Non-aerated cultivation ofHalobacterium cutirubrum and its effect on cellular squalenes

Summary Halobacterium cutirubrum was successfully cultivated under aerobic and microaerobic conditions. The early stationary phase of growth was obtained at 2.2 days and 45–55 days for aerated and non-aerated cultures, respectively. The dry cell yields were 0.7–1.2 gm/l in all preparations grown to early stationary growth phase. The cellular ratio of squalene to dihydro- and tetra-hydrosqualene decreased proportionately with decreased aeration rates.     

Poly(3-hydroxybutyrate) synthesis from glycerol by a recombinant Escherichia coli arcA mutant in fed-batch microaerobic cultures

Poly(3-hydroxybutyrate) (PHB) synthesis was analyzed under microaerobic conditions in a recombinant Escherichia coli arcA mutant using glycerol as the main carbon source. The effect of several additives was assessed in a semi-synthetic medium by the ‘one-factor-at-a-time’ technique. Casein amino acids (CAS) concentration was an important factor influencing both growth and PHB accumulation. Three factors exerting a statistically significant influence on PHB synthesis were selected by using a Plackett–Burman screening design [glycerol, CAS, and initial cell dry weight (CDW) concentrations] and then optimized through a Box–Wilson design. Under such optimized conditions (22.02 g l⁻¹ glycerol, 1.78 g l⁻¹ CAS, and 1.83 g l⁻¹ inoculum) microaerobic batch cultures gave rise to 8.37 g l⁻¹ CDW and 3.52 g l⁻¹ PHB in 48 h (PHB content of 42%) in a benchtop bioreactor. Further improvements in microaerobic PHB accumulation were obtained in fed-batch cultures, in which glycerol was added to maintain its concentration above 5 g l⁻¹. After 60 h, CDW and PHB concentration reached 21.17 and 10.81 g l⁻¹, respectively, which results in a PHB content of 51%. Microaerobic fed-batch cultures allowed a 2.57-fold increase in volumetric productivity when compared with batch cultures. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. An erratum to this article can be found at     

Mineralization of the herbicide 2,3,6-trichlorobenzoic acid by a co-culture of anaerobic and aerobic bacteria

Abstract Bacteria from an anaerobic enrichment reductively removed chlorine from the ortho- position of 2,3,6-trichlorobenzoic acid (2,3,6-TBA) producing 2,5-dichlorobenzoate (2,5-DBA). The strictly aerobic bacterium Pseudomonas aeruginosa JB2 subsequently used 2,5-DBA as a growth substrate in the presence of oxygen. The anaerobic dechlorinating microbial population was grown with P. aeruginosa JB2 in continuous culture. Inside the liquid culture, a nylon netting, on a stainless-steel support, contained vermiculite particles to provide a strictly anaerobic environment within the aerated culture. Complete mineralization of 2,3,6-TBA depended on the extent of oxygen input into the reactor. Under strictly anaerobic conditions 2,5-DBA and Cl⁻ were produced stoichiometrically through the reductive dechlorination of 2,3,6-TBA. This process of reductive dechlorination was not inhibited by (moderate) aeration resulting in an O₂-concentration of 0.3–0.5 μM in the culture liquid.     

Mineralization of the herbicide 2,3,6-trichlorobenzoic acid by a co-culture of anaerobic and aerobic bacteria

Abstract Bacteria from an anaerobic enrichment reductively removed chlorine from the ortho - position of 2,3,6-trichlorobenzoic acid (2,3,6-TBA) producing 2,5-dichlorobenzoate (2,5-DBA). The strictly aerobic bacterium Pseudomonas aeruginosa JB2 subsequently used 2,5-DBA as a growth substrate in the presence of oxygen. The anaerobic dechlorinating microbial population was grown with P. aeruginosa JB2 in continuous culture. Inside the liquid culture, a nylon netting, on a stainless-steel support, contained vermiculite particles to provide a strictly anaerobic environment within the aerated culture. Complete mineralization of 2,3,6-TBA depended on the extent of oxygen input into the reactor. Under strictly anaerobic conditions 2,5-DBA and Cl⁻ were produced stoichiometrically through the reductive dechlorination of 2,3,6-TBA. This process of reductive dechlorination was not inhibited by (moderate) aeration resulting in an O₂-concentration of 0.3–0.5 μM in the culture liquid.     

Poly(3-hydroxybutyrate) synthesis from glycerol by a recombinant <Emphasis Type="Italic">Escherichia coli arcA</Emphasis> mutant in fed-batch microaerobic cultures

Poly(3-hydroxybutyrate) (PHB) synthesis was analyzed under microaerobic conditions in a recombinant Escherichia coli arcA mutant using glycerol as the main carbon source. The effect of several additives was assessed in a semi-synthetic medium by the 'one-factor-at-a-time' technique. Casein amino acids (CAS) concentration was an important factor influencing both growth and PHB accumulation. Three factors exerting a statistically significant influence on PHB synthesis were selected by using a Plackett-Burman screening design [glycerol, CAS, and initial cell dry weight (CDW) concentrations] and then optimized through a Box-Wilson design. Under such optimized conditions (22.02 g l(-1) glycerol, 1.78 g l(-1) CAS, and 1.83 g l(-1) inoculum) microaerobic batch cultures gave rise to 8.37 g l(-1) CDW and 3.52 g l(-1) PHB in 48 h (PHB content of 42%) in a benchtop bioreactor. Further improvements in microaerobic PHB accumulation were obtained in fed-batch cultures, in which glycerol was added to maintain its concentration above 5 g l(-1). After 60 h, CDW and PHB concentration reached 21.17 and 10.81 g l(-1), respectively, which results in a PHB content of 51%. Microaerobic fed-batch cultures allowed a 2.57-fold increase in volumetric productivity when compared with batch cultures.     

Bacterial hemoglobins and flavohemoglobins: versatile proteins and their impact on microbiology and biotechnology

In response to oxygen limitation or oxidative and nitrosative stress, bacteria express three kinds of hemoglobin proteins: truncated hemoglobins (tr Hbs), hemoglobins (Hbs) and flavohemoglobins (flavo Hbs). The two latter groups share a high sequence homology and structural similarity in their globin domain. Flavohemoglobin proteins contain an additional reductase domain at their C-terminus and their expression is induced in the presence of reactive nitrogen and oxygen species. Flavohemoglobins detoxify NO in an aerobic process, termed nitric oxide dioxygenase reaction, which protects the host from various noxious nitrogen compounds. Only a small number of bacteria express hemoglobin proteins and the best studied of these is from Vitreoscilla sp. Vitreoscilla hemoglobin (VHb) has been expressed in various heterologous hosts under oxygen-limited conditions and has been shown to improve growth and productivity, rendering the protein interesting for biotechnology industry. The close interaction of VHb with the terminal oxidases has been shown and this interplay has been proposed to enhance respiratory activity and energy production by delivering oxygen, the ultimate result being an improvement in growth properties.     

Investigation of granulation of a mixture of suspended anaerobic and aerobic cultures under alternating anaerobic/microaerobic/aerobic conditions

This is the first granulation study except Ferguson [Ferguson LN. Anaerobic codigestion of aircraft deicing fluid and microaerobic studies. M.S. Thesis. Milwaukee, WI, USA: Marquette University; 1999] to develop coupled granules by using a mixture of suspended anaerobic and aerobic cultures exposed to alternating cyclic anaerobic/microaerobic/aerobic conditions. Coupled granules with median sizes of 1.28–1.86 mm and settling velocities of 31–39 m/h were developed, which were comparable to those of both anaerobic and aerobic granules. Coupled granules displayed noteworthy specific methanogenic activity (SMA) and specific oxygen uptake rate (SOUR) as 14–42 mL CH₄/g VSS h and 6–47 mg DO/g VSS h, respectively, indicating that they were composed of both anaerobic and aerobic cultures.     

An integrated network analysis identifies how ArcAB enables metabolic oscillations in the nitric oxide detoxification network of Escherichia coli

The virulences of many pathogens depend on their abilities to detoxify the immune antimicrobial nitric oxide (NO?). The functions of bacterial NO? detoxification machinery depend on oxygen (O₂), with O₂ inhibiting some enzymes, whereas others use it as a substrate. Previously, Escherichia coli NO? detoxification was found to be highly attenuated under microaerobic conditions and metabolic oscillations were observed. The oscillations in [NO?] and [O₂] were found to result from the inhibitory action of NO? on aerobic respiration, the catalytic inactivation of NO? by Hmp (an NO? dioxygenase), and an imbalanced competition for O₂ between Hmp and cytochrome terminal oxidase activity. Here the authors investigated the role of the ArcAB two component system (TCS) in microaerobic NO? detoxification. The authors observed that wild‐type, ΔarcA , and ΔarcB had comparable initial NO? clearance times; however, the mutant cultures failed to exhibit [NO?] and [O₂] oscillations. Using an approach that employed experimentation and computational modeling, the authors found that the loss of oscillations in ΔarcA was due to insufficient induction of cytochrome bd ‐I expression. Collectively, these results establish ArcAB as a TCS that influences NO? detoxification in E. coli within the physiologically‐relevant microaerobic regime.     

Measurement of components of proton motive force and related parameters in steady,microaerobic conditions

An experimental system is described for the simultaneous measurement of components of the proton motive force and other energy-related activies in microorganisms under steady defined microaerobic conditions. Oxygen is supplied in a solution containing an O₂-carrier such as myoglobin or leghaemoglobin, to a stirred reaction chamber in which a suspension of the microorganism is required aboce a membrane filter whic is pervious to the carrier. The rate of O₂ consumption is regulated by the rate at which the solution is pumped through the chamber. The concentration of free O₂ in the chamber and the rate of its consumption are calculated from the pumping rate and the decline in the relative oxygenation of the carrier measured spectrophotometrically in the effluent solution. The uptake by the microorganisms of radioactively labelled probes for ΔpH and Δψ is calculated following their injection into the reaction chamber and monitoring of continuously collected fractions of effluent solution, after it has passed through the spectrophometer. An example of the use of the system is given.The use of this system is advocated for many microaerobic applications since most of the measurements can be made without perturbing the steady state until the final shape of the suspension is collected.     

Ethanol Production and Utilization by Aphelenchus avenae and Caenorhabditis sp.

In microaerobic and anaerobic environments the principal glycolytic end-product of A. avenae and Caenorhabditis sp. was lactic acid during the first 12-16 hr, after which it was ethanol. Upon return to aerobiosis, ¹⁴C-labeled ethanol in the medium was utilized by the nematodes; ¹⁴CO₂ and some ¹⁴C-labeled glycogen was detected. Total dry weight loss of non-feeding nematodes was 25% greater in the absence of alcohol than in the presence of ethanol or n-propanol. Physical movement and respiration increased and reproduction was extended by alcohol in the bathing solution.