Glycerol-based sterilization bioindicator system from Bacillus atrophaeus: development, performance evaluation, and cost analysis

The development of new value-added applications for glycerol is of worldwide interest because of the environmental and economic problems that may be caused by an excess of glycerol generated from biodiesel production. A novel use of glycerol as a major substrate for production of a low-cost sterilization biological indicator system (BIS; spores on a carrier plus a recovery medium) was investigated. A sequential experimental design strategy was applied for product development and optimization. The proposed recovery medium enables germination and outgrowth of heat-damaged spores, promoting a D _160 °C value of 6.6?±?0.1 min. Bacillus atrophaeus spores production by solid-state fermentation reached a 2.3?±?1.2?×?10⁸?CFU/g dry matter. Sporulation kinetics results allowed this process to be restricted in 48 h. Germination kinetics demonstrated the visual identification of nonsterile BIS within 24 h. Performance evaluation of the proposed BIS against dry-heat and ethylene oxide sterilization showed compliance with the regulatory requirements. Cost breakdowns were from 41.8 (quality control) up to 72.8 % (feedstock). This is the first report on sterilization BIS production that uses glycerol as a sole carbon source, with significant cost reduction and the profitable use of a biodiesel byproduct.     

蚕豆监测大气SO2污染的指标筛选研究

熏气和暴露试验表明,蚕豆叶片可见伤害症状不宜作为监测指标,超氧化物歧化酶（ＳＯＤ）活性、抗坏血酸（ＡＳＡ）含量和游离氨基酸（ＡＡ）含量作为监测指标,其指示效果优于过氧化物酶（ＰＯＤ）活性和叶绿素含量,其中又以ＳＯＤ活性为最好,而根据以上５个指标综合评价的效果又要优于任何单个指标．     

Comparison of audio recording system performance for detecting and monitoring songbirds

ABSTRACT Acoustic recording systems are being used more frequently to estimate habitat occupancy or relative abundance, and to monitor population trends over time. A potential concern with digital recording systems is that changes in technology could affect detectability of birds and cause bias in trend estimates based on counts of birds detected. We evaluated several currently available commercial recording systems ranging from low‐cost multipurpose digital recorders to custom‐designed wildlife recorders (US$250–$7000 price range) to examine possible differences among systems in species detection. We made recordings during Breeding Bird Surveys (BBS) counts using several units concurrently, and asked several expert birders to listen to the recordings in a factorial design. We found that birders detected, on average, 10% fewer species on some units compared to others, though there was high variance. Analysis of a subset of recordings, using spectrograms and repeated listening, suggested that ～90% of species on each BBS stop could be clearly detected on all units. The remaining species could be identified on at least one unit, but were hard or impossible to detect on others. We found that the recording unit with the lowest empirical signal‐to‐noise‐ratio (SNR) had the lowest number of birds detected on the BBS recordings, and that frequency‐specific SNR differed among units. Missed detections were likely related to variation in internal noise and frequency‐dependent sensitivity of the units, and were an issue for all systems regardless of price. We caution that researchers using recorders need to consider variation among recording systems in their study design, particularly for long‐term monitoring programs.     

Biosensors for process monitoring

Summary A short review about the biosensor research activities for bioprocess monitoring in the F.R.G. after its reunification is given. The principles of biosensor applications are presented. In situ sensors and sensors based on the principles of flow injection analysis are studied. Some applications of a four-channel enzyme thermistor, bio-field effect transistors, and immunoanalysis systems for real process monitoring are presented.     

An in vitro monitoring system for simulated thermal process in cryosurgery

This paper describes a new in vitro experimental model that records temperature changes over a culture plate, which then can be used to assess the biological effects of cryosurgery. A cryoprobe and 16 thermocouples set up by a computer control system were used to monitor the temperature changes during freezing and thawing in a culture plate, and the data were used to create a temperature profile of the entire plate. Location of the thermocouples was confirmed by a digital camera viewing from under the plate, and temperature changes at any point in the interpolated areas were estimated using a curve fitting method. The estimated temperature was checked by sampling with four additional randomly placed thermocouples. Linear regression analysis showed that the estimated temperature and measured temperature were very close (correlation coefficients 0.98-0.99). MBT-2 tumor cells were cultured and then subjected to simulated cryosurgery. The surviving cells were stained with crystal violet and the cell death boundary was detected by image processing. Temperature history at the cell death boundary was retrieved and analyzed. With this system it is possible to recreate the temperature changes that result in a certain biological effect (such as cell death) during the process of simulated cryosurgery.     

Mechanisms underlying the bioindicator notion: spatial association between individual sexual performance and community diversity

The bioindicator notion is an appealing concept that has received more support in applied than in basic ecology, mostly due to the difficulty in deriving general ecological rules applicable to all target organisms. However, recognizing the mechanisms that determine the association between a particular species and the well-being of many other species is important for understanding the functioning of ecosystems and the relationship among different biological levels. We examined here the processes at the individual level that cause an association between species performance and biodiversity value, by analyzing attributes that can be studied in a variety of animals with sexual reproduction, namely breeding site selection and condition-dependent sexual signals. Our study model was the Capercaillie, an indicator of forest functioning and diversity, and the associated bird community, used here as a surrogate of broader forest biodiversity. At a regional scale Capercaillie occurrence was not associated with the most diverse forest patches, but at the scale of male spring territories the sexual display grounds (arenas) were located in the oldest and less disturbed forest portions, which also hosted the richest local bird communities. Social mechanisms and conspecific cueing likely concurred with habitat-driven processes in determining the long-term persistence of traditional display grounds, which were appealing to many other species because of their structural composition. Characteristics of male vocal display that honestly advertize male quality (low frequencies and rapid song rates) were significantly correlated with high diversity values, resulting in a spatial association between individual and community performances. Costly or risky activities such as reproductive or social behaviors, which more than other attributes match gradients in habitat quality, are therefore contributing to functionally connect individuals with ecosystem health.     

蚕豆监测大气SO2污染的指标筛选研究

熏气和暴露试验表明,蚕豆叶片可见伤害症状不宜作为监测指标,超氧化物歧化酶(SOD)活性、抗坏血酸(ASA)含量和游离氨基酸(AA)含量作为监测指标,其指示效果优于过氧化物酶(POD)活性和叶绿素含量,其中又以SOD活性为最好,而根据以上5个指标综合评价的效果又要优于任何单个指标。     

Design and evaluation of a system for thermal decontamination of process air

Aerobiology studies at the Naval Biological Laboratory require use of a vacuum system to provide safe disposal of air contaminated with pathogenic, microorganisms. A system for thermal decontamination of this process air has been installed and tested. The system uses a natural gas burner to heat approximately 550 cfm of air to temperatures exceeding 750°F. Tests showed a reduction in number of acrosolized viable hardy spores (Bacillus subtilis var. niger) of more than 8 logs at design flow rates. The kill rate (D values) measured in this system is somewhat higher than those reported by other workers. The annual owning and operating cost of the system is approximately $9000.     

Hybrid process models for process optimisation,monitoring and control

Hybrid models aim to describe different components of a process in different ways. This makes sense when the corresponding knowledge to be represented is different as well. In this way, the most efficient representations can be chosen and, thus, the model performance can be increased significantly. From the various possible variants of hybrid model, three are selected which were applied for important biotechnical processes, two of them from existing production processes. The examples show that hybrid models are powerful tools for process optimisation, monitoring and control.     

Stability of Bacillus pumilus spore strips used for monitoring radiation sterilization.

Dried Bacillus pumilus spore strips had a stable D-value of 0.17 to 0.18 Mrad after 1 year at 5 or 25 C, but resistance was lost at 35 C. Refrigeration did not have an adverse effect on resistance to irradiation. The presence of water at the time of initial preparation increased this resistance to 0.23 to 0.25 Mrad. Proper drying is essential to achieve reproducible results in sterility tests using USP-type biological indicators.     

On-line near infrared monitoring of glycerol-boosted anaerobic digestion processes: evaluation of process analytical technologies

A study of NIR as a tool for process monitoring of thermophilic anaerobic digestion boosted by glycerol has been carried out, aiming at developing simple and robust Process Analytical Technology modalities for on-line surveillance in full scale biogas plants. Three 5 L laboratory fermenters equipped with on-line NIR sensor and special sampling stations were used as a basis for chemometric multivariate calibration. NIR characterisation using Transflexive Embedded Near Infra-Red Sensor (TENIRS) equipment integrated into an external recurrent loop on the fermentation reactors, allows for representative sampling, of the highly heterogeneous fermentation bio slurries. Glycerol is an important by-product from the increasing European bio-diesel production. Glycerol addition can boost biogas yields, if not exceeding a limiting 5-7 g L(-1) concentration inside the fermenter-further increase can cause strong imbalance in the anaerobic digestion process. A secondary objective was to evaluate the effect of addition of glycerol, in a spiking experiment which introduced increasing organic overloading as monitored by volatile fatty acids (VFA) levels. High correlation between on-line NIR determinations of glycerol and VFA contents has been documented. Chemometric regression models (PLS) between glycerol and NIR spectra needed no outlier removals and only one PLS-component was required. Test set validation resulted in excellent measures of prediction performance, precision: r(2) = 0.96 and accuracy = 1.04, slope of predicted versus reference fitting. Similar prediction statistics for acetic acid, iso-butanoic acid and total VFA proves that process NIR spectroscopy is able to quantify all pertinent levels of both volatile fatty acids and glycerol.     

Designing cross-linked lipase aggregates for optimum performance as biocatalysts

Cross-linked enzyme aggregates (CLEAs) are prepared by precipitation of an enzyme and then chemical cross-linking the precipitate. Three CLEAs of lipase with glutaraldehyde concentrations of 10 mM (CLEA A), 40 mM (CLEA B) and 60 mM (CLEA C) were prepared. Studies show that there is a trade-off between thermal stability vs transesterification/hydrolysis rate vs enantioselectivity. The initial rates for transesterification of β-citronellol for the uncross-linked enzyme and CLEAs A, B and C were 243, 167, 102 and 40 µmol mg^-1 h^-1, respectively. Their thermal stabilities in aqueous media, as reflected by their half-life values at 55°C, were 6, 9, 13 and 16 h, respectively. The enantioselectivity, E values (for kinetic resolution of β-citronellol by transesterification) were 19, 74, 11 and 6, respectively. These results show that CLEA C was the most thermostable; the uncross-linked enzyme was best at obtaining the highest transesterification rate; and CLEA A was best suited for the enantioselective synthesis. Scanning electron microscopy (SEM) showed that the morphology of CLEA was dependent upon the extent of cross-linking.     

Designing cross-linked lipase aggregates for optimum performance as biocatalysts

Cross-linked enzyme aggregates (CLEAs) are prepared by precipitation of an enzyme and then chemical cross-linking the precipitate. Three CLEAs of lipase with glutaraldehyde concentrations of 10 mM (CLEA A), 40 mM (CLEA B) and 60 mM (CLEA C) were prepared. Studies show that there is a trade-off between thermal stability vs transesterification/hydrolysis rate vs enantioselectivity. The initial rates for transesterification of β-citronellol for the uncross-linked enzyme and CLEAs A, B and C were 243, 167, 102 and 40 µmol mg^?1 h^?1, respectively. Their thermal stabilities in aqueous media, as reflected by their half-life values at 55°C, were 6, 9, 13 and 16 h, respectively. The enantioselectivity, E values (for kinetic resolution of β-citronellol by transesterification) were 19, 74, 11 and 6, respectively. These results show that CLEA C was the most thermostable; the uncross-linked enzyme was best at obtaining the highest transesterification rate; and CLEA A was best suited for the enantioselective synthesis. Scanning electron microscopy (SEM) showed that the morphology of CLEA was dependent upon the extent of cross-linking.     

Configuring balanced scorecards for measuring health system performance: evidence from 5 years' evaluation in Afghanistan

Background

In 2004, Afghanistan pioneered a balanced scorecard (BSC) performance system to manage the delivery of primary health care services. This study examines the trends of 29 key performance indicators over a 5-year period between 2004 and 2008.

Methods and Findings

Independent evaluations of performance in six domains were conducted annually through 5,500 patient observations and exit interviews and 1,500 provider interviews in >600 facilities selected by stratified random sampling in each province. Generalized estimating equation (GEE) models were used to assess trends in BSC parameters. There was a progressive improvement in the national median scores scaled from 0–100 between 2004 and 2008 in all six domains: patient and community satisfaction of services (65.3–84.5, p<0.0001); provider satisfaction (65.4–79.2, p<0.01); capacity for service provision (47.4–76.4, p<0.0001); quality of services (40.5–67.4, p<0.0001); and overall vision for pro-poor and pro-female health services (52.0–52.6). The financial domain also showed improvement until 2007 (84.4–95.7, p<0.01), after which user fees were eliminated. By 2008, all provinces achieved the upper benchmark of national median set in 2004.

Conclusions

The BSC has been successfully employed to assess and improve health service capacity and service delivery using performance benchmarking during the 5-year period. However, scorecard reconfigurations are needed to integrate effectiveness and efficiency measures and accommodate changes in health systems policy and strategy architecture to ensure its continued relevance and effectiveness as a comprehensive health system performance measure. The process of BSC design and implementation can serve as a valuable prototype for health policy planners managing performance in similar health care contexts.Please see later in the article for the Editors'' Summary     

Statistical process control for electron beam monitoring

PurposeTo assess the electron beam monitoring statistical process control (SPC) in linear accelerator (linac) daily quality control. We present a long-term record of our measurements and evaluate which SPC-led conditions are feasible for maintaining control.MethodsWe retrieved our linac beam calibration, symmetry, and flatness daily records for all electron beam energies from January 2008 to December 2013, and retrospectively studied how SPC could have been applied and which of its features could be used in the future. A set of adjustment interventions designed to maintain these parameters under control was also simulated.ResultsAll phase I data was under control. The dose plots were characterized by rising trends followed by steep drops caused by our attempts to re-center the linac beam calibration. Where flatness and symmetry trends were detected they were less-well defined. The process capability ratios ranged from 1.6 to 9.3 at a 2% specification level. Simulated interventions ranged from 2% to 34% of the total number of measurement sessions. We also noted that if prospective SPC had been applied it would have met quality control specifications.ConclusionsSPC can be used to assess the inherent variability of our electron beam monitoring system. It can also indicate whether a process is capable of maintaining electron parameters under control with respect to established specifications by using a daily checking device, but this is not practical unless a method to establish direct feedback from the device to the linac can be devised.     

Quantitative evaluation of yeast's requirement for glycerol formation in very high ethanol performance fed-batch process

Background

Glycerol is the major by-product accounting for up to 5% of the carbon in Saccharomyces cerevisiae ethanolic fermentation. Decreasing glycerol formation may redirect part of the carbon toward ethanol production. However, abolishment of glycerol formation strongly affects yeast's robustness towards different types of stress occurring in an industrial process. In order to assess whether glycerol production can be reduced to a certain extent without jeopardising growth and stress tolerance, the yeast's capacity to synthesize glycerol was adjusted by fine-tuning the activity of the rate-controlling enzyme glycerol 3-phosphate dehydrogenase (GPDH). Two engineered strains whose specific GPDH activity was significantly reduced by two different degrees were comprehensively characterized in a previously developed Very High Ethanol Performance (VHEP) fed-batch process.

Results

The prototrophic strain CEN.PK113-7D was chosen for decreasing glycerol formation capacity. The fine-tuned reduction of specific GPDH activity was achieved by replacing the native GPD1 promoter in the yeast genome by previously generated well-characterized TEF promoter mutant versions in a gpd2 Δ background. Two TEF promoter mutant versions were selected for this study, resulting in a residual GPDH activity of 55 and 6%, respectively. The corresponding strains were referred to here as TEFmut7 and TEFmut2. The genetic modifications were accompanied to a strong reduction in glycerol yield on glucose; the level of reduction compared to the wild-type was 61% in TEFmut7 and 88% in TEFmut2. The overall ethanol production yield on glucose was improved from 0.43 g g^-1 in the wild type to 0.44 g g^-1 measured in TEFmut7 and 0.45 g g^-1 in TEFmut2. Although maximal growth rate in the engineered strains was reduced by 20 and 30%, for TEFmut7 and TEFmut2 respectively, strains' ethanol stress robustness was hardly affected; i.e. values for final ethanol concentration (117 ± 4 g L^-1), growth-inhibiting ethanol concentration (87 ± 3 g L^-1) and volumetric ethanol productivity (2.1 ± 0.15 g l^-1 h^-1) measured in wild-type remained virtually unchanged in the engineered strains.

Conclusions

This work demonstrates the power of fine-tuned pathway engineering, particularly when a compromise has to be found between high product yield on one hand and acceptable growth, productivity and stress resistance on the other hand. Under the conditions used in this study (VHEP fed-batch), the two strains with "fine-tuned" GPD1 expression in a gpd2 Δ background showed slightly better ethanol yield improvement than previously achieved with the single deletion strains gpd1 Δ or gpd2 Δ. Although glycerol reduction is known to be even higher in a gpd1 Δ gpd2 Δ double deletion strain, our strains could much better cope with process stress as reflected by better growth and viability.