A new approach to improve the quality of ultrathin cryo-sections; its use for immunogold EM and correlative electron cryo-tomography

Cryo-ultramicrotomy can be used to obtain ultrathin cryo-sections from cryo-fixed or aldehyde-fixed cryo-protected vitreous biologic samples. For immuno-gold EM, cryo-sections are retrieved from the cryo-chamber on a droplet of a pick-up solution (paste-like and almost frozen) to which the sections attach. The sections are then placed on an EM specimen grid at room temperature. This procedure compromises the ultrastructure, resulting in folds, holes, and loss of the original material. In this paper we show the critical influence of humidity, stretching, and relief of compression during thawing of the sections. We show a new lift-up hinge device for semi-automated retrieval of cryo-sections that results in significantly improved section quality. This approach was also applied successfully to vitreous sections from high pressure frozen samples. An important advance is that these vitreous cryo-sections can now successfully be post-fixed and immunolabelled after thawing; this allows cryo-EM comparison with adjacent ribbons of sections still in the frozen hydrated state. These findings call for technical innovations aiming at automated cryo-ultramicrotomy in a fully controlled environment for improved localization of proteins within their 'close to native' cellular context and correlative electron cryo-tomography of consecutive ribbons of sections of one frozen hydrated sample.     

A procedure to deposit fiducial markers on vitreous cryo-sections for cellular tomography

We describe a novel approach for the accurate alignment of images in electron tomography of vitreous cryo-sections. Quantum dots, suspended in organic solvents at cryo-temperatures, are applied directly onto the sections and are subsequently used as fiducial markers to align the tilt series. Data collection can be performed from different regions of the vitreous sections, even when the sections touch the grid only at a few places. We present high-resolution tomograms of some organelles in cryo-sections of human skin cells using this method. The average error in image alignment was about 1nm and the resolution was estimated to be 5-7nm. Thus, the use of section-attached quantum dots as fiducial markers in electron tomography of vitreous cryo-sections facilitates high-resolution in situ 3D imaging of organelles and macromolecular complexes in their native hydrated state.     

A patient immobilization device for prone breast radiotherapy: Dosimetric effects and inclusion in the treatment planning system

PurposeTo assess the dosimetric impact of a patient positioning device for prone breast radiotherapy and assess the accuracy of a treatment planning system (TPS) in predicting this impact.MethodsBeam attenuation and build-up dose perturbations, quantified by ionization chamber and radiochromic film dosimetry, were evaluated for 3 components of the patient positioning device: the carbon fiber baseplate, the support cushions and the support wedge for the contralateral breast. Dose calculations were performed using the XVMC dose engine implemented in the Monaco TPS. All components were included during planning CT acquisition.ResultsBeam attenuation amounted to 7.57% (6 MV) and 5.33% (15 MV) for beams obliquely intersecting the couchtop–baseplate combination. Beams traversing large sections of the support wedge were attenuated by 12.28% (6 MV) and 9.37% (15 MV). For the support cushion foam, beam attenuation remained limited to 0.11% (6 MV) and 0.08% (15 MV) per centimeter thickness. A substantial loss of dose build-up was detected when irradiating through any of the investigated components. TPS dose calculations accurately predicted beam attenuation by the baseplate and support wedge. A manual density overwrite was needed to model attenuation by the support cushion foam. TPS dose calculations in build-up regions differed considerably from measurements for both open beams and beams traversing the device components.ConclusionsIrradiating through the components of the positioning device resulted in a considerable degradation of skin sparing. Inclusion of the device components in the treatment planning CT allowed to accurately model the most important attenuation effect, but failed to accurately predict build-up doses.     

A new tool based on two micromanipulators facilitates the handling of ultrathin cryosection ribbons

A close to native structure of bulk biological specimens can be imaged by cryo-electron microscopy of vitreous sections (CEMOVIS). In some cases structural information can be combined with X-ray data leading to atomic resolution in situ. However, CEMOVIS is not routinely used. The two critical steps consist of producing a frozen section ribbon of a few millimeters in length and transferring the ribbon onto an electron microscopy grid. During these steps, the first sections of the ribbon are wrapped around an eyelash (unwrapping is frequent). When a ribbon is sufficiently attached to the eyelash, the operator must guide the nascent ribbon. Steady hands are required. Shaking or overstretching may break the ribbon. In turn, the ribbon immediately wraps around itself or flies away and thereby becomes unusable. Micromanipulators for eyelashes and grids as well as ionizers to attach section ribbons to grids were proposed. The rate of successful ribbon collection, however, remained low for most operators. Here we present a setup composed of two micromanipulators. One of the micromanipulators guides an electrically conductive fiber to which the ribbon sticks with unprecedented efficiency in comparison to a not conductive eyelash. The second micromanipulator positions the grid beneath the newly formed section ribbon and with the help of an ionizer the ribbon is attached to the grid. Although manipulations are greatly facilitated, sectioning artifacts remain but the likelihood to investigate high quality sections is significantly increased due to the large number of sections that can be produced with the reported tool.     

Statistical analysis of surface electromyographic signal for the assessment of rectus femoris modalities of activation during gait

Aim of the present study was to identify the different modalities of activation of rectus femoris (RF) during gait at self-selected speed, by a statistical analysis of surface electromyographic signal from a large number (hundreds) of strides per subject. The analysis of ten healthy adults showed that RF is characterized by different activation modalities within different strides of the same walk. RF most recurrent modality (observed in 53 ± 6% of total strides) consists of three activations, at the beginning of gait cycle, around foot-off and in the terminal swing. Further two modalities of RF activation differ from the most recurrent one because of the lack of activity around foot-off (26 ± 6%) or the splitting into two (or three) small activations around stance-to-swing transition (17 ± 2%). Despite the large variability, our statistical analysis allowed to identify two patterns of activation that characterize completely the behavior of rectus femoris during gait. The first pattern, around stance-to-swing transition, can be monophasic, biphasic or triphasic and is necessary to control knee extension and hip flexion from pre-swing to initial swing. The second pattern, from terminal swing to following mid-stance, is likely due to the contribution of low-level RF activity and cross-talk from surrounding vastii.     

Development of a modified vitrification strategy suitable for subsequent scale-up for hepatocyte preservation

This work explores the design of a vitrification solution (VS) for scaled-up cryopreservation of hepatocytes, by adapting VS_basic (40% (v/v) ethylene glycol 0.6 M sucrose, i.e. 7.17 M ethylene glycol 0.6 M sucrose), previously proven effective in vitrifying bioengineered constructs and stem cells. The initial section of the scale-up study involved the selection of non-penetrating additives to supplement VS_basic and increase the solution’s total solute concentration. This involved a systematic approach with a step-by-step elimination of non-penetrating cryoprotectants, based on their effect on cells after long/short term exposures to high/low concentrations of the additives alone or in combinations, on the attachment ability of hepatocytes after exposure. At a second stage, hepatocyte suspension was vitrified and functions were assessed after continuous culture up to 5 days.Results indicated Ficoll as the least toxic additive. Within 60 min, the exposure of hepatocytes to a solution composed of 9% Ficoll + 0.6 M sucrose (10⁻³ M Ficoll + 0.6 M sucrose) sustained attachment efficiency of 95%, similar to control. Furthermore, this additive did not cause any detriment to the attachment of these cells when supplementing the base vitrification solution VS_basic. The addition of 9% Ficoll, raised the total solute concentration to 74.06% (w/v) with a negligible 10⁻³ M increase in molarity of the solution. This suggests main factor in inducing detriment to cells was the molar contribution of the additive.Vitrification protocol for scale-up condition sustained hepatocyte suspension attachment efficiency and albumin production. We conclude that although established approach will permit scaling-up of vitrification of hepatocyte suspension, vitrification of hepatocytes which are attached prior to vitrification is more effective by comparison.     

Rapid warming and ostracods mass extinction at the Lower Toarcian (Jurassic) of central Spain

Here we present the results of the study of two Lower Toarcian carbonate sections located in the Iberian Range of central Spain. Analyses of stable isotope on belemnite calcite allowed calculation of seawater palaeotemperature variations, which were compared with the stratigraphical distribution of ostracods. These organisms are particularly sensitive to ratios of temperature and salinity variations and hence are good indicators of climate changes. From a cooling interval, with seawater temperatures of 13.2 °C recorded at the Pliensbachian?Toarcian transition, seawater temperature began to rise in the lowermost Toarcian Tenuicostatum Zone, reaching average temperatures between 14.6 °C and 16.3 °C during the time of deposition of this Zone. Coinciding with this seawater warming, up to 85% of the ostracods species progressively disappeared during a period of approximately 300 kyr, marking the extinction interval. The extinction boundary, located around the Tenuicostatum?Serpentinum zonal boundary, coincides with a marked increase in temperature in the Serpentinum Zone, on which average seawater temperatures of 22 °C have been calculated. Warming continued through part of the Middle Toarcian Bifrons Zone, reaching average temperatures of 24.7 °C. Readjustment of the ostracod population allowed recovery of these faunas in the upper Serpentinum Zone, although the extinction of a major ostracod group, the healdioids, was also recorded. The correlation between mass extinction and warming infers a causal relationship. Comparison of the results with the records of stable isotopes in belemnites and in bulk carbonates, as well as TOC and facies analysis suggests that the anoxia linked to the Early Toarcian oceanic anoxic event was not the main responsible for the ostracod mass extinction.     

The use of spatial ecological modelling as a tool for improving the assessment of geographic range size of threatened species

We analysed endemic threatened tree and reptile species of Socotra Island (Yemen), characterised by different ecological requirements and spatial distribution, in order to evaluate the usefulness of spatial ecological modelling in the estimation of species extent of occurrence (EOO) and area of occupancy (AOO). Point occurrences for the entire species range were used to model their spatial distribution by Random Forest (RF) and Generalised Linear Model (GLM). For each species the suitability area (SA) was obtained by applying the 0% omission error criterion on the probability map, and compared or integrated with EOO and AOO area obtained by topological methods such as the minimum convex polygon (MCP), α-hull and 2 km × 2 km grid.RF showed a lower prediction error than GLM. Higher accuracy was achieved for species with higher number of occurrences and narrower ecological niche. SA was always greater than AOO measured with the 2 km × 2 km grid method. SA was greater than EOO, measured by both MCP and α-hull methods, for species with localised distribution, while it was smaller for widely distributed species. EOO-α-hull area was equal or smaller than that calculated by MCP depending on the spatial distribution of species. AOO measured considering the SA within the EOO-MCP was greater than that measured using the standard 2 km × 2 km grid. Conversely, AOO calculated considering the suitable area within the EOO-α-hull showed variable results, being smaller or greater than the 2 km × 2 km grid AOO depending on the ecological niche and spatial distribution of species. According to our results, SEM does not provide an effective alternative to topological methods for the estimate of EOO and AOO. However, it may be considered a useful tool to estimate AOO within the boundaries of EOO measured by the α-hull method, because it reduces some potential sources of inconsistency and bias.     

The core-microtome: A new tool for surface preparation on cores and time series analysis of varying cell parameters

A microtome designed for the surface preparation of entire increment cores allows cutting plane surfaces on cores up to a length of 40 cm. Compared to the common sanding procedure, the wood cells of the annual rings remain open, not filled with swarf, and the cell walls are smooth and hence clearly visible. This article aims at describing the functionality of the microtome and the procedures needed for an accurate surface preparation to achieve a good contrast for subsequent image analysis. Possible applications for a more detailed analysis of variations in the tracheid structure of conifers and vessel sizes of oak are presented, which can be included in time series analyses.     

Cholesterol microcrystals and cochleate cylinders: Attachment of pyolysin oligomers and domain 4

Using an established organic solvent injection procedure for the preparation of aqueous cholesterol microcrystal suspensions, it has now been shown that a new, hollow, cylindrical, tightly-coiled, multi-bilayer form of cholesterol can be generated, termed the cochleate cylinder. Cholesterol cochleate cylinders are formed in larger numbers at intermediate temperatures (40–75 °C) but are not formed at 100 °C. The structure of the cholesterol microcrystals and cochleate cylinders is shown in negatively stained electron micrographs. Oligomerization and attachment of pyolysin to cholesterol microcrystals and cochleate cylinders is shown, as is the attachment of the pyolysin “cholesterol-binding” domain 4 (D4) fragment. The bound D4 domain forms a linear array on the two planar surfaces and edges of the cholesterol microcrystals and a quasi helical array on the surface of the cochleate cylinders. Little evidence has been obtained to support the possibility that interaction or hetero-oligomerization can occur between intact pyolysin and the pyolysin D4 fragment on the surface of cholesterol microcrystals. Using immobilized cholesterol crystals attached to a carbon support film, single-sided linear labelling of the cholesterol surface with pyolysin D4 has been achieved, which correlates well with the images from the microcrystal suspensions and our earlier data using non-cytolytic streptolysin O mutants.     

Diel variation in movement patterns and habitat use by the Iberian endemic Cabrera vole: Implications for conservation and monitoring

Understanding variations in animal movement and habitat selection behaviour over fine spatial and temporal scales remains a particularly challenging goal in ecology and conservation. Here we document for the first time the diel variations in movement patterns and habitat use by wild-ranging Cabrera voles in fragmented Mediterranean farmland, based on radiotracking data (2006–2008) of 25 adult individuals occupying stable home-ranges in vegetation mosaics dominated by wet grasses and shrubs. Results indicated that the proportion of time animals spent moving, the distance moved, and the selection strength of main vegetation types were closely linked behavioural traits, which varied considerably across different periods of the 24-h cycle. In general, voles moved more frequently and over larger distances during daytime (between 06 h15–22 h00), which was when wet grasses were also used more intensively. These patterns were generally consistent across seasons, though during the dry season there was some tendency for a decrease in movement activity during the hottest hours of the day (between 10 h15–14 h00), with peaks around crepuscular hours (06 h15–10 h00 and 18 h15–22 h00). Overall, our study provides evidence that Cabrera voles may show notable shifts in habitat use and movement patterns on a finer scale than previously considered. This supports the idea that knowledge of the diel variations in species movement-habitat relationships should strongly contribute to improving local habitat management, as well as effective sampling and monitoring programs targeting the species.     

Holdfast aggregation in relation to morphology,age, attachment and drag for the kelp Ecklonia radiata

This study quantified the prevalence of holdfast aggregation (fusion of holdfasts) for the kelp Ecklonia radiata on subtidal reefs in southwestern Australia, and tested whether morphology, age, attachment or drag were different between kelps growing alone (solitary) or in aggregates. Wave-sheltered in-shore reefs consistently had fewer aggregates than wave-exposed off-shore reefs (15–20% versus 20–30%). On average, individual thalli from aggregates were longer (97.8 cm ± 2.2 S.E. versus 88.0 cm ± 2.0 S.E.) and had smaller holdfasts (32.9 g fresh wt ± 1.7 S.E. versus 45.8 g fresh wt ± 2.9 S.E.) than solitary thalli, whereas there were no significant differences in other morphological characters, including total biomass (805.1 g fresh wt ± 38.7 S.E. versus 831.5 g fresh wt ± 38.5 S.E.), stipe length (7.93 cm ± 0.47 S.E. versus 7.65 cm ± 0.40 S.E.) and stipe diameter (12.6 mm ± 0.23 S.E. versus 13.0 mm ± 0.25 S.E.). There was no difference in age between solitary (2.7–3.0 years) and aggregated (2.4–2.8 years) individuals. While the attachment force of whole aggregates (256.5 N ± 21.6 S.E.) was found to be significantly larger than attachment force for solitary individuals (162.5 N ± 12.9 S.E.), attachment areas were also larger for aggregates (90.7 cm² ± 5.40 S.E. versus 64.3 cm² ± 5.54 S.E.) and consequently there were no differences in attachment strength between aggregates (2.92 N cm⁻² ± 0.26 S.E.) and solitary thalli (2.71 N cm⁻² ± 0.22 S.E.). Aggregates had significantly smaller (17%) roughness factors (equivalent to drag coefficients) than solitary individuals and a negative relationship (r = −0.68) between roughness factors and biomass suggested that this was related to the scope for compaction and rearrangement of the thalli. Further, there was no relationship between roughness factors of solitary individuals and the aggregates they produced when combined, suggesting that roughness factors are not additive or multiplicative. The spatial distribution of holdfast aggregates, the morphological differences between solitary and aggregated individuals as well as their attachment and drag characteristics were all consistent with aggregation reducing the rate of fatal kelp dislodgment.     

Correlation between cell attachment areas after 2 h of culture and osteogenic differentiation activity of rat mesenchymal stem cells on hydroxyapatite substrates with various surface properties

The initial attachment of mesenchymal stem cells (MSCs) to substrates and osteogenic differentiation are supported by culture on a hydroxyapatite substrate. Cell attachment areas of rat MSCs after 2 h of culture on hydroxyapatite substrates with various microstructures and the osteogenic differentiation activity thereafter were measured. The perceived outcome was that, after 2 h of culture, rat MSCs with a small attachment area would have a high osteogenic differentiation activity, whereas those with a large attachment area would have a low osteogenic differentiation activity. Furthermore, rat MSCs with a small attachment area had many cytoplasmic processes, while those with a large attachment area revealed clear stress fibers and focal contacts. These results suggest that cell attachment area of rat MSCs after 2 h of culture has a strong effect on the osteogenic differentiation of rat MSCs. Thus, the measurement of cell attachment area after 2 h of culture could become valuable for estimating the osteogenic differentiation activity of rat MSCs thereafter.     

A negative carbon isotope excursion defines the boundary from Cambrian Series 2 to Cambrian Series 3 on the Yangtze Platform,South China

A globally recorded negative carbon isotope excursion characterizes the transition from Cambrian Series 2 to Cambrian Series 3. This transition is also well exposed in sedimentary successions on the Yangtze Platform, and the Wuliu–Zengjiayan section, Guizhou Province, South China has been proposed as a potential Global Stratotype Section and Point (GSSP) for this boundary. Here, we report δ¹³C_carb values for the Jianshan and the Wuliu–Zengjiayan sections. Both sections display a progressive decrease in δ¹³C from values around + 3‰ upwards in stratigraphy to a pronounced δ¹³C minimum with values as low as ? 6.9‰ at the proposed boundary level, and a return to δ¹³C values between 0 and + 1‰ in the upper part of the sections. The δ¹³C minimum is thought to be caused by a transgressive event, flooding the shelf area with ¹³C depleted basinal anoxic bottom water. Our δ¹³C data are in good agreement with carbon isotope profiles recorded elsewhere. These define the so called ROECE event (Redlichiid–Oleneliid Extinction Carbon Isotope Excursion, cf. Zhu et al., 2006, 2007) and may reflect the perturbation of the global carbon cycle during the Cambrian Series 2 to Cambrian Series 3 transition.     

Preparation of a biocatalyst via physical adsorption of lipase from Thermomyces lanuginosus on hydrophobic support to catalyze biolubricant synthesis by esterification reaction in a solvent-free system

Lipase from Thermomyces lanuginosus (TLL) was immobilized on mesoporous hydrophobic poly-methacrylate (PMA) particles via physical adsorption (interfacial activation of the enzyme on the support). The influence of initial protein loading (5–200 mg/g of support) on the catalytic properties of the biocatalysts was determined in the hydrolysis of olive oil emulsion and synthesis of isoamyl oleate (biolubricant) by esterification reaction. Maximum adsorbed protein loading and hydrolytic activity were respectively ≈100 mg/g and ≈650 IU/g using protein loading of 150 mg/g of support. The adsorption process followed the Langmuir isotherm model (R² = 0.9743). Maximum ester conversion around 85% was reached after 30 min of reaction under continuous agitation (200 rpm) using 2500 mM of each reactant in a solvent-free system, 45 °C, 20% m/v of the biocatalyst prepared using 100 mg of protein/g of support. Apparent thermodynamic parameters of the esterification reaction were also determined. Under optimal experimental conditions, reusability tests of the biocatalyst (TLL-PMA) after thirty successive cycles of reaction were performed. TLL-PMA fully retained its initial activity up to twenty two cycles of reaction, followed by a slight decrease around 8.6%. The nature of the product (isoamyl oleate) was confirmed by attenuated total reflection Fourier transform infrared (ATR-FTIR), proton (¹H NMR) and carbon (¹³C NMR) nuclear magnetic resonance spectroscopy analyses.     

Method for Staining and Washing Thin Sections on Support Films

A procedure is described for staining large numbers of thin sections on support films for use with one-hole grids. The film is picked up, carried and protected using easily made plastic blocks. Loop-tipped forceps are then used to transfer tissue ribbons from the knife boat to the support film. A large number of tissue sections can then be stained and washed simultaneously in a modified Pyrex dish without damaging the film. After staining, the slot in the one-hole grid is centered over the tissue ribbon, and the grid is attached to the film. The method is suitable for serial reconstruction and the unobstructed viewing of large thin sections in the TEM.     

Towards whole sheep ovary cryopreservation

Cryopreservation of ovarian tissue aims to assist young women who require treatments that may lead to sterility or infertility. Cryopreservation procedures should therefore be as simple and efficient as possible. This study investigates rapid cooling outcomes for whole sheep ovaries. Ovaries were perfused with VS4 via the ovarian artery, and cooled by quenching in liquid nitrogen in less than a minute (estimated cooling rate above 300 °C/min till the vitreous transition temperature). The ovaries were rewarmed in two stages: slow warming (12–16 °C/min from −196 to −133 °C) in liquid nitrogen vapour, followed by rapid thawing in a 45 °C water bath at about 200 °C/min. DSC measurements showed that under these cryopreservation conditions VS4 would vitrify, but that VS4 perfused ovarian cortex fragments did not vitrify, but formed ice (around 18.4%). Immediately following rewarming, a dye exclusion test indicated that 61.4 ± 2.2% of small follicles were viable while histological analysis showed that 48 ± 3.8% of the primordial follicles were normal. It remains to be clarified whether follicle survival rates will increase if conditions allowing complete tissue vitrification were used.     

Surface dose measurements with GafChromic EBT film for 6 and 18 MV photon beams

The aim of this study was to determine the surface doses using GafChromic EBT films and compare them with plane-parallel ionization chamber measurements for 6 and 18 MV high energy photon beams. The measurements were made in a water equivalent solid phantom in the build-up region of the 6 and 18 MV photon beams at 100 cm SSD for various field sizes. Markus type plane-parallel ion chamber with fixed-separation between collecting electrodes was used to measure the percent depth doses. GafChromic EBT film measurements were performed both on the phantom surface and maximum dose depth at the same geometry with ion chamber measurements. The surface doses found using GafChromic EBT film were 15%, 20%, 29%and 39% ± 2% (1SD) for 6 MV photons, 6%, 11%, 23% and 32% ± 2% (1SD) for 18 MV photons at 5, 10, 20 and 30 cm² field sizes, respectively. GafChromic EBT film provides precise measurements for surface dose in the high energy photons. Agreement between film and plane-parallel chamber measurements was found to be within ±3% for 18 MV photon beams. There was 5% overestimate on the surface doses when compared with the plane-parallel chamber measurements for all field sizes in the 6 MV photon beams.     

A biosensor based on gold nanoparticles,dihexadecylphosphate, and tyrosinase for the determination of catechol in natural water

In this work, a biosensor using a glassy carbon electrode modified with gold nanoparticles (AuNPs) and tyrosinase (Tyr) within a dihexadecylphosphate film is proposed. Cystamine and glutaraldehyde crosslinking agents were used as a support for Tyr immobilization. The proposed biosensor was characterized by scanning electron microscopy (SEM), transmission electron microscopy (TEM), and cyclic voltammetry in the presence of catechol. The determination of catechol was carried out by amperometry and presented a linear concentration range from 2.5 × 10⁻⁶ to 9.5 × 10⁻⁵ mol L⁻¹ with a detection limit of 1.7 × 10⁻⁷ mol L⁻¹. The developed biosensor showed good repeatability and stability. Moreover, this novel amperometric method was successfully applied in the determination of catechol in natural water samples. The results were in agreement with a 95% confidence level for those obtained using the official spectrophotometric method.     

Development of Halomonas TD01 as a host for open production of chemicals

Genetic engineering of Halomonas spp. was seldom reported due to the difficulty of genetic manipulation and lack of molecular biology tools. Halomonas TD01 can grow in a continuous and unsterile process without other microbial contaminations. It can be therefore exploited for economic production of chemicals. Here, Halomonas TD01 was metabolically engineered using the gene knockout procedure based on markerless gene replacement stimulated by double-strand breaks in the chromosome. When gene encoding 2-methylcitrate synthase in Halomonas TD01 was deleted, the conversion efficiency of propionic acid to 3-hydroxyvalerate (3HV) monomer fraction in random PHBV copolymers of 3-hydroxybutyrate (3HB) and 3HV was increased from around 10% to almost 100%, as a result, cells were grown to accumulate 70% PHBV in dry weight (CDW) consisting of 12 mol% 3HV from 0.5 g/L propionic acid in glucose mineral medium. Furthermore, successful deletions on three PHA depolymerases eliminate the possible influence of PHA depolymerases on PHA degradation in the complicated industrial fermentation process even though significant enhanced PHA content was not observed. In two 500 L pilot-scale fermentor studies lasting 70 h, the above engineered Halomonas TD01 grew to 112 g/L CDW containing 70 wt% P3HB, and to 80 g/L CDW with 70 wt% P(3HB-co-8 mol% 3HV) in the presence of propionic acid. The cells grown in shake flasks even accumulated close to 92% PHB in CDW with a significant increase of glucose to PHB conversion efficiency from around 30% to 42% after 48 h cultivation when pyridine nucleotide transhydrogenase was overexpressed. Halomonas TD01 was also engineered for producing a PHA regulatory protein PhaR which is a robust biosurfactant.