H2 production by photofermentation in an innovative plate-type photobioreactor with meandering channels.

Hydrogen production by Rhodobacter capsulatus is an anaerobic, photobiological process requiring specific mixing conditions. In this study, an innovative design of a photobioreactor is proposed. The design is based on a plate-type photobioreactor with an interconnected meandering channel to allow culture mixing and H2 degassing. The culture flow was characterized as a quasi-plug-flow with radial mixing caused by a turbulent-like regime achieved at a low Reynolds number. The dissipated volumetric power was decreased 10-fold while maintaining PBR performances (production and yields) when compared with a magnetically stirred tank reactor. To increase hydrogen production flow rate, several bacterial concentrations were tested by increasing the glutamate concentration using fed-batch cultures. The maximum hydrogen production flow rate (157.7 ± 9.3 ml H2 /L/h) achieved is one of the highest values so far reported for H2 production by R. capsulatus. These first results are encouraging for future scale-up of the plate-type reactor.

Selection of Cyanobacterial (Synechococcus sp. Strain PCC 6301) RubisCO Variants with Improved Functional Properties That Confer Enhanced CO2-Dependent Growth of Rhodobacter capsulatus, a Photosynthetic Bacterium.

Ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO) is a ubiquitous enzyme that catalyzes the conversion of atmospheric CO2 into organic carbon in primary producers. All naturally occurring RubisCOs have low catalytic turnover rates and are inhibited by oxygen. Evolutionary adaptations of the enzyme and its host organisms to changing atmospheric oxygen concentrations provide an impetus to artificially evolve RubisCO variants under unnatural selective conditions. A RubisCO deletion strain of the nonsulfur purple photosynthetic bacterium Rhodobacter capsulatus was previously used as a heterologous host for directed evolution and suppressor selection studies that led to the identification of a conserved hydrophobic region near the active site where amino acid substitutions selectively impacted the enzyme's sensitivity to O2 In this study, structural alignments, mutagenesis, suppressor selection, and growth complementation with R. capsulatus under anoxic or oxygenic conditions were used to analyze the importance of semiconserved residues in this region of Synechococcus RubisCO. RubisCO mutant substitutions were identified that provided superior CO2-dependent growth capabilities relative to the wild-type enzyme. Kinetic analyses of the mutant enzymes indicated that enhanced growth performance was traceable to differential interactions of the enzymes with CO2 and O2 Effective residue substitutions also appeared to be localized to two other conserved hydrophobic regions of the holoenzyme. Structural comparisons and similarities indicated that regions identified in this study may be targeted for improvement in RubisCOs from other sources, including crop plants.IMPORTANCE RubisCO catalysis has a significant impact on mitigating greenhouse gas accumulation and CO2 conversion to food, fuel, and other organic compounds required to sustain life. Because RubisCO-dependent CO2 fixation is severely compromised by oxygen inhibition and other physiological constraints, improving RubisCO's kinetic properties to enhance growth in the presence of atmospheric O2 levels has been a longstanding goal. In this study, RubisCO variants with superior structure-functional properties were selected which resulted in enhanced growth of an autotrophic host organism (R. capsulatus), indicating that RubisCO function was indeed growth limiting. It is evident from these results that genetically engineered RubisCO with kinetically enhanced properties can positively impact growth rates in primary producers.

Transcriptome analysis of Rhodobacter capsulatus grown on different nitrogen sources.

This study investigated the effect of different nitrogen sources, namely, ammonium chloride and glutamate, on photoheterotrophic metabolism of Rhodobacter capsulatus grown on acetate as the carbon source. Genes that were significantly differentially expressed according to Affymetrix microarray data were categorized into Clusters of Orthologous Groups functional categories and those in acetate assimilation, hydrogen production, and photosynthetic electron transport pathways were analyzed in detail. Genes related to hydrogen production metabolism were significantly downregulated in cultures grown on ammonium chloride when compared to those grown on glutamate. In contrast, photosynthetic electron transport and acetate assimilation pathway genes were upregulated. In detail, aceA encoding isocitrate lyase, a unique enzyme of the glyoxylate cycle and ccrA encoding the rate limiting crotonyl-CoA carboxylase/reductase enzyme of ethylmalonyl-coA pathway were significantly upregulated. Our findings indicate for the first time that R. capsulatus can operate both glyoxylate and ethylmalonyl-coA cycles for acetate assimilation.

Differing isoforms of the cobalamin binding photoreceptor AerR oppositely regulate photosystem expression.

Phototrophic microorganisms adjust photosystem synthesis in response to changes in light intensity and wavelength. A variety of different photoreceptors regulate this process. Purple photosynthetic bacteria synthesize a novel photoreceptor AerR that uses cobalamin (B12) as a blue-light absorbing chromophore to control photosystem synthesis. AerR directly interacts with the redox responding transcription factor CrtJ, affecting CrtJ's interaction with photosystem promoters. In this study, we show that AerR is translated as two isoforms that differ by 41 amino acids at the amino terminus. The ratio of these isoforms was affected by light and cell growth phase with the long variant predominating during photosynthetic exponential growth and the short variant predominating in dark conditions and/or stationary phase. Pigmentation and transcriptomic analyses show that the short AerR variant represses, while long variant activates, photosynthesis genes. The long form of AerR also activates many genes involved in cellular metabolism and motility.

Inhibition of bacteriochlorophyll biosynthesis in the purple phototrophic bacteria Rhodospirillumrubrum and Rhodobacter capsulatus grown in the presence of a toxic concentration of selenite.

Background In many works, the chemical composition of bacterially-produced elemental selenium nanoparticles (Se0-nanoparticles) was investigated using electron dispersive X-ray analysis. The results suggest that these particles should be associated with organic compounds. However, a complete analysis of their chemical composition is still missing. Aiming at identifying organic compounds associated with the Se0-nanoparticles produced by the purple phototrophic bacteria Rhodospirillum rubrum and Rhodobacter capsulatus (α group of the proteobacteria), we used MALDI-TOF spectrometry.Results This technic revealed that numerous signals obtained from particles produced by both species of bacteria were from metabolites of the photosynthetic system. Furthermore, not only bacteriochlorophyll a, bacteriopheophytin a, and bacteriopheophorbide a, which are known to accumulate in stationary phase cultures of these bacteria grown phototrophically in the absence of selenite, were identified. The particles were also associated with intermediary metabolites of the bacteriochlorophyll a biosynthesis pathway such as protoporphyrin IX, protoporphyrin IX monomethyl ester, bacteriochlorophyllide a and, most likely, Mg-protoporphyrin IX-monomethyl ester, as well as with oxidation products of the substrates of protochlorophyllide reductase and chlorin reductase.Conclusion Accumulation of intermediary metabolites of the bacteriochlorophyll biosynthesis pathway in these purple phototrophic bacteria was attributed to inhibition of oxygen-sensitive enzymes involved in this pathway. Consistent with this interpretation it has been reported that these bacteria reduce selenite intracellularly, that they contain high levels of glutathione and that the reduction of selenite with glutathione is a very fast reaction accompanied by the production of reactive oxygen species. As many enzymes involved in the biosynthesis of bacteriochlorophyll contain [Fe-S] clusters in their active site, which are known to be degraded in the presence of reactive oxygen species as well as in the presence of molecular oxygen, we concluded that the substrates of these enzymes accumulate in cells during selenite reduction.Association of metabolites of bacteriochlorophyll biosynthesis and degradation with the Se0-nanoparticles produced by Rhodospirillum rubrum and Rhodobacter capsulatus is proposed to result from coating of the nanoparticles with the intracytoplasmic membrane of these bacteria, where the photochemical apparatus is concentrated.

Functional flexibility of electron flow between quinol oxidation Qo site of cytochrome bc1 and cytochrome c revealed by combinatory effects of mutations in cytochrome b, iron-sulfur protein and cytochrome c1.

Transfer of electron from quinol to cytochrome c is an integral part of catalytic cycle of cytochrome bc1. It is a multi-step reaction involving: i) electron transfer from quinol bound at the catalytic Qo site to the Rieske iron-sulfur ([2Fe-2S]) cluster, ii) large-scale movement of a domain containing [2Fe-2S] cluster (ISP-HD) towards cytochrome c1, iii) reduction of cytochrome c1 by reduced [2Fe-2S] cluster, iv) reduction of cytochrome c by cytochrome c1. In this work, to examine this multi-step reaction we introduced various types of barriers for electron transfer within the chain of [2Fe-2S] cluster, cytochrome c1 and cytochrome c. The barriers included: impediment in the motion of ISP-HD, uphill electron transfer from [2Fe-2S] cluster to heme c1 of cytochrome c1, and impediment in the catalytic quinol oxidation. The barriers were introduced separately or in various combinations and their effects on enzymatic activity of cytochrome bc1 were compared. This analysis revealed significant degree of functional flexibility allowing the cofactor chains to accommodate certain structural and/or redox potential changes without losing overall electron and proton transfers capabilities. In some cases inhibitory effects compensated one another to improve/restore the function. The results support an equilibrium model in which a random oscillation of ISP-HD between the Qo site and cytochrome c1 helps maintaining redox equilibrium between all cofactors of the chain. We propose a new concept in which independence of the dynamics of the Qo site substrate and the motion of ISP-HD is one of the elements supporting this equilibrium and also is a potential factor limiting the overall catalytic rate.

Utilization of distillery wastewater for hydrogen production in one-stage and two-stage processes involving photofermentation.

In this study, distillery wastewater was treated by dark fermentation or photofermentation alone, and by sequential dark and photofermentation processes using anaerobic saccharolytic consortium and purple nonsulfur bacteria. Combination of dark and photofermentation resulted in the maximal H2 yield of 17.6L/L of distillery waste with chemical oxygen demand 40g/L. It is equivalent to 205kJ/L distillery wastewater and corresponds to recovery of approximately 4-8% of energy consumed during ethanol production. Optimal performance of photofermentation was observed at 20% concentration of pre-fermented distillery waste. In photofermentation, the range of the suitable distillery waste concentrations was extended and the H2 yield was improved by choosing the tolerant strain of purple bacteria Rhodobacter sphaeroides B-3059. After two stages, organic acids and sugars were completely consumed that means wastewater treatment concomitant to H2 production.

Implementation and analysis of temperature control strategies for outdoor photobiological hydrogen production.

For outdoor photobiological hydrogen production, the effective control of temperature in photobioreactors is a challenge. In this work, an internal cooling system for outdoor tubular photobioreactors was designed, built, and tested. The temperatures in the reactors with bacteria were consistently higher than those without bacteria, and were also strongly influenced by solar irradiation and ambient air temperature. The cooling protocol applied successfully kept the reactor temperatures below the threshold limit (38 °C) required for the bioprocess and provided a uniform distribution of temperature along the reactor tube length. The biomass growth and hydrogen production were similar in the reactors cooled co-currently and counter-currently. The biomass growth rate was 0.1 l/h, the maximum hydrogen production rate was 1.28 mol/m3/h, and the overall hydrogen yield obtained was 20 %. The change in the biomass was fitted using the logistic model while cumulative hydrogen production was fitted using the modified Gompertz equation.

Species differences in unlocking B-side electron transfer in bacterial reaction centers.

The structure of the bacterial photosynthetic reaction center (RC) reveals symmetry-related electron transfer (ET) pathways, but only one path is used in native RCs. Analogous mutations have been made in two Rhodobacter (R.) species. A glutamic acid at position 133 in the M subunit increases transmembrane charge separation via the naturally inactive (B-side) path through impacts on primary ET in mutant R. sphaeroidesRCs. Prior work showed that the analogous substitution in the R. capsulatusRC also increases B-side activity, but mainly affects secondary ET. The overall yields of transmembrane ET are similar, but enabled in fundamentally different ways.

Zinc biosorption by the purple non-sulfur bacterium Rhodobacter capsulatus.

This paper presents the first report providing information on the zinc (Zn) biosorption potentialities of the purple non-sulfur bacterium Rhodobacter capsulatus. The effects of various biological, physical, and chemical parameters on Zn biosorption were studied in both the wild-type strain B10 and a strain, RC220, lacking the endogenous plasmid. At an initial Zn concentration of 10 mg·L(-1), the Zn biosorption capacity at pH 7 for bacterial biomass grown in synthetic medium containing lactate as carbon source was 17 and 16 mg Zn·(g dry mass)(-1) for strains B10 and RC220, respectively. Equilibrium was achieved in a contact time of 30-120 min, depending on the initial Zn concentration. Zn sorption by live biomass was modelled, at equilibrium, according to the Redlich-Peterson and Langmuir isotherms, in the range of 1-600 mg Zn·L(-1). The wild-type strain showed a maximal Zn uptake capacity (Qm) of 164 ± 8 mg·(g dry mass)(-1) and an equilibrium constant (Kads) of 0.017 ± 0.00085 L·(mg Zn)(-1), compared with values of 73.9 mg·(g dry mass)(-1) and 0.361 L·mg(-1) for the strain lacking the endogenous plasmid. The Qm value observed for R. capsulatus B10 is one of the highest reported in the literature, suggesting that this strain may be useful for Zn bioremediation. The lower Qm value and higher equilibrium constant observed for strain RC220 suggest that the endogenous plasmid confers an enhanced biosorption capacity in this bacterium, although no genetic determinants for Zn resistance appear to be located on the plasmid, and possible explanations for this are discussed.

Hybrid fusions show that inter-monomer electron transfer robustly supports cytochrome bc1 function in vivo.

Electronic connection between Qo and Qi quinone catalytic sites of dimeric cytochrome bc1 is a central feature of the energy-conserving Q cycle. While both the intra- and inter-monomer electron transfers were shown to connect the sites in the enzyme, mechanistic and physiological significance of the latter remains unclear. Here, using a series of mutated hybrid cytochrome bc1-like complexes, we show that inter-monomer electron transfer robustly sustains the function of the enzyme in vivo, even when the two subunits in a dimer come from different species. This indicates that minimal requirement for bioenergetic efficiency is to provide a chain of cofactors for uncompromised electron flux between the catalytic sites, while the details of protein scaffold are secondary.

Variation in composition and relative content of accumulated photopigments in a newly isolated Rhodobacter capsulatus strain XJ-1 in response to arsenic.

This study aimed to isolate and characterize a new arsenic (As)-tolerant bacterial strain (XJ-1) from the Halosol soil, to evaluate its As tolerance, and to examine the variation in composition and relative content of accumulated photosynthetic pigments in response to As. The experiments were performed with high-performance liquid chromatography (HPLC), inductively-coupled plasma mass spectrometry (ICP-MS), liquid chromatography/mass spectrometry (LC/MS), thin-layer chromatography (TLC) and grayscale intensity image analysis using Gel-Pro analyzer software. Strain XJ-1 was identified as Rhodobacter (R.) capsulatus based on 16S rRNA gene sequencing and physiological characteristics. Strain XJ-1 was able to grow when exposed to arsenite [As(III)] and arsenate [As(V)] under anaerobic-light conditions. The median effective concentrations (EC50) of As(III) and As(V) were 0.61 mM and 2.03 mM, respectively. Strain XJ-1 could reduce As(V) to As(III), but As(III) could not be transformed back to As(V) or other organic As compounds. Accumulation of bacteriochlorophylls and carotenoids in strain XJ-1 varied in the presence of 0.2-1.2 mM As(III) and 0-2.5 mM As(V). As exposure resulted in pronounced variation in compositions and contents of photosynthetic pigments, especially hydroxyspheroidene, bacteriophaeophytin, the ratio of tetrahydrogeranylgeranyl to phytylated BChl a, and the ratio of spheroidene to spheroidenone. This research highlights the adaptative response of R. capsulatus strain XJ-1 photosystems to environmental As, and demonstrates the potential of utilizing the sensitivity of its photosynthetic pigments to As(III) and As(V) for the biodetection of As in the environment.

Identification of a predicted partner-switching system that affects production of the gene transfer agent RcGTA and stationary phase viability in Rhodobacter capsulatus.

BACKGROUND: Production of the gene transfer agent RcGTA in the α-proteobacterium Rhodobacter capsulatus is dependent upon the response regulator protein CtrA. Loss of this regulator has widespread effects on transcription in R. capsulatus, including the dysregulation of numerous genes encoding other predicted regulators. This includes a set of putative components of a partner-switching signaling pathway with sequence homology to the σ-regulating proteins RsbV, RsbW, and RsbY that have been extensively characterized for their role in stress responses in gram-positive bacteria. These R. capsulatus homologues, RbaV, RbaW, and RbaY, have been investigated for their possible role in controlling RcGTA gene expression. RESULTS: A mutant strain lacking rbaW showed a significant increase in RcGTA gene expression and production. Mutation of rbaV or rbaY led to a decrease in RcGTA gene expression and production, and these mutants also showed decreased viability in the stationary phase and produced unusual colony morphologies. In vitro and in vivo protein interaction assays demonstrated that RbaW and RbaV interact. A combination of gene disruptions and protein-protein interaction assays were unsuccessful in attempts to identify a cognate σ factor, and the genetic data support a model where the RbaV protein that is the determinant regulator of RcGTA gene expression in this system. CONCLUSIONS: These findings provide new information about RcGTA regulation by a putative partner-switching system and further illustrate the integration of RcGTA production into R. capsulatus physiology.

Electrochemical communication between heterotrophically grown Rhodobacter capsulatus with electrodes mediated by an osmium redox polymer.

The metabolically versatile purple bacteria Rhodobacter capsulatus was investigated to check its possible applicability in biofuel cells and electrochemical microbial biosensors. The wild type strain ATCC 17015 and mutant strain 37b4 lacking the lipopolysaccharide capsule was compared for their ability to communicate with electrodes modified with an osmium redox polymer. In this work, aerobic heterotrophically grown R. capsulatus were used to screen for efficient cell-electrode communication for later implementation using photoheterotrophically grown bacteria. The bacterial cells embedded in the osmium polymer matrix demonstrated efficient electrical "wiring" with the electrodes and were able to generate a noticeable current with succinate as substrate. Interestingly, at 2mM succinate the wild type strain showed much better bioelectrocatalytic current generation (4.25 µA/cm(2)) than the strain lacking capsule (1.55 µA/cm(2)). The wild type strain also exhibited a stable current response for longer time, demonstrating that the bacterial lipopolysaccharide in fact enhances the stability of the polymer matrix layer of the modified electrode. Control experiments with R. capsulatus without any mediator did not show any current irrespective of the capsule presence. This demonstrates that development of photosensors and other light driven bioelectrochemical devices could be feasible using R. capsulatus and will be at focus for future studies.

Missense mutations in cytochrome c maturation genes provide new insights into Rhodobacter capsulatus cbb3-type cytochrome c oxidase biogenesis.

The Rhodobacter capsulatus cbb(3)-type cytochrome c oxidase (cbb(3)-Cox) belongs to the heme-copper oxidase superfamily, and its subunits are encoded by the ccoNOQP operon. Biosynthesis of this enzyme is complex and needs dedicated biogenesis genes (ccoGHIS). It also relies on the c-type cytochrome maturation (Ccm) process, which requires the ccmABCDEFGHI genes, because two of the cbb(3)-Cox subunits (CcoO and CcoP) are c-type cytochromes. Recently, we reported that mutants lacking CcoA, a major facilitator superfamily type transporter, produce very small amounts of cbb(3)-Cox unless the growth medium is supplemented with copper. In this work, we isolated "Cu-unresponsive" derivatives of a ccoA deletion strain that exhibited no cbb(3)-Cox activity even upon Cu supplementation. Molecular characterization of these mutants revealed missense mutations in the ccmA or ccmF gene, required for the Ccm process. As expected, Cu-unresponsive mutants lacked the CcoO and CcoP subunits due to Ccm defects, but remarkably, they contained the CcoN subunit of cbb(3)-Cox. Subsequent construction and examination of single ccm knockout mutants demonstrated that membrane insertion and stability of CcoN occurred in the absence of the Ccm process. Moreover, while the ccm knockout mutants were completely incompetent for photosynthesis, the Cu-unresponsive mutants grew photosynthetically at lower rates and produced smaller amounts of cytochromes c(1) and c(2) than did a wild-type strain due to their restricted Ccm capabilities. These findings demonstrate that different levels of Ccm efficiency are required for the production of various c-type cytochromes and reveal for the first time that maturation of the heme-Cu-containing subunit CcoN of R. capsulatus cbb(3)-Cox proceeds independently of that of the c-type cytochromes during the biogenesis of this enzyme.

Enhanced photo-fermentative hydrogen production by Rhodobacter capsulatus with pigment content manipulation.

High content of pigment in purple nonsulfur photosynthetic bacteria hinders its photo-hydrogen production rate under intense light irradiation. In order to alleviate the light shielding effect and improve its photo-fermentative hydrogen production performance, pufQ, which is the regulatory gene of bacteriochlorophyll biosynthesis in Rhodobacter capsulatus, was cloned and relocated in the genome under cbb3 promoter by homologous recombination. The UV-vis spectra indicated that the light absorption of the mutant between 300 and 900 nm was reduced. Photo-hydrogen production experiments by the recombinant and wild type strain were carried out in 350 mL photo bioreactors using acetic and butyric acid as substrate. The results showed that the hydrogen production of recombinant with reduced pigment was 27% higher than that of its parental strain, indicating that it is effective on enhancing photo-fermentative hydrogen production by manipulating pigment biosynthesis in purple nonsulfur photosynthetic bacteria.

Tyrosine triad at the interface between the Rieske iron-sulfur protein, cytochrome c1 and cytochrome c2 in the bc1 complex of Rhodobacter capsulatus.

A triad of tyrosine residues (Y152-154) in the cytochrome c(1) subunit (C1) of the Rhodobacter capsulatus cytochrome bc(1) complex (BC1) is ideally positioned to interact with cytochrome c(2) (C2). Mutational analysis of these three tyrosines showed that, of the three, Y154 is the most important, since its mutation to alanine resulted in significantly reduced levels, destabilization, and inactivation of BC1. A second-site revertant of this mutant that regained photosynthetic capacity was found to have acquired two further mutations-A181T and A200V. The Y152Q mutation did not change the spectral or electrochemical properties of C1, and showed wild-type enzymatic C2 reduction rates, indicating that this mutation did not introduce major structural changes in C1 nor affect overall activity. Mutations Y153Q and Y153A, on the other hand, clearly affect the redox properties of C1 (e.g. by lowering the midpoint potential as much as 117 mV in Y153Q) and the activity by 90% and 50%, respectively. A more conservative Y153F mutant on the other hand, behaves similarly to wild-type. This underscores the importance of an aromatic residue at position Y153, presumably to maintain close packing with P184, which modeling indicates is likely to stabilize the sixth heme ligand conformation.

Photomixotrophic growth of Rhodobacter capsulatus SB1003 on ferrous iron.

This study investigates the role iron oxidation plays in the purple non-sulfur bacterium Rhodobacter capsulatus SB1003. This organism is unable to grow photoautotrophically on unchelated ferrous iron [Fe(II)] despite its ability to oxidize chelated Fe(II). This apparent paradox was partly resolved by the discovery that SB1003 can grow photoheterotrophically on the photochemical breakdown products of certain ferric iron-ligand complexes, yet whether it could concomitantly benefit from the oxidation of Fe(II) to fix CO(2) was unknown. Here, we examine carbon fixation by stable isotope labeling of the inorganic carbon pool in cultures growing phototrophically on acetate with and without Fe(II). We show that R. capsulatus SB1003, an organism formally thought incapable of phototrophic growth on Fe(II), can actually harness the reducing power of this substrate and grow photomixotrophically, deriving carbon both from organic sources and from fixation of inorganic carbon. This suggests the possibility of a wider occurrence of photoferrotrophy than previously assumed.

Coordinated, long-range, solid substrate movement of the purple photosynthetic bacterium Rhodobacter capsulatus.

The long-range movement of Rhodobacter capsulatus cells in the glass-agar interstitial region of borosilicate Petri plates was found to be due to a subset of the cells inoculated into plates. The macroscopic appearance of plates indicated that a small group of cells moved in a coordinated manner to form a visible satellite cluster of cells. Satellite clusters were initially separated from the point of inoculation by the absence of visible cell density, but after 20 to 24 hours this space was colonized by cells apparently shed from a group of cells moving away from the point of inoculation. Cell movements consisted of flagellum-independent and flagellum-dependent motility contributions. Flagellum-independent movement occurred at an early stage, such that satellite clusters formed after 12 to 24 hours. Subsequently, after 24 to 32 hours, a flagellum-dependent dispersal of cells became visible, extending laterally outward from a line of flagellum-independent motility. These modes of taxis were found in several environmental isolates and in a variety of mutants, including a strain deficient in the production of the R. capsulatus acyl-homoserine lactone quorum-sensing signal. Although there was great variability in the direction of movement in illuminated plates, cells were predisposed to move toward broad spectrum white light. This predisposition was increased by the use of square plates, and a statistical analysis indicated that R. capsulatus is capable of genuine phototaxis. Therefore, the variability in the direction of cell movement was attributed to optical effects on light waves passing through the plate material and agar medium.