Improvement of mixing time,mass transfer,and power consumption in an external loop airlift photobioreactor for microalgae cultures

Longer mixing times and higher power consumption are common problems in the design of photobioreactors. In this study, a vertical triangular external airlift loop photobioreactor was designed, constructed and operated for microalgae production studies. Gas feeding was performed by two spargers: one at the bottom of the hypotenuse (downcomer) and another at the bottom of the vertical side (riser). This configuration provided more effective countercurrent liquid–gas flow in the hypotenuse. The mass transfer coefficient, gas hold-up, mixing time, circulation time, dimensionless mixing time, bubble size, and volumetric power consumption were measured and optimized using response surface methodology. Investigations were carried out on the performance of the riser (the vertical side), downcomer (the hypotenuse), and separator. The countercurrent flow in the hypotenuse provided sufficient contact between gas and liquid phases, and increased mixing and mass transfer rates, in contrast to the results of previous studies. The promising results of this geometry were shorter mixing time and a significant decrease in volumetric power consumption in comparison with other configurations for photobioreactors.     

Hydrodynamics and mass transfer in a tubular airlift photobioreactor

In photobioreactors, which are usually operated under light limitation,sufficient dissolved inorganic carbon must be provided to avoid carbonlimitation. Efficient mass transfer of CO₂ into the culture mediumisdesirable since undissolved CO₂ is lost by outgassing. Mass transferof O₂ out of the system is also an important consideration, due tothe need to remove photosynthetically-derived O₂ before it reachesinhibitory concentrations. Hydrodynamics (mixing characteristics) are afunctionof reactor geometry and operating conditions (e.g. gas and liquid flow rates),and are a principal determinant of the light regime experienced by the culture.This in turn affects photosynthetic efficiency, productivity, and cellcomposition. This paper describes the mass transfer and hydrodynamics within anear-horizontal tubular photobioreactor. The volume, shape and velocity ofbubbles, gas hold-up, liquid velocity, slip velocity, axial dispersion,Reynoldsnumber, mixing time, and mass transfer coefficients were determined intapwater,seawater, and algal culture medium. Gas hold-up values resembled those ofvertical bubble columns, and the hydraulic regime could be characterized asplug-flow with medium dispersion. The maximum oxygen mass transfer coefficientis approximately 7 h^–1. A regime analysisindicated that there are mass transfer limitations in this type ofphotobioreactor. A methodology is described to determine the mass transfercoefficients for O₂ stripping and CO₂ dissolution whichwould be required to achieve a desired biomass productivity. This procedure canassist in determining design modifications to achieve the desired mass transfercoefficient.     

Applicability of airlift draft-tube fluidized bioreactors for binary protein mixture bioseparation

An airlift draft-tube fluidized bioreactor has been designed and tested for applications in protein bioseparation. Operating parameters and geometrical dimensions of the bioreactor were optimized to ensure fluid circulation in a defined cyclic pattern between the riser and the downcomer. The overall directionality of liquid flow generates homogeneous field of low shear and achieves good mixing efficiency. Bioseparation of proteins was achieved from solutions containing both BSA and BHb at different initial concentrations and at pH 7. Similar adsorption capacities of both proteins were observed in single protein adsorption experiments at pH 7. Compressibility of BHb allowed for high adsorption capacity, in addition to the hydrophobic interaction forces. Apparently the homogeneous and lower shear generated by the airlift bioreactor reduces the compressibility of adsorbed BHb. This allowed for higher BSA adsorption from solutions containing BSA and BHb mixtures. Conventional batch adsorption experiments showed more adsorption of BHb, which reduces bioseparation efficiency.     

Hydrodynamics, mass transfer and rheological studies of gibberellic acid production in an airlift bioreactor

Although a lot of research has been done into modelling microbial processes, the applicability of these concepts to problems specific for bioreactor design and optimization of process conditions is limited. This is partly due to the tendency to separate the two essential factors of bioreactor modelling, i.e. physical transport processes and microbial kinetics. The deficiencies of these models become especially evident in industrial production processes where O₂ supply is likely to become the limiting factor, e.g. production of gibberellic acid and other organic acids. Hydrodynamics, mass transfer and rheology of gibberellic acid production by Gibberella fujikuroi in an airlift bioreactor is presented in this work. Important hydrodynamic parameters such as gas holdup, liquid velocity in the riser and in the downcomer, and mixing time were determined and correlated with superficial gas velocity in the riser. Mass transfer was studied evaluating the volumetric mass transfer coefficient, which was determined as a function of superficial gas velocity in the riser and as a function of fermentation time. Culture medium rheology was studied through fermentation time and allowed to explain the volumetric mass transfer coefficient behaviour. Rheological behaviour was explained in terms of changes in the morphology of the fungus. Finally, rheological studies let us obtain correlations for gas holdup and volumetric mass transfer coefficient estimation using the superficial gas velocity in the riser and the culture medium apparent viscosity.     

Biotransformation of glucose to gluconic acid by Aspergillus niger—study of mass transfer in an airlift bioreactor

A glucose–gluconic acid biotransformation system was suggested for the experimental study of oxygen transfer in bioreactors. This biosystem was used for the investigation of the effect of the flow rate and biomass concentration on the volumetric oxygen transfer coefficient k_La in a 10 dm³ internal-loop airlift bioreactor. For this purpose, the fermentation broth of the mycelial strain Aspergillus niger was employed, representing a three-phase system, where bubbles come into contact with dense rigid pellets. The results showed that the presented biotransformation system can be successfully utilised for the determination of the oxygen transfer rate in airlift bioreactors. The experiments showed a strong positive influence of the air flow rate on the rate (r_Glu), specific rate of gluconic acid production (k_Glu/X) as well as on the volumetric oxygen transfer coefficient (k_La). This confirmed an expected limitation of production rate by the oxygen transport from the gas to the liquid phase in the whole range of air flow rates applied. Moreover, consistent curves of the production rate r_Glu and k_La values vs. biomass concentration c_X (amount of enzymes) were observed. These exhibited a local maximum for c_X equal to 6.68 g dm⁻³. On the other hand, the specific production rate monotonously decreased with increasing biomass concentration. A decline of k_La values at higher c_X values was attributed to a bubble coalescence promoting effect of mycelial pellets.     

Large-scale cultivation of adventitious roots of Echinacea purpurea in airlift bioreactors for the production of chichoric acid, chlorogenic acid and caftaric acid

Adventitious roots of Echinacea purpurea were cultured in airlift bioreactors (20 l, 500 l balloon-type, bubble bioreactors and 1,000 l drum-type bubble bioreactor) using Murashige and Skoog (MS) medium with 2 mg indole butyric acid l⁻¹ and 50 g sucrose l⁻¹ for the production of chichoric acid, chlorogenic acid and caftaric acid. In the 20 l bioreactor (containing 14 l MS medium) a maximum yield of 11 g dry biomass l⁻¹ was achieved after 60 days. However, the amount of total phenolics (57 mg g⁻¹ DW), flavonoids (34 mg g⁻¹ DW) and caffeic acid derivatives (38 mg g⁻¹ DW) were highest after 50 days. Based on these studies, pilot-scale cultures were established and 3.6 kg and 5.1 kg dry biomass were achieved in the 500 l and 1,000 l bioreactors, respectively. The accumulation of 5 mg chlorogenic acid g⁻¹ DW, 22 mg chichoric acid g⁻¹ DW and 4 mg caftaric acids g⁻¹ DW were achieved with adventitious roots grown in 1,000 l bioreactors.     

Influence of sparger on energy dissipation, shear rate, and mass transfer to sea water in a concentric-tube airlift bioreactor

Data on volumetric mass-transfer coefficient, K_La_L, in a 12 × 10⁻³ m³ airlift bioreactor are reported. Measurements were made in sea water. The superficial gas velocity ranged up to 0.21 m/s. Four cylindrical spargers (60–1000 μm pore size) were tested. In bubbly flow, the sparger pore size strongly influenced the K_La_L; the highest K_La_L values were obtained with the smallest pore size. In contrast, in the transition and heterogeneous flow regimes, the pore size had little influence on K_La_L. The best correlation of the mass transfer data was obtained when both gas holdup and liquid superficial velocity were taken as independent variables. Shear rates were estimated in the different zones of the reactor. The highest values were found in the bottom zone of the reactor and in the gas-liquid separator. The penetration and isotropic turbulence models were used to develop a semi-theoretical equation relating the volumetric mass-transfer coefficient to shear rate; hence providing a better understanding of how the operational variables may be manipulated to attain a moderate shear rate and an appropriate level of mass transfer, two extremely important parameters for the growth of sensible microorganisms as those used in marine biotechnology.     

Design principles of photo‐bioreactors for cultivation of microalgae

The present hype in microalgae biotechnology has shown that the topic of photo‐bioreactors has to be revisited with respect to availability in really large scale measured in hectars footprint area, minimization of cost, auxiliary energy demand as well as maintenance and life span. This review gives an overview about present designs and the basic limiting factors which include light distribution to avoid saturation kinetics, mixing along the light gradient to make use of light/dark cycles, aeration and mass transfer along the vertical or horizontal main axis for carbon dioxide supply and oxygen removal and last but not least the energy demand necessary to fulfil these tasks. To make comparison of the performance of different designs easier, a commented list of performance parameters is given. Based on these critical points recent developments in the areas of membranes for gas transfer and optical structures for light transfer are discussed. The fundamental starting point for the optimization of photo‐bioprocesses is a detailed understanding of the interaction between the bioreactor in terms of mass and light transfer as well as the microalgae physiology in terms of light and carbon uptake kinetics and dynamics.     

Hydrodynamic characteristics and mixing behaviour of Sclerotium glucanicum culture fluids in an airlift reactor with an internal loop used for scleroglucan production

The filamentous fungus, Sclerotium glucanicum NRRL 3006, was cultivated in a 0.008 m³ airlift bioreactor with internal recirculation loop (ARL-IL) for production of the biopolymer, scleroglucan. The rheological behaviour of the culture fluid was characterised by measurement of the fluid consistency coefficient (K) and the flow behaviour index (n). Based on these measurements, the culture fluid changed from a low viscosity Newtonian system early in the process, to a viscous non-Newtonian (pseudoplastic) system. In addition, reactor hydrodynamics and mixing behaviour were characterised by measurement of whole mean gas hold-up (ɛ _g), liquid re-circulation velocity (U _ld) and mixing time (t _m). Under identical process conditions, the effects of the viscosity of the culture fluid and air flow rate on ɛ _g, U _ld and t _m were examined and empirical correlations for ɛ _g, U _ld and t _m with both superficial velocity U _g and consistency coefficient K were obtained and expressed separately. The correlations obtained are likely to describe the behaviour of real fungal culture fluids more accurately than previous correlations based on Newtonian or simulated non-Newtonian systems. Journal of Industrial Microbiology & Biotechnology (2001) 27, 208–214. Received 05 June 2000/ Accepted in revised form 18 March 2001     

Enzyme, β-galactosidase immobilized on membrane surface for galacto-oligosaccharides formation from lactose: Kinetic study with feed flow under recirculation loop

The work focuses on producing galacto-oligosaccharides (GOS) through an enzymatic reaction with lactose under a partial recirculation loop by utilizing membrane-immobilized β-galactosidase. Cross-linking through covalent bonding, using gluteraldehyde, was employed to immobilize enzyme on a microporous polyvinylidene fluoride membrane. GOS synthesis was carried out in a laboratory fabricated reaction cell, whereby three immobilized membranes were housed in series. The reaction was conducted at varying initial lactose concentrations (ILCs) and feed flow rates at pH 6 and 40 °C. A maximum GOS of 30% (dry basis) was obtained after 60 h of reaction time, 50 g/L ILC, 241 U of enzyme (specific loading of 600 U/g-membrane), and 0.5 mL/min of feed flow rate at 56% lactose conversion. The GOS yield increased with increased ILC and decreased feed flow rate. The selectivity of GOS formation increased by increasing both the ILC and the feed flow rate, whereas the reverse was true for mono-saccharides. The immobilized enzyme retained ∼50% of its initial activity after 30 days of storage at 20 °C, while the native enzyme lost 100% of its activity within 21 days. Furthermore, a five-step, nine-parameter model was developed, and simulated results showed excellent agreement with the experimental data.     

A micro-jet array for economic intensification of gas transfer in bioreactors

Bioreactors are of interest for gas-to-liquid conversion of stranded or waste industrial gases, such as CO, CH₄, or syngas. Process economics requires reduction of bioreactor cost and size while maintaining intense production via rapid delivery of gases to the liquid phase (i.e., high k_La). Here, we show a novel bioreactor design that outperforms all known technology in terms of gas transfer energy efficiency (k_La per power density) while operating at high k_La (i.e., near 0.8 s⁻¹). The reactor design uses a micro-jet array to break feedstock gas into a downward microbubble flow. Hydrodynamic and surfactant measurements show the reactor's advanced performance arises from its bubble breakage mechanism, which limits fluid shear to a thin plane located at an optimal location for bubble breakage. Power dissipation and k_L are shown to scale with micro-jet diameter rather than reactor diameter, and the micro-jet array achieves improved performance compared to classical impinging-jets, ejector, or U-loop reactors. The hydrodynamic mechanism by which the micro-jets break bubbles apart is shown to be shearing the bubbles into filaments then fragmentation by surface tension rather than “cutting in half” of bubbles. Guided by these hydrodynamic insights, strategies for industrial design are given. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2710, 2019     

Recent advances in two-phase partitioning bioreactors for the treatment of volatile organic compounds

Biological processes are considered to be the most cost-effective technology for the off-gas treatment of volatile organic compounds (VOC) at low concentrations. Two-phase partitioning bioreactors (TPPBs) emerged in the early 1990s as innovative multiphase systems capable of overcoming some of the key limitations of traditional biological technologies such as the low mass transfer rates of hydrophobic VOCs and microbial inhibition at high VOC loading rates. Intensive research carried out in the last 5 years has helped to provide a better understanding of the mass transfer phenomena and VOC uptake mechanisms in TPPBs, which has significantly improved the VOC biodegradation processes utilizing this technology platform. This work presents an updated state-of-the-art review on the advances of TPPB technology for air pollution control. The most recent insights regarding non-aqueous phase (NAP) selection, microbiology, reactor design, mathematical modeling and case studies are critically reviewed and discussed. Finally, the key research issues required to move towards the development of efficient and stable full-scale VOC biodegradation processes in TPPBs are identified.     

A comparative study of solid and liquid non‐aqueous phases for the biodegradation of hexane in two‐phase partitioning bioreactors

A comparative study of the performance of solid and liquid non‐aqueous phases (NAPs) to enhance the mass transfer and biodegradation of hexane by Pseudomonas aeruginosa in two‐phase partitioning bioreactors (TPPBs) was undertaken. A preliminary NAP screening was thus carried out among the most common solid and liquid NAPs used in pollutant biodegradation. The polymer Kraton G1657 (solid) and the liquid silicone oils SO20 and SO200 were selected from this screening based on their biocompatibility, resistance to microbial attack, non‐volatility and high affinity for hexane (low partition coefficient: K = C_g/C_NAP, where C_g and C_NAP represent the pollutant concentration in the gas phase and NAP, respectively). Despite the three NAPs exhibited a similar affinity for hexane (K ≈ 0.0058), SO200 and SO20 showed a superior performance to Kraton G1657 in terms of hexane mass transfer and biodegradation enhancement. The enhanced performance of SO200 and SO20 could be explained by both the low interfacial area of this solid polymer (as a result of the large size of commercial beads) and by the interference of water on hexane transfer (observed in this work). When Kraton G1657 (20%) was tested in a TPPB inoculated with P. aeruginosa, steady state elimination capacities (ECs) of 5.6 ± 0.6 g m^?3 h^?1 were achieved. These values were similar to those obtained in the absence of a NAP but lower compared to the ECs recorded in the presence of 20% of SO200 (10.6 ± 0.9 g m^?3 h^?1). Finally, this study showed that the enhancement in the transfer of hexane supported by SO200 was attenuated by limitations in microbial activity, as shown by the fact that the ECs in biotic systems were far lower than the maximum hexane transfer capacity recorded under abiotic conditions. Biotechnol. Bioeng. 2010;106: 731–740. © 2010 Wiley Periodicals, Inc.     

Improved production of phenylethanoid glycosides by Cistanche deserticola cells cultured in an internal loop airlift bioreactor with sifter riser

An internal loop airlift bioreactor with sifter riser (ILABSR) was composed of a bubble column and a draught-tube rolled with 40-mesh sifter that placed 5 cm above the bottom at the center of the column. A 2 L ILABSR was used for the suspension cultivation of Cistanche deserticola cells and its performance was compared with shake flask culture and a bubble column. Under the optimum culture conditions with the air flowrate of 0.075 m³/h and the inoculation size of 4.7%, about one-fifth cells were attached to the sifter draught-tube. PeG content in these cells was 16.3%, which was 104% higher than that of suspension cells. The production of phenylethanoid glycosides reached 0.85 g/L, which was 102 and 4% higher than those cultured in a 2 L bubble column and shake flasks respectively under their optimal culture conditions.     

Systematic Approach to Solvent Selection for Biphasic Systems with a Combination of COSMO‐RS and a Dynamic Modeling Tool

Biphasic aqueous‐organic systems are important reaction systems for catalytic processes. This is especially true for biocatalysis where the range of accessible products can be significantly extended. In such systems, the aqueous phase is the reactive phase in which the biocatalyst is dissolved and the organic phase is nonreactive and acts as substrate reservoir and as in situ product extraction solvent. Here, the choice of the nonreactive phase is highly important for the overall performance of the system. In this contribution, a systematic approach to solvent selection for biphasic aqueous‐organic systems is presented with respect to partition coefficients. The model reaction is the stereoselective carbon‐carbon coupling of two 3,5‐dimethoxy‐benzaldehyde molecules to (R)‐3,3',5,5'‐tetramethoxy‐benzoin catalyzed by benzaldehyde lyase (EC 4.1.2.38) from Pseudomonas fluorescens. A systematic approach to solvent selection consisting of two steps is proposed: Firstly, the conductor‐like screening model for real solvents (COSMO‐RS) is used to facilitate a fast solvent screening. Since this is an ab initio approach it allows a pre‐screening without laborious experimental input. The proposed ranking of solvents, based on the ratio of partition coefficients at infinite dilution, is a sound basis for the successive steps. Secondly, a dynamic model is fitted to experimental data in order to obtain detailed and reliable results for mass transfer and partition coefficients. Therefore, the method makes efficient use of the experimental data and substantiates quantitative results with guided experiments.     

A system of miniaturized stirred bioreactors for parallel continuous cultivation of yeast with online measurement of dissolved oxygen and off‐gas

Chemostat cultivation is a powerful tool for physiological studies of microorganisms. We report the construction and application of a set of eight parallel small‐scale bioreactors with a working volume of 10 mL for continuous cultivation. Hungate tubes were used as culture vessels connected to multichannel‐peristaltic pumps for feeding fresh media and removal of culture broth and off‐gas. Water saturated air is sucked into the bioreactors by applying negative pressure, and small stirrer bars inside the culture vessels allow sufficient mixing and oxygen transfer. Optical sensors are used for non‐invasive online measurement of dissolved oxygen, which proved to be a powerful indicator of the physiological state of the cultures, particularly of steady‐state conditions. Analysis of culture exhaust‐gas by means of mass spectrometry enables balancing of carbon. The capacity of the developed small‐scale bioreactor system was validated using the fission yeast Schizosaccharomyces pombe, focusing on the metabolic shift from respiratory to respiro‐fermentative metabolism, as well as studies on consumption of different substrates such as glucose, fructose, and gluconate. In all cases, an almost completely closed carbon balance was obtained proving the reliability of the experimental setup. Biotechnol. Bioeng. 2013; 110: 535–542. © 2012 Wiley Periodicals, Inc.     

A comparison of the gas, solution, and solid state coordination environments for the Cu(II) complexes of a series of linear aminopyridine ligands with varying ratios of 5- and 6-membered chelate rings

The synthesis and characterization of the copper(II) complexes of a series of tetradentate, pentadentate and hexadentate aminopyridine ligands that contain ethylenediamine and/or propylenediamine groups are described. The ligands include: 1,12-bis(2-pyridyl)-2,5,8,11-tetraazadodecane, TRIEN-pyr; 1,13-bis(2-pyridyl)-2,5,9,12-tetraazatridecane, DIEN-PN-pyr; 1,14-bis(2-pyridyl)-2,6,9,13-tetraazatetradecane, DIPN-EN-pyr; 1,15-bis(2-pyridyl)-2,6,l0,14-tetraazapentadecane, TRIPN-pyr; 1,9-bis(2-pyridyl)-2,5,8-triazanonane, DIEN-pyr; 1,11-bis(2-pyridyl)-2,6,10-triazaundecanenane, DIPN-pyr; 1,6-bis(2-pyridyl)-2,5-diazahexane, EN-pyr; and 1,7-bis(2-pyridyl)-2,6-diazaheptane, PN-pyr. The following methods were used to determine the binding geometries of the copper(II) complexes in the solid, solution, and gas phases: magnetic susceptibility measurements, absorption spectroscopy, EPR spectroscopy, electrochemistry, and electrospray ionization mass spectrometry. An X-ray structure was determined for the DIPN-pyr complex. The solid state structures were all found to be monomeric Cu(II) complexes with the coordination number set by the denticity of the ligand while the solution structures of all of the complexes except those with TRIPN-pyr and DIPN-pyr were found to be square pyramidal or elongated octahedral. The TRIPN-pyr and DIPN-pyr complexes showed considerable trigonal bipyramidal distortions. The gas phase data showed that the substitution of 6-membered for 5-membered chelate rings helped the ligand span more coordination sites. The TRIEN-pyr complex was 4- or 5-coordinate compared to the 5- or 6-coordination seen with the other three hexadentate ligands, and the DIPN-pyr complex was weakly 5-coordinate as compared to the 4-coordinate DIEN-pyr complex. The preferred structures of the ligands were consistent with their electrochemical behavior which showed the stability of the Cu(II) complex decreased in the order: DIPN-EN-pyr, TRIEN-pyr, DIEN-PN-pyr > DIEN-pyr > DIPN-pyr > TRIPN-pyr >  PN-pyr > EN-pyr.     

A comparison of the gas, solution, and solid state coordination environments for the Ni(II) complexes of a series of linear penta- and hexadentate aminopyridine ligands with accessible Ni(III) oxidation states

The synthesis and characterization of the nickel(II) complexes of a series of pentadentate and hexadentate aminopyridine ligands that contain ethylenediamine and/or propylenediamine groups are described. The ligands include: 1,12-bis(2-pyridyl)-2,5,8,11-tetraazadodecane, TRIEN-pyr; 1,13-bis(2-pyridyl)-2,5,9,12-tetraazatridecane, DIEN-PN-pyr; 1,14-bis(2-pyridyl)-2,6,9,13-tetraazatetradecane, DIPN-EN-pyr; 1,15-bis(2-pyridyl)-2,6,10,14-tetraazapentadecane, TRIPN-pyr; 1,9-bis(2-pyridyl)-2,5,8-triazanonane, DIEN-pyr; and 1,11-bis(2-pyridyl)-2,6,10-triazaundecane, DIPN-pyr. The following methods were used to determine the binding geometries of the nickel(II) complexes in the solid, solution, and gas phases: magnetic susceptibility measurements, absorption spectroscopy, EPR spectroscopy, electrochemistry, and electrospray ionization mass spectrometry. All of the ligands form 6-coordinate compounds in the solid, liquid, and gas states, with the exception of the TRIEN-pyr, DIEN-PN-pyr(partially), DIPN-pyr, and DIEN-pyr ligands which form 5-coordinate species in the gas state. All of the complexes could be oxidized to Ni(III) species electrochemically, although the Ni(III) complexes of TRIPN-pyr and DIPN-pyr were much less stable than the other four ligands. EPR spectra of the frozen solutions showed the low spin d⁷ Ni(III) complexes of TRIEN-pyr and DIPN-EN-pyr to be similar to those that have been found for poly-aza macrocyclic compounds.     

Negatively charged residues in the H loop of PsaB subunit in Photosystem I from Synechocystis sp. PCC 6803 appear to be responsible for electrostatic repulsions with plastocyanin*

Wild-type plastocyanin from the cyanobacterium Synechocystis sp. PCC 6803 does not form any kinetically detectable transient complex with Photosystem I (PS I) during electron transfer, but the D44R/D47R double mutant of copper protein does [De la Cerda et al. (1997) Biochemistry 36: 10125–10130]. To identify the PS I component that is involved in the complex formation with the D44R/D47R plastocyanin, the kinetic efficiency of several PS I mutants, including a PsaF–PsaJ-less PS I and deletion mutants in the lumenal H and J loops of PsaB, were analyzed by laser flash absorption spectroscopy. The experimental data herein suggest that some of the negative charges at the H loop of PsaB are involved in electrostatic repulsions with mutant plastocyanin. Mutations in the J loop demonstrate that this region of PsaB is also critical. The interaction site of PS I is thus not as defined as first expected but much broader, thereby revealing how complex the evolution of intermolecular electron transfer mechanisms in photosynthesis has been. This revised version was published online in June 2006 with corrections to the Cover Date.