High-pressure, high-temperature bioreactor for comparing effects of hyperbaric and hydrostatic pressure on bacterial growth.

We describe a high-pressure reactor system suitable for simultaneous hyperbaric and hydrostatic pressurization of bacterial cultures at elevated temperatures. For the deep-sea thermophile ES4, the growth rate at 500 atm (1 atm = 101.29 kPa) and 95 degrees C under hydrostatic pressure was ca. three times the growth rate under hyperbaric pressure and ca. 40% higher than the growth rate at 35 atm.     

Subsampling technique for measuring growth of bacterial cultures under high hydrostatic pressure.

A method is presented for measuring growth of bacteria under high hydrostatic pressure in subsamples taken without pressure change in the incubation vessel. Subsamples may be withdrawn rapidly (5 s) and are not subjected to shear forces. Vice versa, nutrient media, labeled substrates, etc., may be introduced into the culture while under pressure. Chemical fixation of subsamples for electron microscopy or adenosine 5'-triphosphate determinations under pressure is also possible without affecting the growing culture. Data are given of growth experiments demonstrating the feasibility of the method. Problems of oxygen depletion are discussed.     

Pneumatic pressure bioreactor for cyclic hydrostatic stress application: mechanobiology effects on periodontal ligament cells

A bioreactor system was developed to provide high-amplitude cyclic hydrostatic compressive stress (cHSC) using compressed air mixed commercially as needed to create partial pressures of oxygen and carbon dioxide appropriate for the cells under investigation. Operating pressures as high as 300 psi are achievable in this system at cyclic speeds of up to 0.2 Hz. In this study, ligamentous fibroblasts from human periodontal ligaments (n = 6) were compressed on two consecutive days at 150 psi for 3 h each day, and the mRNA for families of extracellular matrix protein and protease isoforms was evaluated by real-time PCR array. Several integrins were significantly upregulated, most notably alpha-3 (6.4-fold), as was SPG7 (12.1-fold). Among the collagens, Col8a1 was highly upregulated at 53.5-fold, with Col6a1, Col6a2, and Col7a1 also significantly upregulated 4.4- to 8.5-fold. MMP-1 was the most affected at 122.9-fold upregulation. MMP-14 likewise increased 17.8-fold with slight reductions for the gelatinases and a significant increase of TIMP-2 at 5.8-fold. The development of this bioreactor system and its utility in characterizing periodontal ligament fibroblast mechanobiology in intermediate-term testing hold promise for better simulating the conditions of the musculoskeletal system and the large cyclic compressive stresses joints may experience in gait, exertion, and mastication.     

Influence of hydrostatic pressure on the effects of the heavy metal cations of manganese, copper, cobalt, and nickel on the growth of three deep-sea bacterial isolates.

Increases hydrostatic pressure varied the 72-h growth yield of three bacterial isolates from the deep sea in the presence of heavy metal cations of Mn, Cu, Co, and Ni, depending on the bacterial isolate, the metal cation and its concentration, and the level of hydrostatic pressure. Above atmospheric, hydrostatic pressure was found to have one of the following four effects on the response of culture growth to a heavy metal cation. (i) It could be without effect; (ii) it could enhance inhibition by a metal cation; (iii) it could increase the 72-h growth yield by a metal cation; or (iv) it could protect against a growth inhibitory effect noted at a lower pressure. Possible reasons for these varied responses are discussed.     

Effects of high hydrostatic pressure on bacterial growth on human ossicles explanted from cholesteatoma patients

Background

High hydrostatic pressure (HHP) treatment can eliminate cholesteatoma cells from explanted human ossicles prior to re-insertion. We analyzed the effects of HHP treatment on the microbial flora on ossicles and on the planktonic and biofilm states of selected isolates.

Methodology

Twenty-six ossicles were explanted from cholesteatoma patients. Five ossicles were directly analyzed for microbial growth without further treatment. Fifteen ossicles were cut into two pieces. One piece was exposed to HHP of 350 MPa for 10 minutes. Both the treated and untreated (control) pieces were then assessed semi-quantitatively. Three ossicles were cut into two pieces and exposed to identical pressure conditions with or without the addition of one of two different combinations of antibiotics to the medium.Differential effects of 10-minute in vitro exposure of planktonic and biofilm bacteria to pressures of 100 MPa, 250 MPa, 400 MPa and 540 MPa in isotonic and hypotonic media were analyzed using two patient isolates of Staphylococcus epidermidis and Neisseria subflava. Bacterial cell inactivation and biofilm destruction were assessed by colony counting and electron microscopy.

Principal Findings

A variety of microorganisms were isolated from the ossicles. Irrespective of the medium, HHP treatment at 350 MPa for 10 minutes led to satisfying but incomplete inactivation especially of Gram-negative bacteria. The addition of antibiotics increased the efficacy of elimination. A comparison of HHP treatment of planktonic and biofilm cells showed that the effects of HPP were reduced by about one decadic logarithmic unit when HPP was applied to biofilms.High hydrostatic pressure conditions that are suitable to inactivate cholesteatoma cells fail to completely sterilize ossicles even if antibiotics are added. As a result of the reduced microbial load and the viability loss of surviving bacteria, however, there is a lower risk of re-infection after re-insertion.     

Energetics of streptococcal growth inhibition by hydrostatic pressure.

Growth of Streptococcus faecalis in complex media with various fuel sources appeared to be limited by the rate of supply of adenosine-5' -triphosphate (ATP) at 1 atm and also under 408 atm of hydrostatic pressure. Growth under pressure was energetically inefficient, as indicated by an average cell yield for exponentially growing cultures of only 10.7 g (dry weight) per mol of ATP produced compared with a 1-atm value of 15.6. Use of ATP for pressure-volume work or for turnover of protein, peptidoglycan, or stable ribonucleic acid (RNA) did not appear to be significant causes of growth inefficiency under pressure. In addition, there did not seem to be an increased ATP requirement for ion uptake because cells growing at 408 atm had significantly lower internal K(+) levels than did those growing at 1 atm. Pressure did stimulate the membrane adenosine triphosphatase (ATPase) or S. faecalis at ATP concentrations greater than 0.5 mM. Intracellular ATP levels were found to vary during the culture cycle from about 2.5 mumol/ml of cytoplasmic water for lag-phase or stationary-phase cells to maxima for exponentially growing cells of about 7.5 mumol/ml at 1 atm and 5.5 mumol/ml at 408 atm. N,N'-dicyclohexylcarbodiimide at a 10 muM concentration improved growth efficiency under pressure, as did Mg(2+) or Ca(2+) ions at 50 mM concentration. These agents also enhanced ATP pooling, and it seemed that at least part of the growth inefficiency under pressure was due to increased ATPase activity. In all, it appeared that S. faecalis growing under pressure has somewhat reduced ATP supply but significantly increased demand and that the inhibitory effects of pressure can be interpreted largely in terms of ATP supply and demand.     

Exploration of the effects of high hydrostatic pressure on microbial growth, physiology and survival: perspectives from piezophysiology

The discovery of piezophiles (previously referred to as barophiles) prompted researchers to investigate the survival strategies they employ in high-pressure environments. There have been innovative high-pressure studies on biological processes applying modern techniques of genetics and molecular biology in bacteria and yeasts as model organisms. Recent advanced studies in this field have shown unexpected outcomes in microbial growth, physiology and survival when living cells are subjected to high hydrostatic pressure. The effects are conceptually dependent on the sign and magnitude of volume changes associated with any chemical reaction in the cells. Nevertheless, it is difficult to explain the pressure effects on complex metabolic networks based on a simple volume law. The challenges in piezophysiology are to discover whether the physiological responses of living cells to high pressure are relevant to their growth and to identify the critical factors in cell viability and lethality under high pressure from the general and organism-specific viewpoints.     

Antagonistic effects of hydrostatic pressure and osmotic pressure on cytochrome P-450cam spin transition.

The combined effects of hydrostatic pressure and osmotic pressure, generated by polyols, on the spin equilibrium of fenchone-bound cytochrome P-450cam were investigated. Hydrostatic pressure indices a high spin to low spin transition, whereas polyols induce the reversed reaction. Of the four solutes used, glycerol, glucose, stachyose, and sucrose, only the last two would act on the spin transition by osmotic stress. The spin volume changes measured by both techniques are different, 29 and -350 ml/mol for hydrostatic pressure and osmotic pressure, respectively. It suggests that even if the two are perturbing water molecules, different properties are probed. From the volume change induced by osmotic stress, 19 water molecules are deduced that would be implicated in the spin transition of the fenchone-bound protein. This result suggests that water molecules other than the well defined ones located in the active site play a key role in modulating the spin equilibrium of cytochrome P-450cam.     

Effect of high hydrostatic pressure on the bacterial mechanosensitive channel MscS

We have investigated the effect of high hydrostatic pressure on MscS, the bacterial mechanosensitive channel of small conductance. Pressure affected channel kinetics but not conductance. At negative pipette voltages (corresponding to membrane depolarization in the inside-out patch configuration used in our experiments) the channel exhibited a reversible reduction in activity with increasing hydrostatic pressure between 0 and 900 atm (90 MPa) at 23°C. The reduced activity was characterized by a significant reduction in the channel opening probability resulting from a shortening of the channel openings with increasing pressure. Thus high hydrostatic pressure generally favoured channel closing. Cooling the patch by approximately 10°C, intended to order the bilayer component of the patch by an amount similar to that caused by 50 MPa at 23°C, had relatively little effect. This implies that pressure does not affect channel kinetics via bilayer order. Accordingly we postulate that lateral compression of the bilayer, under high hydrostatic pressure, is responsible. These observations also have implications for our understanding of the adaptation of mechanosensitive channels in deep-sea bacteria.A Proceeding of the 28th Annual Meeting of the Australian Society for Biophysics.     

An improved sampling technique for bacterial cultures under high hydrostatic pressure

A sampling technique for bacterial cultures subjected to high hydrostatic pressure is described. A sample-receiving vessel with a motor driven interface-piston is employed. By precisely matching the pressures in the bulk culture and the sample-receiving vessel, none of the sample is subjected to the high shear forces common to other desings of high pressure sampler. The use of the technique was illustrated by the growth of an anaerobic culture at 300 bar and 75°C.     

High-pressure-temperature bioreactor for studying pressure-temperature relationships in bacterial growth and productivity

Thermophilic organisms offer many potential advantages for biotechnological processes; however, realization of the promise of thermophiles will require extensive research on bacterial thermophily and high-temperature cultivation systems. This article describes a novel bioreactor suitable for precise studies of microbial growth and productivity at temperatures up to 260 degrees C and pressures up to 350 bar. The apparatus is versatile and corrosion resistant, and enables direct sampling of both liquids and gases from a transparent culture vessel without altering the reaction conditions. Gas recirculation through the culture can be controlled through the action of a magnetically driven pump. Initial studies in this bioreactor of Methanococcus jannaschii, an extremely thermophilic methanogen isolated from a deep-sea hydrothermal vent, revealed that increasing the pressure from 7.8 to 100 bar accelerated the production of methane and cellular protein by this archaebacterium at 90 degrees C, and raised the maximum temperature allowing growth from 90 to 92 degrees C. Further increases in pressure had little effect on the growth rate at 90 degrees C.