Increasing cell biomass in Saccharomyces cerevisiae increases recombinant protein yield:the use of a respiratory strain as a microbial cell factory

BACKGROUND: Recombinant protein production is universally employed as a solution to obtain the milligram to gram quantities of a given protein required for applications as diverse as structural genomics and biopharmaceutical manufacture. Yeast is a well-established recombinant host cell for these purposes. In this study we wanted to investigate whether our respiratory Saccharomyces cerevisiae strain, TM6*, could be used to enhance the productivity of recombinant proteins over that obtained from corresponding wild type, respiro-fermentative strains when cultured under the same laboratory conditions. RESULTS: Here we demonstrate at least a doubling in productivity over wild-type strains for three recombinant membrane proteins and one recombinant soluble protein produced in TM6* cells. In all cases, this was attributed to the improved biomass properties of the strain. The yield profile across the growth curve was also more stable than in a wild-type strain, and was not further improved by lowering culture temperatures. This has the added benefit that improved yields can be attained rapidly at the yeast's optimal growth conditions. Importantly, improved productivity could not be reproduced in wild-type strains by culturing them under glucose fed-batch conditions: despite having achieved very similar biomass yields to those achieved by TM6* cultures, the total volumetric yields were not concomitantly increased. Furthermore, the productivity of TM6* was unaffected by growing cultures in the presence of ethanol. These findings support the unique properties of TM6* as a microbial cell factory. CONCLUSIONS: The accumulation of biomass in yeast cell factories is not necessarily correlated with a proportional increase in the functional yield of the recombinant protein being produced. The respiratory S. cerevisiae strain reported here is therefore a useful addition to the matrix of production hosts currently available as its improved biomass properties do lead to increased volumetric yields without the need to resort to complex control or cultivation schemes. This is anticipated to be of particular value in the production of challenging targets such as membrane proteins.

An analysis of the effectiveness of a behavioural based safety intervention in a UK paper mill

This research examines a behavioural based safety (BBS) intervention within a paper mill in the South East of England. Further to this intervention two other mills are examined for the purposes of comparison — one an established BBS programme and the other an improving safety management system through management ownership. BBS programmes have become popular within the UK, but most of the research about their efficacy is carried out by the BBS providers themselves. This thesis aims to evaluate a BBS intervention from a standpoint which is not commercially biased in favour of BBS schemes. The aim of a BBS scheme is to either change personnel behaviours or attitudes, which in turn will positively affect the organisation's safety culture. The research framework involved a qualitative methodology in order to examine the effects of the intervention on the paper mill's safety culture. The techniques used were questionnaires and semi structured interviews, in addition to observation and discussions which were possible because of the author's position as participant observer. The results demonstrated a failure to improve any aspect of the mill's safety culture, which worsened following the BBS intervention. Issues such as trust, morale, communication and support of management showed significant signs of negative workforce response. The paper mill where the safety management system approach was utilised demonstrated a significantly improved safety culture and achieved site ownership from middle managers and supervisors. Research has demonstrated that a solid foundation is required prior to successfully implementing a BBS programme. For a programme to work there must be middle management support in addition to senior management commitment. If a trade union actively distances itself from BBS, it is also unlikely to be effective. This thesis proposes that BBS observation programmes are not suitable for the papermaking industry, particularly when staffing levels are low due to challenging economic conditions. Observers are not available when there are high hazard situations and this suggests that BBS implementation is not the correct intervention for the paper industry.

The ecology of paper mill by-product and its evaluation as a casing medium in the culture of Agaricus bisporus (Lange) Pilat

Effluent from pulp and paper production at the Kemsley mill of Bowaters U.K. Paper Company Limited passes through two treatment stages before its discharge into the Swale estuary. Suspended material removed during treatment is deposited on wasteground as a thin sludge. The solids it contains are mainly wood components lost during pulp production, whilst it also has a high salt content, derived from chemicals used in pulping processes. After deposition the sludge undergoes an ageing process during which it dries out and its salt content is reduced. This ageing can be reproduced and accelerated by improved drainage under controlled conditions. The paper mill sludge was investigated as a casing medium in the culture of Agaricus bisporus (Lange) Pilat, the cultivated mushroom. It was unsuitable up to one year from deposition due largely to the inhibitory effect of its salt content on fruiting. Material eighteen months or more in age gave yields comparable to standard peat casing. Before use as a casing the material must be shredded to a satisfactory structure, neutralised with chalk, and pasteurised to eliminate organisms harmful to the crop. The prepared medium has a high water holding capacity and a structure resilient to management procedures, important requirements of a good casing. A passive movement of salts from the compost to the casing was shown to occur during culture, capable of enhancing the natural decline in cropping if sufficiently great. The ions chloride, potassium, sodium and sulphate were shown to be responsible, their damaging effects being due to high conductivity created in the casing. Studies of elements available during culture suggested phosphate availability in the compost could limit crop potential, whilst iron released by mycelium of A.bisporus in the casing may be utilised by associated micro-organisms.

Some aspects of system design for production management in the rolling-mill industry

Case studies in copper-alloy rolling mill companies showed that existing planning systems suffer from numerous shortcomings. Where computerised systems are in use, these tend to simply emulate older manual systems and still rely heavily on modification by experienced planners on the shopfloor. As the size and number of orders increase, the task of process planners, while seeking to optimise the manufacturing objectives and keep within the production constraints, becomes extremely complicated because of the number of options for mixing or splitting the orders into batches. This thesis develops a modular approach to computerisation of the production management and planning functions. The full functional specification of each module is discussed, together with practical problems associated with their phased implementation. By adapting the Distributed Bill of Material concept from Material Requirements Planning (MRP) philosophy, the production routes generated by the planning system are broken down to identify the rolling stages required. Then to optimise the use of material at each rolling stage, the system generates an optimal cutting pattern using a new algorithm that produces practical solutions to the cutting stock problem. It is shown that the proposed system can be accommodated on a micro-computer, which brings it into the reach of typical companies in the copper-alloy rolling industry, where profit margins are traditionally low and the cost of widespread use of mainframe computers would be prohibitive.

Cadburys' new factory system, 1879-1919

This thesis examines the reasons for Cadburys' move from a city centre site to a greenfield site in Bournville in 1879 and the subsequent development of the factory and the Bournville community. The founding of the Bournville Village Trust by George Cadbury is discussed in relation to the Garden City movement. The welfare and personnel management policies which Cadburys adopted in the 1900s are considered in relation to welfarism in general, especially in the United States. The extent to which the idea of a `Quaker employer' can explain Cadburys policies is questioned both methodologically and empirically. The early use of scientific management at Bournville is described and related to Edward Cadbury's writings on the subject. Finally, the institution of a Works Council Scheme in 1918 is described and its uses are discussed. It is concluded that Cadburys instituted a new factory system in this period which consisted of a synthesis of ideas borrowed from elsewhere and that for a variety of reasons Cadburys was an appropriate site for their implementation.

Identification of key temperature measurement technologies for the enhancement of product and equipment integrity in the light controlled factory

Thermal effects in uncontrolled factory environments are often the largest source of uncertainty in large volume dimensional metrology. As the standard temperature for metrology of 20°C cannot be achieved practically or economically in many manufacturing facilities, the characterisation and modelling of temperature offers a solution for improving the uncertainty of dimensional measurement and quantifying thermal variability in large assemblies. Technologies that currently exist for temperature measurement in the range of 0-50°C have been presented alongside discussion of these temperature measurement technologies' usefulness for monitoring temperatures in a manufacturing context. Particular aspects of production where the technology could play a role are highlighted as well as practical considerations for deployment. Contact sensors such as platinum resistance thermometers can produce accuracy closest to the desired accuracy given the most challenging measurement conditions calculated to be ∼0.02°C. Non-contact solutions would be most practical in the light controlled factory (LCF) and semi-invasive appear least useful but all technologies can play some role during the initial development of thermal variability models.

Large volume metrology technologies for the light controlled factory

In the Light Controlled Factory part-to-part assembly and reduced weight will be enabled through the use of predictive fitting processes; low cost high accuracy reconfigurable tooling will be made possible by active compensation; improved control will allow accurate robotic machining; and quality will be improved through the use of traceable uncertainty based quality control throughout the production system. A number of challenges must be overcome before this vision will be realized; 1) controlling industrial robots for accurate machining; 2) compensation of measurements for thermal expansion; 3) Compensation of measurements for refractive index changes; 4) development of Embedded Metrology Tooling for in-tooling measurement and active tooling compensation; and 5) development of Software for the Planning and Control of Integrated Metrology Networks based on Quality Control with Uncertainty Evaluation and control systems for predictive processes. This paper describes how these challenges are being addressed, in particular the central challenge of developing large volume measurement process models within an integrated dimensional variation management (IDVM) system.

A hybrid measurement systems analysis and uncertainty of measurement approach for industrial measurement in the light controlled factory

The uncertainty of measurements must be quantified and considered in order to prove conformance with specifications and make other meaningful comparisons based on measurements. While there is a consistent methodology for the evaluation and expression of uncertainty within the metrology community industry frequently uses the alternative Measurement Systems Analysis methodology. This paper sets out to clarify the differences between uncertainty evaluation and MSA and presents a novel hybrid methodology for industrial measurement which enables a correct evaluation of measurement uncertainty while utilising the practical tools of MSA. In particular the use of Gage R&R ANOVA and Attribute Gage studies within a wider uncertainty evaluation framework is described. This enables in-line measurement data to be used to establish repeatability and reproducibility, without time consuming repeatability studies being carried out, while maintaining a complete consideration of all sources of uncertainty and therefore enabling conformance to be proven with a stated level of confidence. Such a rigorous approach to product verification will become increasingly important in the era of the Light Controlled Factory with metrology acting as the driving force to achieve the right first time and highly automated manufacture of high value large scale products such as aircraft, spacecraft and renewable power generation structures.

Integrated Dimensional Variation Management in the Digital Factory

This paper describes how dimensional variation management could be integrated throughout design, manufacture and verification, to improve quality while reducing cycle times and manufacturing cost in the Digital Factory environment. Initially variation analysis is used to optimize tolerances during product and tooling design and also results in the creation of a simplified representation of product key characteristics. This simplified representation can then be used to carry out measurability analysis and process simulation. The link established between the variation analysis model and measurement processes can subsequently be used throughout the production process to automatically update the variation analysis model in real time with measurement data. This ‘live’ simulation of variation during manufacture will allow early detection of quality issues and facilitate autonomous measurement assisted processes such as predictive shimming. A study is described showing how these principles can be demonstrated using commercially available software combined with a number of prototype applications operating as discrete modules. The commercially available modules include Catia/Delmia for product and process design, 3DCS for variation analysis and Spatial Analyzer for measurement simulation. Prototype modules are used to carry out measurability analysis and instrument selection. Realizing the full potential of Metrology in the Digital Factory will require that these modules are integrated and software architecture to facilitate this is described. Crucially this integration must facilitate the use of realtime metrology data describing the emerging assembly to update the digital model.