Production of Recombinant Trichoderma Reesei Endoglucanase Protein CEL7B by Using Kluyveromyces Lactis

This research is about producing recombinant Trichoderma reesei endoglucanase Cel7B by using Kluyveromyces lactis, transformed with chromosomally integrated Cel7B cDNA, as a host cell (K. lactis Cel7B). Cel7B is one of the glycoside hydrolyze family of proteins that are produced by T. reesei. Cel7B together with other endoglucanases, exoglucanases, and â-glucosidases hydrolyze cellulose to glucose, which can then be fermented to biofuels or other value-added products. The research objective of this MS project is to examine favorable fermentation conditions for recombinant Cel7B enzyme production and improved activity. Production of enzyme on different types of media was examined, and the activity of the enzyme was measured by using different tools or procedures. The first condition tested for was using different concentrations of galactose as a carbon and energy source; however galactose also acts as a potent promoter of recombinant Cel7B expression in K. lactis Cel7B. The purpose of this method is to determine the relationship between production of enzyme with increasing sugar concentration. The second culture condition test was using different types of media: a complex medium-yeast extract, peptone, galactose (YPGal); a minimal medium-yeast nitrogen base (YNB) with galactose; and a minimal medium with supplement-yeast nitrogen base with casamino acid (YBC), a nitrogen source, with galactose. The third condition was using different types of reactors or fermenters: a small reactor (shake flask) and a larger automated bioreactor (BioFlo 3000 fermenter). The purpose of this method is to determine the quantity of the protein produced by using different environments of production. Different tools to determine the presence and activity of Cel7B enzyme were used. For the presence of enzyme, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was used. Secondly, to detect enzyme activity, the carboxymethyl cellulose- 3,5-dinitrosalicylic acid (CMC- DNS) assay was employed. SDS-PAGE showed that the enzyme band was at 67 kDa, which is larger than native Cel7B (52 kDa.), likely due to over glycolylation during post-translational processing in K. lactis. For the different types of media used in our fermentation, recombinant Cel7B was produced from yeast extract peptone galactose (YPGal), and yeast nitrogen base with casamino acid (YBC), but was not produced and no activity was detected from yeast nitrogen base (YNB). This experiment concluded that the Cel7B production requires the amino acid resources as part of fermentation medium. In experiments where recombinant Cel7B net activity was measured at 1% galactose initial concentration in YPGal and YBC media, higher enzyme activity was detected for the complex medium YPGal. Higher activity of recombinant Cel7B was detected for flask culture in 2% galactose compared to 1% galactose for YBC medium. Two bioreactor experiments were conducted under these culture conditions at 30°C, pH 7.0, dissolved oxygen of 50% of saturation, and 250 rpm agitation (variable depending on DO control) K. lactis-Cel7B yeast growth curves were quite reproducible with maximum optical density (O.D) at 600 nm of between 7 and 8 (when factoring dilution of 10:1). Galactose was consumed rapidly during the first 15 hours of bioreactor culture and recombinant Cel7B started to appear in the culture at 10-15 hours and increased thereafter up to a maximum of between 0.9 and 1.6 mg/mL/hr in these experiments. These bioreactor enzyme activity results are much higher than comparable experiments conducted with flask-scale culture (0.5 mg/mL/hr). In order to achieve the highest recombinant Cel7B activity from batch culture of K. lactis-Cel7B, based on this research it is best to use a complex medium, 2% initial galactose concentration, and an automated bioreactor where good control of temperature, pH, and dissolved oxygen can be achieved.

Callus cell culture of Pothomorphe umbellata (L.) under stress condition leads to high content of peroxidase enzyme

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Effect of the environment on condition and quality of cultured blue mussels (Mytilus edulis) with reference to culture depth and post-harvest practices

The condition and quality of cultured blue mussels (Mytilus edulis) are affected by various environmental characteristics including temperature, salinity, food concentration, composition and year-to-year variability, waves, tides, and currents. Mussels are a keystone species in the ecosystem, affecting the surrounding environment through filtration, biodeposition and nutrient recycling. This study evaluated the effects of culture depth and post-harvest handling on cultured blue mussels in Newfoundland, Canada. Depth was examined over two years; three shallow water (5 m depth) and three deep water sites (15 m depth) were compared for environmental characteristics, mussel physiological stress response, growth, and biochemical composition. The area examined presented complex hydrodynamic characteristics; deep water sites appeared to be located more often near or within the pycnocline than shallow water sites. Deep water sites presented lower temperatures than shallow sites from spring to fall. Physiological stress response varied seasonally, but was unaffected by culture depth. In Year 1 shallow and deep water mussels presented similar growth, while in Year 2 deep water mussels showed better final condition. Lipid and glycogen showed seasonal variation, but no significant differences between shallow and deep water were noted. Fatty acid profiles showed a higher content of omega-3s PUFA in deep water sites at the end of Year 2. Under extreme weather conditions, deep water appeared to provide a more stable environment for mussel growth than shallow water. Harvested mussels were kept under ambient live-holding conditions for one month during the fall, winter, and spring seasons. They were compared to freshly harvested mussels for condition, biochemical profile and palatability. A progressive loss of dry tissue weight and an increase in water content were shown over the holding period during the fall and spring seasons, when compared to field controls. The biochemical analysis suggested seasonal changes; differences in triacylglycerol content were found in the spring season when compared with controls. The palatability data indicated that the panellists were unable to determine a difference between mussels kept in holding and those freshly harvested from the site. This study presents new knowledge for mussel farming, especially in terms of environmental interactions and deep water culture.

Development of a 3D culture system to study the skeletal metastasis of prostate cancer

In the cancer research field, most in vitro studies still rely on two-dimensional (2D) cultures. However, the trend is rapidly shifting towards using a three-dimensional (3D) culture system. This is because 3D models better recapitulate the microenvironment of cells, and therefore, yield cellular and molecular responses that more accurately describe the pathophysiology of cancer. By adopting technology platforms established by the tissue engineering discipline, it is now possible to grow cancer cells in extracellular matrix (ECM)-like environments and dictate the biophysical and biochemical properties of the matrix. In addition, 3D models can be modified to recapitulate different stages of cancer progression for instance from the initial development of tumor to metastasis. Inevitably, to recapitulate a heterotypic condition, comprising more than one cell type, it requires a more complex 3D model. To date, 3D models that are available for studying the prostate cancer (CaP)-bone interactions are still lacking. Therefore, the aim of this study is to establish a co-culture model that allows investigation of direct and indirect CaP-bone interactions. Prior to that, 3D polyethylene glycol (PEG)-based hydrogel cultures for CaP cells were first developed and growth conditions were optimised. Characterization of the 3D hydrogel cultures show that LNCaP cells form a multicellular mass that resembles avascular tumor. In comparison to 2D cultures, besides the difference in cell morphology, the response of LNCaP cells to the androgen analogue (R1881) stimulation is different compared to the cells in 2D cultures. This discrepancy between 2D and 3D cultures is likely associated with the cell-cell contact, density and ligand-receptor interactions. Following the 3D monoculture study, a 3D direct co-culture model of CaP cells and the human tissue engineered bone (hTEBC) construct was developed. Interactions between the CaP cells and human osteoblasts (hOBs) resulted in elevation of Matrix Metalloproteinase 9 (MMP9) for PC-3 cells and Prostate Specific Antigen (PSA) for LNCaP cells. To further investigate the paracrine interaction of CaP cells and (hOBs), a 3D indirect co-culture model was developed, where LNCaP cells embedded within PEG hydrogels were co-cultured with hTEBC. It was found that the cellular changes observed reflect the early event of CaP colonizing the bone site. In the absence of androgens, interestingly, up-regulation of PSA and other kallikreins is also detected in the co-culture compared to the LNCaP monoculture. This non androgenic stimulation could be triggered by the soluble factors secreted by the hOB such as Interleukin-6. There are also decrease in alkaline phosphatase (ALP) activity and down-regulation of genes of the hOB when co-cultured with LNCaP cells that have not been previously described. These genes include transforming growth factor β1 (TGFβ1), osteocalcin and Vimentin. However, no changes to epithelial markers (e.g E-cadherin, Cytokeratin 8) were observed in both cell types from the co-culture. Some of these intriguing changes observed in the co-cultures that had not been previously described have enriched the basic knowledge of the CaP cell-bone interaction. From this study, we have shown evidence of the feasibility and versatility of our established 3D models. These models can be adapted to test various hypotheses for studies pertaining to underlying mechanisms of bone metastasis and could provide a vehicle for anticancer drug screening purposes in the future.

Development of zonal cartilage constructs : effects of chondrocyte subpopulation, compressive stimulation, and culture models

Articular cartilage is a highly resilient tissue located at the ends of long bones. It has a zonal structure, which has functional significance in load-bearing. Cartilage does not spontaneously heal itself when damaged, and untreated cartilage lesions or age-related wear often lead to osteoarthritis (OA). OA is a degenerative condition that is highly prevalent, age-associated, and significantly affects patient mobility and quality of life. There is no cure for OA, and patients usually resort to replacing the biological joint with an artificial prosthesis. An alternative approach is to dynamically regenerate damaged or diseased cartilage through cartilage tissue engineering, where cells, materials, and stimuli are combined to form new cartilage. However, despite extensive research, major limitations remain that have prevented the wide-spread application of tissue-engineered cartilage. Critically, there is a dearth of information on whether autologous chondrocytes obtained from OA patients can be used to successfully generate cartilage tissues with structural hierarchy typically found in normal articular cartilage. I aim to address these limitations in this thesis by showing that chondrocyte subpopulations isolated from macroscopically normal areas of the cartilage can be used to engineer stratified cartilage tissues and that compressive loading plays an important role in zone-dependent biosynthesis of these chondrocytes. I first demonstrate that chondrocyte subpopulations from the superficial (S) and middle/deep (MD) zones of OA cartilage are responsive to compressive stimulation in vitro, and that the effect of compression on construct quality is zone-dependent. I also show that compressive stimulation can influence pericelluar matrix production, matrix metalloproteinase secretion, and cytokine expression in zonal chondrocytes in an alginate hydrogel model. Subsequently, I focus on recreating the zonal structure by forming layered constructs using the alginate-released chondrocyte (ARC) method either with or without polymeric scaffolds. Resulting zonal ARC constructs had hyaline morphology, and expressed cartilage matrix molecules such as proteoglycans and collagen type II in both scaffold-free and scaffold-based approaches. Overall, my findings demonstrate that chondrocyte subpopulations obtained from OA joints respond sensitively to compressive stimulation, and are able to form cartilaginous constructs with stratified organization similar to native cartilage using the scaffold-free and scaffold-based ARC technique. The ultimate goal in tissue engineering is to help provide improved treatment options for patients suffering from debilitating conditions such as OA. Further investigations in developing functional cartilage replacement tissues using autologous chondrocytes will bring us a step closer to improving the quality of life for millions of OA patients worldwide.

Analyzing the literature for the link between the conservative Islamic culture of Saudi Arabia and the design of sustainable housing

Saudi Arabia experiences housing shortage for mid and low-income families, which is caused by rapid population growth. This condition is worsened by the fact that the current housing supply has problems in meeting both sustainable requirements and cultural needs of those families. This paper aims to investigate the link between the unique conservative Saudi culture and the design of sustainable housing, while keeping the housing cost affordable for mid and low-income families. The paper is based on a review of literatures on the issues of the Islamic culture and how can they be integrated into the design process of a Saudi house. Findings from literature reveiw suggest several design requirements for accommodating the conservative Saudi Culture in low cost sustainable houses. Such requirements include the implementation of proper usage of windows, and house orientation with a courtyard inside rather than facing the main street will provide natural ventilation while maintaining privacy. The main contribution to the body of knowledge is that this is a new approach to sustainable housing in Saudi Arabia considering not only energy use and architectural design issues but also socio-cultural issues as an essential part of sustainability.

Organisational culture in airworthiness management programs : developing a measurement model

All civil and private aircraft are required to comply with the airworthiness standards set by their national airworthiness authority and throughout their operational life must be in a condition of safe operation. Aviation accident data shows that over twenty percent of all fatal accidents in aviation are due to airworthiness issues, specifically aircraft mechanical failures. Ultimately it is the responsibility of each registered operator to ensure that their aircraft remain in a condition of safe operation, and this is done through both effective management of airworthiness activities and the effective program governance of safety outcomes. Typically, the projects within these airworthiness management programs are focused on acquiring, modifying and maintaining the aircraft as a capability supporting the business. Program governance provides the structure through which the goals and objectives of airworthiness programs are set along with the means of attaining them. Whilst the principal causes of failures in many programs can be traced to inadequate program governance, many of the failures in large scale projects can have their root causes in the organisational culture and more specifically in the organisational processes related to decision-making. This paper examines the primary theme of project and program based enterprises, and introduces a model for measuring organisational culture in airworthiness management programs using measures drawn from 211 respondents in Australian airline programs. The paper describes the theoretical perspectives applied to modifying an original model to specifically focus it on measuring the organisational culture of programs for managing airworthiness; identifying the most important factors needed to explain the relationship between the measures collected, and providing a description of the nature of these factors. The paper concludes by identifying a model that best describes the organisational culture data collected from seven airworthiness management programs.

Organizational culture in airworthiness management programmes : extending an existing measurement model

All civil and private aircraft are required to comply with the airworthiness standards set by their national airworthiness authority and throughout their operational life must be in a condition of safe operation. Aviation accident data shows that over 20% of all fatal accidents in aviation are due to airworthiness issues, specifically aircraft mechanical failures. Ultimately it is the responsibility of each registered operator to ensure that their aircraft remain in a condition of safe operation, and this is done through both effective management of airworthiness activities and the effective programme governance of safety outcomes. Typically, the projects within these airworthiness management programmes are focused on acquiring, modifying and maintaining the aircraft as a capability supporting the business. Programme governance provides the structure through which the goals and objectives of airworthiness programmes are set along with the means of attaining them. Whilst the principal causes of failures in many programmes can be traced to inadequate programme governance, many of the failures in large-scale projects can have their root causes in the organizational culture and more specifically in the organizational processes related to decision-making. This paper examines the primary theme of project and programme-based enterprises, and introduces a model for measuring organizational culture in airworthiness management programmes using measures drawn from 211 respondents in Australian airline programmes. The paper describes the theoretical perspectives applied to modifying an original model to specifically focus it on measuring the organizational culture of programmes for managing airworthiness; identifying the most important factors needed to explain the relationship between the measures collected, and providing a description of the nature of these factors. The paper concludes by identifying a model that best describes the organizational culture data collected from seven airworthiness management programmes.

Developments in controlled green-water larval culture technologies for estuarine fishes in Queensland, Australia and elsewhere

Since 1989, researchers with the Department of Primary Industries and Fisheries (DPI&F) in Queensland, Australia, have successfully used controlled low-water exchange green-water cultures to rear the larvae of estuarine fishes and crustaceans through to metamorphosis. High survivals and excellent fry condition have been achieved for several commercially important endemic species produced for various projects. They include barramundi or sea bass, Lates calcarifer, Australian bass, Macquaria novemaculeata, dusky flathead, Platycephalus fuscus, sand whiting, Sillago ciliata, red sea bream or snapper, Pagrus auratus, banana prawn, Fenneropenaeus merguiensis, and others. The consistent success of our standardised and relatively simple approach at different localities has led to it being incorporated into general fingerling production practices at several establishments in Australia. Although post-metamorphosis rearing methods have differed for each species investigated, due to various biological and behavioural traits and project requirements, these larval rearing methods have been successful with few species-specific modifications. Initially modelled on the Taiwanese approach to rearing Penaeids in aerated low-water exchange cultures, the approach similarly appears to rely on a beneficial assemblage of micro-organisms. Conceptually, these micro-organisms may include a mixture of the air-borne primary invaders of pure phytoplankton cultures when exposed to outdoor conditions. Whilst this would vary with different sites, our experiences with these methods have consistently been favourable. Mass microalgal cultures with eco-physiological youth are used to regularly augment larval fish cultures so that rearing conditions simulate an exponential growth-phase microalgal bloom. Moderate to heavy aeration prevents settlement of particulate matter and encourages aerobic bacterial decomposition of wastes. The green-water larval rearing approach described herein has demonstrated high practical utility in research and commercial applications, and has greatly simplified marine finfish hatchery operations whilst generally lifting production capacities for metamorphosed fry in Australia. Its potential uses in areas of aquaculture other than larviculture are also discussed.

Assessing organizational culture in complex sociotechnical systems : Methodological evidence from studies in nuclear power plant maintenance organizations

Failures in industrial organizations dealing with hazardous technologies can have widespread consequences for the safety of the workers and the general population. Psychology can have a major role in contributing to the safe and reliable operation of these technologies. Most current models of safety management in complex sociotechnical systems such as nuclear power plant maintenance are either non-contextual or based on an overly-rational image of an organization. Thus, they fail to grasp either the actual requirements of the work or the socially-constructed nature of the work in question. The general aim of the present study is to develop and test a methodology for contextual assessment of organizational culture in complex sociotechnical systems. This is done by demonstrating the findings that the application of the emerging methodology produces in the domain of maintenance of a nuclear power plant (NPP). The concepts of organizational culture and organizational core task (OCT) are operationalized and tested in the case studies. We argue that when the complexity of the work, technology and social environment is increased, the significance of the most implicit features of organizational culture as a means of coordinating the work and achieving safety and effectiveness of the activities also increases. For this reason a cultural perspective could provide additional insight into the problem of safety management. The present study aims to determine; (1) the elements of the organizational culture in complex sociotechnical systems; (2) the demands the maintenance task sets for the organizational culture; (3) how the current organizational culture at the case organizations supports the perception and fulfilment of the demands of the maintenance work; (4) the similarities and differences between the maintenance cultures at the case organizations, and (5) the necessary assessment of the organizational culture in complex sociotechnical systems. Three in-depth case studies were carried out at the maintenance units of three Nordic NPPs. The case studies employed an iterative and multimethod research strategy. The following methods were used: interviews, CULTURE-survey, seminars, document analysis and group work. Both cultural analysis and task modelling were carried out. The results indicate that organizational culture in complex sociotechnical systems can be characterised according to three qualitatively different elements: structure, internal integration and conceptions. All three of these elements of culture as well as their interrelations have to be considered in organizational assessments or important aspects of the organizational dynamics will be overlooked. On the basis of OCT modelling, the maintenance core task was defined as balancing between three critical demands: anticipating the condition of the plant and conducting preventive maintenance accordingly, reacting to unexpected technical faults and monitoring and reflecting on the effects of maintenance actions and the condition of the plant. The results indicate that safety was highly valued at all three plants, and in that sense they all had strong safety cultures. In other respects the cultural features were quite different, and thus the culturally-accepted means of maintaining high safety also differed. The handicraft nature of maintenance work was emphasised as a source of identity at the NPPs. Overall, the importance of safety was taken for granted, but the cultural norms concerning the appropriate means to guarantee it were little reflected. A sense of control, personal responsibility and organizational changes emerged as challenging issues at all the plants. The study shows that in complex sociotechnical systems it is both necessary and possible to analyse the safety and effectiveness of the organizational culture. Safety in complex sociotechnical systems cannot be understood or managed without understanding the demands of the organizational core task and managing the dynamics between the three elements of the organizational culture.

The History, Present Condition, and Future of the Molluscan Fisheries of North and Central America and Europe: Volume 2, Pacific Coast and Supplemental Topics

Over 100 molluscan species are landed in Mexico. About 30% are harvested on the Pacific coast and 70% on the Atlantic coast. Clams, scallops, and squid predominate on the Pacific coast (abalone, limpets, and mussels are landed there exclusively). Conchs and oysters predominate on the Atlantic coast. In 1988, some 95,000 metric tons (t) of mollusks were landed, with a value of $33 million. Mollusks were used extensively in prehispanic Mexico as food, tools, and jewelry. Their use as food and jewelry continues. Except in the States of Baja California and Baja California Sur, where abalone, clams, and scallops provide fishermen with year-round employment, mollusk fishing is done part time. On both the Pacific and Atlantic coasts, many fishermen are nomads, harvesting mollusks wherever they find abundant stocks. Upon finding such beds, they build camps, begin harvesting, and continue until the mollusks become so scarce that it no longer pays to continue. They then look for productive beds in other areas and rebuild their camps. Fishermen harvest abalones, mussels, scallops, and clams by free-diving and using scuba and hooka. Landings of clams and cockles have been growing, and 22,000 t were landed in 1988. Fishermen harvest intertidal clams by hand at wading depths, finding them with their feet. In waters up to 5 m, they harvest them by free-diving. In deeper water, they use scuba and hooka. Many species of gastropods have commercial importance on both coasts. All species with a large detachable muscle are sold as scallops. On the Pacific coast, hatchery culture of oysters prevails. Oyster culture in Atlantic coast lagoons began in the 1950's, when beds were enhanced by spreading shells as cultch for spat. (PDF file contains 228 pages.)

The History, Present Condition, and Future of the Molluscan Fisheries of North and Central American and Europe: Volume 1, Atlantic and Gulf Coasts

This three-volume monograph represents the first major attempt in over a century to provide, on regional bases, broad surveys of the history, present condition, and future of the important shellfisheries of North and Central America and Europe. It was about 100 years ago that Ernest Ingersoll wrote extensively about several molluscan fisheries of North America (1881, 1887) and about 100 years ago that Bashford Dean wrote comprehensively about methods of oyster culture in Europe (1893). Since those were published, several reports, books, and pamphlets have been written about the biology and management of individual species or groups ofclosely related mollusk species (Galtsoff, 1964; Korringa, 1976 a, b, c; Lutz, 1980; Manzi and Castagna, 1989; Shumway, 1991). However, nothing has been written during the past century that is comparable to the approach used by Ingersoll in describing the molluscan fisheries as they existed in his day in North America or, for that matter, in Europe. (PDF file contains 224 pages.)

Prawn culture and policy options: technology import and culture through fishermen vis a vis industry

Recent developments in aquaculture has created an awareness that prawn culture is a dollar spinner, in which industry can step in to earn foreign exchange by producing an expensive food iten which has a high market demand abroad. The Government has to take a policy decision whether the prawn culture should be done through small fishermen to improve their socio-economic condition or through private industry with the high technology input and predefined objectives of export trade. Perhaps a simultaneous operation of the two could be allowed best in the interest of India. Perhaps in the interest of quick development and adoption of high production technology, through fishermen organization, the development is encouraged through the implimentation of welfare and area development schemes. In some selected areas private industry may be encoureged to use high production technology to develop prawns.

Field trials on culture of Clarias batrachus

Culture of Magur, Clarias batrachus on an experimental basis was carried out for a total of 159 days in 6 earthen ponds each of 0.02 ha in area, singly in 2 ponds and in combination with Rohu, Labeo rohita in 4 ponds. Three different artificial feeds were used. Growth of Magur varied from 89 to 110 and survival form 93 to 100%. Out of the 3 feeds used, feed formulation 2 yielded better results. Length-weight relationship parameters were found as log W=0.4979067+1.878346 log L; -1.1116438+2.3511497 log L; and -1.238157+2.433125 log L indicating growth to be not isometric. Relative condition factor (K u) was close to or higher than 1.0 only in fishes higher than 200 mm of total length; K. values which were less than 1.0 up to January, reached values greater than 1.0 by March-April. Condition appears to be influenced by spawning rather than feeding.