved new capabilities for physiological and systems biology research.

• The system provides a well-defined growth environment under both batch and continuous operating conditions while mitigating specific challenges associated with phototrophic cultivation.

• The ability to carry out fast measurements of pigment-specific light absorption, programmable light control, and real-time monitoring has transformed this cultivation tool into an automated analytical device with non-invasive sampling.

• High level of control allows for large quantities of sample material to be obtained with low heterogeneity, using photosynthetic microorganisms grown under repeatable defined conditions.