Effects of glycine on cell growth and pigment biosynthesis in Rhodobacter azotoformans.

The effect of exogenous glycine (a precursor for the biosynthesis of bacteriochlorophyll) on the cell growth and photopigment accumulation was investigated in phototrophic growing Rhodobacter azotoformans 134K20. The growth rate and the biomass of strain 134K20 were significantly inhibited by glycine addition when ammonium sulfate or glutamate were used as nitrogen sources and acetate or succinate as carbon sources. A characteristic absorption maximum at approximately 423 nm was present in the absorption spectra of glutamate cultures while it was absent by the addition of high-concentration glycine of 15 mM. The component account for the 423 nm peak was eventually identified as magnesium protoporphyrin IX monomethyl ester, a precursor of bacteriochlorophyll a (BChl a). Comparative analysis of pigment composition revealed that the amount of BChl a precursors was significantly decreased by the addition of 15-mM glycine while the BChl a accumulation was increased. Moreover, glycine changed the carotenoid compositions and stimulated the accumulation of spheroidene. The A850 /A875 in the growth-inhibited cultures was increased, indicating an increased level of the light-harvesting complex 2 compared to the reaction center. The exogenous glycine possibly played an important regulation role in photosynthesis of purple bacteria.

Evaluation of Lighting Systems, Carbon Sources, and Bacteria Cultures on Photofermentative Hydrogen Production.

Fluorescent and incandescent lighting systems were applied for batch photofermentative hydrogen production by four purple non-sulfur photosynthetic bacteria (PNSB). The hydrogen production efficiency of Rhodopseudomonas palustris, Rhodobacter sphaeroides, Rhodobacter capsulatus, and Rhodospirillum rubrum was evaluated using different carbon sources (acetate, butyrate, lactate, and malate). Incandescent light was found to be more effective for bacteria cell growth and hydrogen production. It was observed that PNSB followed substrate selection criteria for hydrogen production. Only R. palustris was able to produce hydrogen using most carbon sources. Cell density was almost constant, but cell growth rate and hydrogen production were significantly varied under the different lighting systems. The kinetics study suggested that initial substrate concentration had a positive correlation with lag phase duration. Among the PNSB, R. palustris grew faster and had higher hydrogen yields of 1.58, 4.92, and 2.57 mol H2/mol using acetate, butyrate, and lactate, respectively. In the integrative approach with dark fermentation effluents rich in organic acids, R. palustris should be enriched in the phototrophic microbial consortium of the continuous hydrogen production system.

Single-stage photofermentative biohydrogen production from sugar beet molasses by different purple non-sulfur bacteria.

Biohydrogen production via fermentative routes offers considerable advantages in waste recycling and sustainable energy production. This can be realized by single-stage dark or photofermentative processes, or by a two-stage integrated process; the latter offering the higher production yields due to complete conversion of sugar substrates into H2 and CO2. However, problems arising from the integration of these two processes limit its scale-up and implementation. Hence, high efficiency one-step fermentative biohydrogen production processes from sugar-rich wastes are preferable. In this study, different strains of purple non-sulfur bacteria were investigated for their biohydrogen production capacity on pure sucrose and sugar beet molasses, and the feasibility of single-stage photofermentative biohydrogen production was evaluated. A single-stage photofermentation process was carried out using four different strains of purple non-sulfur bacteria (Rhodobacter capsulatus DSM 1710, R. capsulatus YO3, Rhodobacter sphaeroides O.U.001, and Rhodopseudomonas palustris DSM 127) on different initial sucrose concentrations. The highest hydrogen yield obtained was 10.5 mol H2/mol of sucrose and the maximum hydrogen productivity was 0.78 mmol/L h by Rp. palustris on 5 mM sucrose. A hydrogen yield of 19 mol H2/mol sucrose, which represents 79% of theoretical yield, and a maximum hydrogen productivity of 0.55 mmol/L h were obtained by Rp. palustris from sugar beet molasses. The yield was comparable to those values obtained in two-stage processes. The present study demonstrates that single-stage photofermentation using purple non-sulfur bacteria on sucrose-based wastes is promising.

Effects of aeration and internal recycle flow on nitrous oxide emissions from a modified Ludzak-Ettinger process fed with glycerol.

Nitrous oxide (N2O) is emitted from a modified Ludzak-Ettinger (MLE) process, as a primary activated sludge system, which requires mitigation. The effects of aeration rates and internal recycle flow (IRF) ratios on N2O emission were investigated in an MLE process fed with glycerol. Reducing the aeration rate from 1.5 to 0.5 L/min increased gaseous the N2O concentration from the aerobic tank and the dissolved N2O concentration in the anoxic tank by 54.4 and 53.4 %, respectively. During the period of higher aeration, the N2O-N conversion ratio was 0.9 % and the potential N2O reducers were predominantly Rhodobacter, which accounted for 21.8 % of the total population. Increasing the IRF ratio from 3.6 to 7.2 decreased the N2O emission rate from the aerobic tank and the dissolved N2O concentration in the anoxic tank by 56 and 48 %, respectively. This study suggests effective N2O mitigation strategies for MLE systems.

Biodegradation of Various Aromatic Compounds by Enriched Bacterial Cultures: Part A-Monocyclic and Polycyclic Aromatic Hydrocarbons.

Present study focused on the screening of bacterial consortium for biodegradation of monocyclic aromatic hydrocarbon (MAH) and polycyclic aromatic hydrocarbons (PAHs). Target compounds in the present study were naphthalene, acenaphthene, phenanthrene (PAHs), and benzene (MAH). Microbial consortia enriched with the above target compounds were used in screening experiments. Naphthalene-enriched consortium was found to be the most efficient consortium, based on its substrate degradation rate and its ability to degrade other aromatic pollutants with significantly high efficiency. Substrate degradation rate with naphthalene-enriched culture followed the order benzene > naphthalene > acenaphthene > phenanthrene. Chryseobacterium and Rhodobacter were discerned as the predominant species in naphthalene-enriched culture. They are closely associated to the type strain Chryseobacterium arthrosphaerae and Rhodobacter maris, respectively. Single substrate biodegradation studies with naphthalene (PAH) and benzene (MAH) were carried out using naphthalene-enriched microbial consortium (NAPH). Phenol and 2-hydroxybenzaldehyde were identified as the predominant intermediates during benzene and naphthalene degradation, respectively. Biodegradation of toluene, ethyl benzene, xylene, phenol, and indole by NAPH was also investigated. Monod inhibition model was able to simulate biodegradation kinetics for benzene, whereas multiple substrate biodegradation model was able to simulate biodegradation kinetics for naphthalene.

[Characterization of a new strain of a purple nonsulfur bacterium from a thermal spring].

A new budding nonsulfur purple bacterium of the genus Rhodobacter (strain Ku-2) was isolated from a mat of a moderately thermal spring (Baikal rift zone, Buryat Republic, Russia). The bacterium had lamellar photosynthetic membranes, which are typica of only one Rhodobacter species, Rba. blasticus. The cells contined spheroidene carotenoids and bacteriochlorophyll a (Bchl a). In vivo absorption spectrum of the cells had the major maximum at 863 nm and an additional peak at 887 nm, which is characteristic of the pigment-protein complexes of Bchl a-containing membranes. The previously described Rba. blasticus strains did not exhibit a 887-nm maximum. The new isolate was photoheterotrophic, with optimal growth occurring at 35 degrees C, 3 g/L NaCl, and pH 7-8. The DNA G+C content was 64.4 mol %. The similarity between the 16S rRNA gene sequences of strain Ku-2 and the Rba. blasticus type strain was 98.7%. The similarity between the PufM amino acid sequences of strain Ku-2 and the previously studied Rba. blasticus strain was 89.0%. Thus, the bacterial strain Ku-2 belonged to the genus Rhodobacter and was phylogenetically related to Rba. blasticus.

Continuous cultivation of photosynthetic bacteria for fatty acids production.

In the present work, we introduced a novel approach for microbial fatty acids (FA) production. Photosynthetic bacteria, Rhodobacter sphaeroides KD131, were cultivated in a continuous-flow, stirred-tank reactor (CFSTR) at various substrate (lactate) concentrations. At hydraulic retention time (HRT) 4d, cell concentration continuously increased from 0.97 g dcw/L to 2.05 g dcw/L as lactate concentration increased from 30 mM to 60mM. At 70 mM, however, cell concentration fluctuated with incomplete substrate degradation. By installing a membrane unit to CFSTR, a stable performance was observed under much higher substrate loading (lactate 100mM and HRT 1.5d). A maximum cell concentration of 16.2g dcw/L, cell productivity of 1.9 g dcw/L/d, and FA productivity of 665 mg FA/L/d were attained, and these values were comparable with those achieved using microalgae. The FA content of R. sphaeroides was around 35% of dry cell weight, mainly composed of vaccenic acid (C18:1, omega-7).

Transient response of microbial communities in a water well field to application of an impressed current.

Deterioration of water wells due to clogging and corrosion over time is a common problem where solutions may be costly and ineffective. Pilot studies have suggested that impressed current or cathodic protection may be used to reduce microbially-induced declines in water well performance. Two water wells in an alluvial aquifer close to the North Saskatchewan River were selected to study the response of subsurface microbial communities to the application of an impressed current as an anti-fouling technology. The treated well was exposed to an impressed current while the untreated well was used as a reference site. Biofilms grown on in situ coupons under the influence of the impressed current were significantly (p < 0.05) thicker (mean thickness = 67.3 µm) when compared to the biofilms (mean thickness = 19.3 µm) grown outside the electric field. Quantitative PCR analyses showed significantly (p < 0.05) higher numbers of total bacteria, iron- and nitrate-reducers in the electrified zone. Molecular analysis revealed that the predominant bacteria present in biofilms grown under the influence of the impressed current belonged to Rhodobacter spp., Sediminibacterium spp. and Geobacter spp. In addition to favouring the growth of biofilms, direct microscopic and ICP-AES analyses revealed that the impressed current also caused the deposition of iron and manganese on, and in the vicinity of, the well screen. Together, these factors contributed to rapid clogging leading to reduced specific pumping capacities of the treated well. The study revealed that the impressed current system was not effective as an anti-fouling technology but actually promoted both microbial growth and physical clogging in this aquifer.

Rhodobacter megalophilus sp. nov., a phototroph from the Indian Himalayas possessing a wide temperature range for growth.

Two strains of phototrophic, purple non-sulfur bacteria capable of growing at low temperatures (5 degrees C) were isolated from the Himalayas. The two strains showed positive phototaxis and grew over a relatively wide temperature range (5-40 degrees C). Phylogenetic analysis based on 16S rRNA gene sequences showed that strain JA194T clustered with members of the genus Rhodobacter. Strain JA194T showed highest 16S rRNA gene sequence similarity with Rhodobacter sphaeroides DSM 158T (99 %). However, DNA-DNA hybridization experiments between Rba. sphaeroides DSM 158T and strain JA194T revealed a level of relatedness of only 67 %. The DNA base composition of strain JA194T was 66.67 mol% G+C (by HPLC). Based on 16S rRNA gene sequence analysis, morphological, physiological, Fourier transform infrared fingerprinting and DNA-DNA hybridization studies, strain JA194T (=KCTC 5602T =JCM 14598T) is sufficiently different from other Rhodobacter species to merit its description as the type strain of a novel species, for which the name Rhodobacter megalophilus sp. nov. is proposed.

Rhodobacter ovatus sp. nov., a phototrophic alphaproteobacterium isolated from a polluted pond.

A novel ovoid, phototrophic, purple non-sulfur bacterium was isolated in pure culture from a sample of sediment from an industrially polluted pond (Noor Mohammad Kunta) in Hyderabad, India. Strain JA234(T) was found to be Gram-negative and non-motile and grew photoheterotrophically with a number of organic compounds serving as carbon source/electron donor. Photo-organoheterotrophic and chemo-organoheterotrophic growth were demonstrated. Biotin and thiamine were required for growth of strain JA234(T). Phylogenetic analysis on the basis of 16S rRNA gene sequences showed that strain JA234(T) is most closely related to Rhodobacter azotoformans (97.3 % sequence similarity) and clusters with other species of the genus Rhodobacter of the family Rhodobacteraceae. However, DNA-DNA hybridization with Rba. azotoformans JCM 9340(T) showed a relatedness of only 31.3 % with respect to strain JA234(T). On the basis of 16S rRNA gene sequence analysis, DNA-DNA hybridization data and morphological and physiological characteristics, strain JA234(T) represents a novel species of the genus Rhodobacter, for which the name Rhodobacter ovatus sp. nov. is proposed. The type strain is JA234(T) (=JCM 14779(T)=CCUG 55049(T)).

Rhodobacter changlensis sp. nov., a psychrotolerant, phototrophic alphaproteobacterium from the Himalayas of India.

A Gram-negative, non-motile, oval to rod-shaped, psychrotolerant, phototrophic, purple non-sulfur bacterium (designated strain JA139T) was isolated from a snow sample from Changla Pass in the Indian Himalayas. Strain JA139T had vesicular-type intracytoplasmic membrane structures and contained bacteriochlorophyll a and most probably spheroidene-like carotenoids. Biotin, niacin and thiamine were required for growth of strain JA139T. Phylogenetic analysis on the basis of 16S rRNA gene sequences showed that the strain clustered with species of the genus Rhodobacter but was distinctly separate from all recognized members of the family Rhodobacteraceae. Based on the genotypic and phenotypic differences observed between strain JA139T and recognized Rhodobacter species, strain JA139T is considered to represent a novel species of the genus, for which the name Rhodobacter changlensis sp. nov. is proposed. The type strain is JA139T (=DSM 18774T=CCUG 53722T=JCM 14338T).

Characterization of phototrophic purple nonsulfur bacteria forming colored microbial mats in a swine wastewater ditch.

The community structure of pink-colored microbial mats naturally occurring in a swine wastewater ditch was studied by culture-independent biomarker and molecular methods as well as by conventional cultivation methods. The wastewater in the ditch contained acetate and propionate as the major carbon nutrients. Thin-section electron microscopy revealed that the microbial mats were dominated by rod-shaped cells containing intracytoplasmic membranes of the lamellar type. Smaller numbers of oval cells with vesicular internal membranes were also found. Spectroscopic analyses of the cell extract from the biomats showed the presence of bacteriochlorophyll a and carotenoids of the spirilloxanthin series. Ubiquinone-10 was detected as the major quinone. A clone library of the photosynthetic gene, pufM, constructed from the bulk DNA of the biomats showed that all of the clones were derived from members of the genera Rhodobacter and Rhodopseudomonas. The dominant phototrophic bacteria from the microbial mats were isolated by cultivation methods and identified as being of the genera Rhodobacter and Rhodopseudomonas by studying 16S rRNA and pufM gene sequence information. Experiments of oxygen uptake with lower fatty acids revealed that the freshly collected microbial mats and the Rhodopseudomonas isolates had a wider spectrum of carbon utilization and a higher affinity for acetate than did the Rhodobacter isolates. These results demonstrate that the microbial mats were dominated by the purple nonsulfur bacteria of the genera Rhodobacter and Rhodopseudomonas, and the bioavailability of lower fatty acids in wastewater is a key factor allowing the formation of visible microbial mats with these phototrophs.

The response of Vibrio- and Rhodobacter-related populations of the NW Mediterranean Sea to additions of dissolved organic matter, phages, or dilution.

We investigated the growth response of the heterotrophic prokaryotic community focusing on Vibrio- and Rhodobacter-related populations (SRF3) to variation in the availability of dissolved organic matter (DOM), population density-dependent effects, and prokaryotic virus (phage) infection in coastal and offshore waters of the NW Mediterranean Sea. We tested the response of the prokaryotic community to three different DOM fractions prepared by ultrafiltration. One of the DOM fractions contained phages (<0.2 m), a second was virus-free (<100 kDa), and a third contained only low molecular weight (<1 kDa). The proportion of Vibrio and SRF3 populations as determined by fluorescent in situ hybridization in the community ranged from <1 to 6.2% and from 3.2 to 6.3%, respectively. Based on changes in cell numbers, growth rates ranged from 2.1 to 3.1 day(-1) for Vibrio and from 0.8 to 1.2 day(-1) for SRF3. Growth rates of Vibrio were similar or higher than those of the total prokaryotic community, whereas the ability of Vibrio to use high molecular weight (HMW) DOM and the responses to additions of phage-rich material were lower. Growth rates of SRF3 were lower than that of the community. Susceptibility to infection of SRF3 was sometimes lower than in the community, whereas the growth stimulation of HMW DOM was similar or lower. Reducing the cell concentrations of the prokaryotic community by dilution stimulated the overall growth of the community, including that of its constituent Vibrio and SRF3 populations, but the effect was smaller on the SRF3 and greater on Vibrio populations than for the total community. Comparisons with the community also revealed that life strategy traits of bacterial populations differed between coastal and offshore waters. Overall, our data suggest that Vibrio is an r-strategist or opportunistic population in the NW Mediterranean Sea, whereas SRF3 is a K-strategist or equilibrium population.

Investigation of Oscillatoria spongeliae-dominated bacterial communities in four dictyoceratid sponges.

Certain species of marine sponges in the order Dictyoceratida harbor large populations of the cyanobacterial symbiont Oscillatoria spongeliae in the mesohyl (interior) of the sponge. We show that in four of these sponge species (Lamellodysidea herbacea, Lamellodysidea chlorea, Lendenfeldia chondrodes, and Phyllospongia papyracea) from Palau there is a consistent community of alpha-proteobacteria in addition to O. spongeliae that fall within the Rhodobacter group based on 16S rRNA gene analysis. Some of the alpha-proteobacteria in Lendenfeldia chondrodes and P. papyracea but not in the Lamellodysidea spp. contained site-specific insertions in the 16S rRNA gene. Reverse transcription-PCR experiments demonstrated that the largest insertion found in this study (63 bp) is present in the mature rRNA. Lendenfeldia chondrodes was the only sponge found to have another cyanobacterium in the tissue, a Synechocystis sp. We found that the Synechocystis sp. was present in both the pinacoderm (surface epithelial tissue) and mesohyl, in contrast to O. spongeliae, which was only found in the mesohyl through the use of specific fluorescence in situ hybridization experiments. Of the four sponge species, only P. papyracea was found to contain a significant number of gamma-proteobacteria. These results demonstrate that O. spongeliae-dominated bacterial communities in different sponge species can vary considerably and increase our understanding of the bacterial communities found in marine invertebrates.

[Consumption of organic carbon sources and biosynthesis of lactic acid by the photosynthetic bacterium Rhodobacter sp. D-4]. / Usvoenie organicheskikh istochnikov ugleroda i biosintez molochnoi kisloty fotosinteziruiushchei bakteriei Rhodobacter sp., sht. D-4.

Nonsulfur photosynthetic purple bacteria isolated from the Dzhermuk mineral springs (Armenia) were grown on sugar-containing media and found to be capable of synthesizing L(+)-lactic acid. Various organic compounds were tested as possible sole sources of carbon and an electron donors required to support bacterial growth and biosynthesis of lactic acid under various growth conditions.

Proposal of Pseudorhodobacter ferrugineus gen nov, comb nov, for a non-photosynthetic marine bacterium, Agrobacterium ferrugineum, related to the genus Rhodobacter.

The marine gram-negative non-photosynthetic bacterium, Agrobacterium ferrugineum IAM 12616(T) forms one cluster with the species of the photosynthetic genus Rhodobacter in phylogenetic trees based on molecules of 16S rRNA, 23S rRNA and DNA gyrases. Agrobacterium ferrugineum and Rhodobacter species are similar in that growth occurs without NaCl in the culture medium (optimal NaCl concentration for growth of P. ferrugineus is 1%) and their major hydroxy fatty acid compositions are 3-hydroxy decanoic acids (3-OH 10:0) and 3-hydroxy tetradecanoic acids (3-OH 14:1). However, A. ferrugineum differs from Rhodobacter species in G+C content (58 mol% in A. ferrugineum versus 64-73 mol% in Rhodobacter species), in having an insertion in its 16S rRNA gene sequence, and in lacking photosynthetic abilities, bacteriochlorophyll a and intracytoplasmic membrane systems. Furthermore, experiments using PCR and Southern hybridization show that A. ferrugineum does not have puhA gene and puf genes localized near the opposite ends of the photosynthesis gene cluster of Rhodobacter capsulatus. It suggests that A. ferrugineum may not have any genes for photosynthesis. We propose the transfer of A. ferrugineum IAM 12616(T) to the genus Pseudorhodobacter gen. nov. as Pseudorhodobacter ferrugineus comb. nov. Although Pseudorhodobacter ferrugineus disturbs the phylogenetic monophyly of the genus Rhodobacter, this taxonomic proposal seems adequate until it has been clarified whether P. ferrugineus possesses an incomplete photosynthetic apparatus.

Growth and production of biomass of Rhodovulum sulfidophilum in sardine processing wastewater.

AIMS: Rhodovulum sulfidophilum was grown in sardine processing wastewater to assess growth characteristics for the production of bacterial biomass with simultaneous reduction of chemical oxygen demand. METHODS AND RESULTS: Growth characteristics were compared in diluted and undiluted, settled and non-settled wastewater growing in anaerobic light and aerobic dark conditions; and also at different agitation speeds. The highest biomass (8.75 g l(-1)) and a reduction in chemical oxygen demand of 71% were obtained in unsettled, undiluted wastewater after 120 h culture with 15% inoculum. In settled wastewater, highest biomass (7.64 g l(-1)) and a COD reduction of 77% was also obtained after 120 h. Total biomass was higher (4.34 g l(-1)) after 120 h culture in anaerobic light compared to (3.23 g l(-1)) in aerobic dark growth. CONCLUSIONS, SIGNIFICANCE AND IMPACT OF THE STUDY: Better performance, mean of total biomass (6.97 g l(-1) after 96 h), total carotenoids (4.24 mg g(-1) dry cell from 24 h) and soluble protein (431 microg ml(-1) after 96 h) were obtained from aerobic dark culture at 300 rev min(-1). The COD reduction, however, was lower (69%) after 96 h culture. Thus, the benefits in the production of bacterial biomass in non-sterilized sardine processing wastewater with the reduction of chemical oxygen demand could be achieved.

Influence of sulfate-reducing bacteria on outdoor hydrogen production by photosynthetic bacterium with seawater.

The application of seawater for bacterial fermentative production is a cost-effective technology. Hydrogen production by marine photosynthetic bacterium with seawater failed to continue after more than 10 days, and was accompanied by the formation of hydrogen sulfide and a change in culture color from red to black. However, substrate consumption in the blackish culture was comparable to that in a hydrogen-producing culture. A decrease in hydrogen production occurred upon the addition of sodium sulfide at concentrations of 1.5 mM or higher. PCR analysis targeted at the 16S rDNA sequence selective for sulfate-reducing bacteria revealed the existence of sulfate-reducing bacteria in inoculation cultures of the phototrophic bacterium and medium for hydrogen production. Hence, the high sulfate concentration of seawater, the low oxidation-reduction potential under hydrogen-producing conditions, and the presence of electron donors such as acetate might promote the metabolic activities of sulfate-reducing bacteria, resulting in the deterioration of hydrogen production with seawater.

MELISSA: global control strategy of the artificial ecosystem by using first principles models of the compartments.

MELISSA is a micro-organisms based ecosystem conceived as a tool for understanding the behaviour of artificial ecosystems, and developing the technology for a future biological life support system for long term space mission. The driving element of MELISSA is the recovering of oxygen and edible biomass from waste (faeces, urea). Due to its intrinsic instability and the safety requirements of manned missions, an important control strategy is developed to pilot this system and to optimize its recycling performance. This is a hierarchical control strategy. Each MELISSA compartment has its local control system, and taking into account the states of other compartments and a global desired functioning point, the upper level determines the setpoints for each compartment. The developed approach is based on first principles models of each compartment (physico chemical equations, stoichiometries, kinetic rates, ...). Those models are used to develop a global simulator of the system (in order to study the global functioning). They are also used in the control strategy, which is a non linear predictive model based strategy. This paper presents the general approach of the control strategy of the loop from the compartment level up to the overall loop. At the end, some simulation and experimental results are presented.

Biosynthesis of the 3-acetyl and 13(1)-oxo groups of bacteriochlorophyll a in the facultative aerobic bacterium, Rhodovulum sulfidophilum--the presence of both oxygenase and hydratase pathways for isocyclic ring formation.

Using (18)O-labelling and mass spectrometry, we have examined bacteriochlorophyll a formation in Rhodovulum sulfidophilum, formerly known as Rhodobacter sulfidophilus, which forms large amounts of BCh1 a both aerobically in the dark and anaerobically in the light. R. sulfidophilum, growing under strict anaerobiosis in the light, possesses hydratases which incorporate (18)O label from H2(18)O into both the 13(1)-oxo and 3-acetyl oxygens; in addition, the four carboxyl oxygens at C13(3) and C17(3) were labelled by H2(18)O. Under aerobic conditions in the dark, the labelling of the 13(1)-oxo group by H2(18)O was reduced indicating that (16)O was being incorporated into this group from air. R. sulfidophilum, grown in the dark under an atmosphere initially containing 50% (18)O2 in Ar, possessed an oxygenase which incorporated (18)O label from (18)O2 specifically into the 13(1)-oxo group; under these conditions the acetyl and carboxyl groups remained unlabelled. Thus, both an oxygenase and hydratase operate in R. sulfidophilum to form the 13(1)-oxo group of ring E of BCh1 a; the 3-acetyl group oxygen, however, arises only from water via a hydratase.