Biotechnology report. Solid state fermentations

A unique method is described by which large yields of secondary metabolites arc produced on solid substrates. The process involves the use of moist substrates which are continuously agitated in appropriate fermentation equipment. The amount of agitation, aeration, and moisture can be varied. Extremely high yields of secondary metabolites such as ochratoxin and aflatoxin were obtained using Aspergillus and Penicillium species. The process prevents sporulation of the fungus and because of the nature of the solid substrate makes recovery of the product easier than in conventional liquid media. The substrates include rice, corn, wheat, and other cereals.     

Solid state approach in biophoton research

Main characteristics of the delayed luminescence (DL) emitted in the seconds range from biological systems is analyzed. The correlation between change in DL and cell's organization, and similarity with some characteristics of DL from solid state system suggest to connect DL in biological system to decay of collective electron states, formed during energy and charge transport along the macromolecular ordered structures which form the cell. Results of a proposed soliton model are discussed, together with some phenomenological evidence which emphasize the possibility of using DL measurements as an intrinsic probe in biophysical investigations.     

Solid state fermentation of banana wastes

Summary Using a strain ofAspergillus niger, the protein content of banana wastes was raised from 6 to 18 % by solid state fermentation. A meal of green bananas was obtained by solar drying, and fermentation was conducted in a 15 kg (dry weight) capacity stirred reactor. As the substrate consumption was 24 % of initial weight after 43 h. of fermentation, protein production was calculated to be 150 % of the initial content. According to its composition (50 % total sugars, 13 % reducing sugars, 18 % proteins) the fermented banana waste could be used as cattle feed.     

Solid state photochemistry of 1,4-dihydropyridines

Photochemical behavior of some 1,4-dihydropyridines has been investigated in the solid state. Whereas upon irradiation of 1,4-dihydropyridines in solution phase, their photo-oxidation and formation of pyridine derivatives have been observed, irradiation of these compounds in the solid state decreases their light sensitivity. In many cases photo-oxidation has been observed only in very low yields.     

Solid state fluorescence of lyophilized proteins

Fluorescence spectroscopy has been used to measure changes in the tertiary structure of proteins in the solution state. The sensitivity of fluorescence to the protein tryptophan environment has made it a useful tool for studying protein conformation and stability. Using fluorescence spectroscopy to probe structural alterations in lyophilized proteins has been limited due to technical challenges and overwhelming background light scattering. We have investigated the possibility of analyzing lyophilized proteins using the Cary-Eclipse spectrofluorometer by monitoring the fluorescence of the protein therapeutic after subjecting the lyophilized cake to heat-induced accelerated degradation. We have been able to obtain reproducible fluorescence spectra, detecting possible structural changes under these conditions. Fluorescence and circular dichroism spectroscopic analyses of the reconstituted proteins indicated that changes in fluorescence intensities observed in the solid state could be correlated to that in solution and to possible tertiary structural changes. Size exclusion chromatography analysis of protein Y subject to accelerated degradation showed a correlation between decreasing fluorescence intensity and increasing protein Y tetramer in solution, consistent with long-term stability. This suggests that solid state, intrinsic protein fluorescence measurements using the Cary-Eclipse holder may be feasible for long-term stability studies and formulation development.     

The mechanical behaviour of cancellous bone

Cancellous bone has a cellular structure: it is made up of a connected network of rods and plates. Because of this, its mechanical behaviour is similar to that of other cellular materials such as polymeric foams. A recent study on the mechanisms of deformation in such materials has led to an understanding of how their mechanical properties depend on their relative density, cell wall properties and cell geometry. In this paper, the results of this previous study are applied to cancellous bone in an attempt to further understand its mechanical behaviour. The results of the analysis agree reasonably well with experimental data available in the literature.     

Stiffness behaviour of trabecular bone specimens

Trabecular bone specimens were tested by non-destructive technique with the purpose of investigating stiffness behaviour and optimizing stiffness determination. Cylindrical specimens (n = 25) were loaded repetitively (0.1 Hz, 30 cycles) by axial compression to 50% of predicted ultimate strength and finally compressed to failure. Analyses of single compression curves showed increasing stiffness (E') until a stress level about 50% of ultimate stress followed by decreasing stiffness. Curve fit analysis of the elastic part of the compression curve showed the best fit, when a second order polynomial was used (r = 0.94, p less than 0.001). The stiffness determined non-destructively at the 25% level of ultimate strength increased significantly to the tenth loading cycle followed by a steady state. The precision of stiffness determination as an average of five consecutive measurements at steady state was E' +/- less than 5% (95% confidence limits). A reproducibility test by repetition of the test sequence after 3 h rest showed qualitatively the same stiffness behaviour. The variation of stiffness determination between the two test sequences was +/- 27% at the first loading cycle falling to +/- 12% at steady state.     

Stress relaxation behaviour of trabecular bone specimens

The present study defines several conditions under which stress relaxation tests can be performed and investigates the viscoelastic behaviour of trabecular bone in compression through a series of stress relaxation tests at three strain levels and in three loading directions of each cubic specimen. A visoelastic model is proposed to characterize the behaviour of trabecular bone and a spectrum of relaxation times is determined. Trabecular bone from the femoral head is non-linearly viscoelastic and displays anisotropic behaviour, which cannot be more symmetric elastically than orthotropic.     

Age-dependent fatigue behaviour of human cortical bone

Despite a general understanding that bone quality contributes to skeletal fragility, very little information exits on the age-dependent fatigue behavior of human bone. In this study four-point bending fatigue tests were conducted on aging bone in conjunction with the analysis of stiffness loss and preliminary investigation of nanoindentation based measurements of local tissue stiffness and histological evaluation of resultant tensile and compressive damage to identify the damage mechanism responsible for the increase in age-related bone fragility. The results obtained show that there is an exponential decrease in fatigue life with age, and old bone exhibits different modulus degradation profiles than young bone. In addition, this study provides preliminary evidence indicating that during fatigue loading, younger bone formed diffuse damage, lost local tissue stiffness on the tensile side. Older bone, in contrast, formed linear microcracks lost local tissue stiffness on the compressive side. Thus, the propensity of aging human bone to form more linear microcracks than diffuse damage may be a significant contributor to bone quality, and age related fragility in bone.     

Functional behaviour of bone around dental implants

Achieving a long-term stable implant interface is a significant clinical issue when there is insufficient cortical bone stabilisation at implant placement. Clinical outcomes studies suggest that the higher risk implants are those placed in compromised cortical bone (thin, porous, etc.) in anatomical sites with minimal existing trabecular bone (characterised as type IV bone). In establishing and maintaining an implant interface in such an environment, one needs to consider the impact of masticatory forces, the response of bone to these forces and the impact of age on the adaptive capacity of bone. These forces, in turn, have the potential to create localised changes in interfacial stiffness through viscoelastic changes at the interface. Changes in bone as a function of age (e.g. localised hypermineralised osteopetrosis and localised areas of osteopenia) will alter the communication between osteocytes and osteoblasts creating the potential for differences in response of osteoblastic cells in the older population. A key to understanding the biomechanical and functional behaviour of implants in the older population is to control the anticipated modelling and remodelling behaviour through implant design that takes into account how tissues respond to the mechanically active environment.     

The behaviour of microcracks in compact bone

This paper summarises four separate studies carried out by our group over the past number of years in the area of bone microdamage. The first study investigated the manner by which microcracks accumulate and interact with bone microstructure during fatigue testing of compact bone specimens. In a series of fatigue tests carried out at four different stress ranges between 50 and 80 MPA, crack density increased with loading cycles at a rate determined by the applied stress. Variations in the patterns of microdamage accumulation suggest that that at low stress levels, larger amounts of damage can build up without failure occurring. In a second study using a series of four-pont bending tests carried out on ovine bone samples, it was shown that bone microstructure influenced the ability of microcracks to propagate, with secondary osteons acting as barriers to crack growth. In a third study, the manner by which crack growth disrupts the canalicular processes connecting osteocytes was investigated. Analysis of individual cracks showed that disruption of the canalicular processes connecting osteocytes occurred due to shear displacement at the face of propagating microcracks, suggesting that this may play some role in the mechanism that signals bone remodelling. In a fourth in vivo study, it was shown that altering the mechanical load applied to the long bones of growing rats causes microcrack formation. In vivo microdamage was present in rats subjected to hindlimb suspension with a higher microcrack density found in the humeri than the femora. Microdamage was also found in control animals. This is the first study to demonstrate in vivo microcracks in normally loaded bones in a rat model.     

Solid state fermentation of dried citrus peel byAspergillus niger

Summary Solid state fermentation of dried citrus peel was studied in a packed bed reactor withAspergillus niger QH-2. When no nitrogen source was added the growth was limited. When urea and/or ammonium sulphate were added in the proportion of 0.63 g N/100 g solid dry substrate a diauxie was observed and the final content of crude protein was higher than 10%. The results of a factorial design show that ammonium sulphate has a significant effect on the specific growth rate.     

Solid state biosurfactant production in a fixed-bed column bioreactor

Biosurfactants are surface active substances which reduce interfacial tension and are produced or excreted at the microbial cell surface. We evaluated the biosurfactant production by Aspergillus fumigatus and Phialemonium sp. in solid state processes using fixed-bed column reactors. We evaluated two media, rice husks alone (simple support) and rice husks plus defatted rice bran (complex support), both enriched with either soy oil or diesel oil. The highest water-in-oil emulsifying activity (EAw/o) obtained was 7.36 EU g(-1) produced by A. fumigatus growing on complex support enriched with soy oil and supplied with air at a rate of 60 mL g(-1) h(-1), while Phialemonium sp. had a maximum production of 6.11 EU g(-1) using the simple support with diesel oil and an aeration rate of 120 mL g(-1) h(-1). The highest oil-in-water emulsifying activity (EAo/w) was 12.21 EU g(-1) produced by Phialemonium sp. on the complex support enriched with diesel oil and at an aeration rate of 60 mL g(-1) h(-1), while A. fumigatus produced a maximum EAo/w of 10.98 EU g(-1) when growing on the complex support with no additional carbon source and an aeration rate of 60 mL g(-1) 1 h(-1).     

Tailoring broadband inversion pulses for MAS Solid state NMR

A simple approach is demonstrated for designing optimised broadband inversion pulses for MAS solid state NMR studies of biological systems. The method involves a two step numerical optimisation procedure and takes into account experimental requirements such as the pulse length, resonance offset range and extent of H₁ inhomogeneity compensation needed. A simulated annealing protocol is used initially to find appropriate values for the parameters that define the well known tanh/tan adiabatic pulse such that a satisfactory spin inversion is achieved with minimum RF field strength. This information is then used in the subsequent stage of refinement where the RF pulse characteristics are further tailored via a local optimisation procedure without imposing any restrictions on the amplitude and frequency modulation profiles. We demonstrate that this approach constitutes a generally applicable tool for obtaining pulses with good inversion characteristics. At moderate MAS frequencies the efficacy of the method is experimentally demonstrated for generating double-quantum NMR spectra via the zero-quantum dipolar recoupling scheme RFDR.     

Corresponding state behaviour of capillary condensation of confined alkanes

The corresponding state behaviour of capillary condensation of confined alkanes is studied by means of GC-TMMC simulations. In this study, alkane is considered under graphite and mica slit pore confinements of various slit widths. Corresponding state phase coexistence envelopes of capillary condensation display insignificant effect of surface chemistry for slit pore confinements of 6 Å for methane and 15 Å for n-butane and n-octane. On the other hand, the significant effect of surface chemistry on the corresponding state coexistence envelope is observed for larger slit pore width. Moreover, at a given slit width, more shrinking in the corresponding state coexistence envelope of alkane is observed with the mica slit pore as compared to graphite slit pore. The local behaviour of capillary condensation of corresponding state is also investigated in some typical cases using local density profiles, to understand the overall behaviour of corresponding state capillary condensation. Moreover, for a given slit pore width smaller size alkane has shown more shrinking of the corresponding state coexistence envelope, irrespective of surface chemistry. However, the effect of surface chemistry on the corresponding state coexistence envelope of different alkanes becomes insignificant in smaller slit pore width. Fluctuating positive and negative deviations in the corresponding state vapour–liquid equilibrium pressure with respect to corresponding bulk values are observed in studied slit pore confinements. The corresponding state vapour?liquid interfacial tension of alkanes is less in mica slit pore as compared to graphite slit pore for the studied range of reduced temperatures.     

Fracture behaviour of commercial surgical acrylic bone cements

The resistance to slow growth of three commerical acrylic bone cements has been evaluated. It was found that small additions of 1μm diameter barium sulphate particles reduced the resistance to slow crack growth of Simplex P and CMW acrylic bone cements, while a small addition of the antibiotic Gentamicin had no detectable effect on cracking in Palacos R bone cement.     

Feeding behaviour and bone utilization by theropod dinosaurs

Hone, D.W.E. & Rauhut, O.W.M. 2009: Feeding behaviour and bone utilization by theropod dinosaurs. Lethaia, Vol. 43, pp. 232–244. Examples of bone exploitation by carnivorous theropod dinosaurs are relatively rare, representing an apparent waste of both mineral and energetic resources. A review of the known incidences and possible ecological implications of theropod bone use concludes that there is currently no definitive evidence supporting the regular deliberate ingestion of bone by these predators. However, further investigation is required as the small bones of juvenile dinosaurs missing from the fossil record may be absent as a result of theropods preferentially hunting and consuming juveniles. We discuss implications for both hunting and feeding in theropods based on the existing data. We conclude that, like modern predators, theropods preferentially hunted and ate juvenile animals leading to the absence of small, and especially young, dinosaurs in the fossil record. The traditional view of large theropods hunting the adults of large or giant dinosaur species is therefore considered unlikely and such events rare. □Behaviour, carnivory, palaeoecology, predation, resource utilization.     

Anisotropy of the fatigue behaviour of cancellous bone

The fatigue behaviour of materials is of particular interest for the failure prediction of materials and structures exposed to cyclic loading. For trabecular bone structures only a few sets of lifetime data have been reported in the literature and structural measures are commonly not considered. The influence of load contributions which are not aligned with the main physiological axis remains unclear. Furthermore site and species dependent relationships are not well described. In this study five different groups of trabecular bone, defined in terms of orientation, species and site were exposed to cyclic compression. In total, 108 fatigue tests were analysed. The lifetimes were found to decrease drastically when off-axis loads were applied. Additionally, species and site strongly affect fatigue lifetimes. Strains at failure were also found to be a function of orientation.     

Solid state fermentation for the production of lipolytic fungal enzymes

The production of lypolitic moulds in solid state fermentation was studied.Aspergillus oryzae andRhizopus oryzae were grown on rice hulls using different media. Tween 80 and olive oil were employed as main carbon sources; the whole solid cultures were lyophilised and employed in heptane for catalysing the formation of various geranyl esters with molar conversions ranging from 40 to 95%, under optimised conditions, starting from 50 mM geraniol and equimolar amount of the acid.