Agitation conditions for the culture and detachment of hMSCs from microcarriers in multiple bioreactor platforms

In our recent work in different bioreactors up to 2.5L in scale, we have successfully cultured hMSCs using the minimum agitator speed required for complete microcarrier suspension, N JS. In addition, we also reported a scaleable protocol for the detachment from microcarriers in spinner flasks of hMSCs from two donors. The essence of the protocol is the use of a short period of intense agitation in the presence of enzymes such that the cells are detached; but once detachment is achieved, the cells are smaller than the Kolmogorov scale of turbulence and hence not damaged. Here, the same approach has been effective for culture at N JS and detachment in-situ in 15mL ambr™ bioreactors, 100mL spinner flasks and 250mL Dasgip bioreactors. In these experiments, cells from four different donors were used along with two types of microcarrier with and without surface coatings (two types), four different enzymes and three different growth media (with and without serum), a total of 22 different combinations. In all cases after detachment, the cells were shown to retain their desired quality attributes and were able to proliferate. This agitation strategy with respect to culture and harvest therefore offers a sound basis for a wide range of scales of operation.

Systematic microcarrier screening and agitated culture conditions improves human mesenchymal stem cell yield in bioreactors

Production of human mesenchymal stem cells for allogeneic cell therapies requires scalable, cost-effective manufacturing processes. Microcarriers enable the culture of anchorage-dependent cells in stirred-tank bioreactors. However, no robust, transferable methodology for microcarrier selection exists, with studies providing little or no reason explaining why a microcarrier was employed. We systematically evaluated 13 microcarriers for human bone marrow-derived MSC (hBM-MSCs) expansion from three donors to establish a reproducible and transferable methodology for microcarrier selection. Monolayer studies demonstrated input cell line variability with respect to growth kinetics and metabolite flux. HBM-MSC1 underwent more cumulative population doublings over three passages in comparison to hBM-MSC2 and hBM-MSC3. In 100 mL spinner flasks, agitated conditions were significantly better than static conditions, irrespective of donor, and relative microcarrier performance was identical where the same microcarriers outperformed others with respect to growth kinetics and metabolite flux. Relative growth kinetics between donor cells on the microcarriers were the same as the monolayer study. Plastic microcarriers were selected as the optimal microcarrier for hBM-MSC expansion. HBM-MSCs were successfully harvested and characterised, demonstrating hBM-MSC immunophenotype and differentiation capacity. This approach provides a systematic method for microcarrier selection, and the findings identify potentially significant bioprocessing implications for microcarrier-based allogeneic cell therapy manufacture. Large-scale production of human bone-marrow derived mesenchymal stem cells (hBM-MSCs) requires expansion on microcarriers in agitated systems. This study demonstrates the importance of microcarrier selection and presents a systematic methodology for selection of an optimal microcarrier. The study also highlights the impact of an agitated culture environment in comparison to a static system, resulting in a significantly higher hBM-MSC yield under agitated conditions.

Sedimentary record of the western Amundsen Sea Embayment after the last glacial maximum

Reliable dating of glaciomarine sediments deposited on the Antarctic shelf since the Last Glacial Maximum (LGM) is very challenging because of the general absence of calcareous (micro-) fossils and the recycling of fossil organic matter. As a consequence, radiocarbon (14C) ages of the acid-insoluble organic fraction (AIO) of the sediments bear uncertainties that are very difficult to quantify. In this paper we present the results of three different chronostratigraphic methods to date a sedimentary unit consisting of diatomaceous ooze and diatomaceous mud that was deposited following the last deglaciation at five core sites on the inner shelf in the western Amundsen Sea (West Antarctica). In three cores conventional 14C dating of the AIO in bulk sediment samples yielded age reversals down-core, but at all sites the AIO 14C ages obtained from diatomaceous ooze within the diatom-rich unit yielded similar uncorrected 14C ages ranging from 13,517±56 to 11,543±47 years before present (yr BP). Correction of these ages by subtracting the core-top ages, which are assumed to reflect present-day deposition (as indicated by 21044 Pb dating of the sediment surface at one core site), yielded ages between ca. 10,500 and 8,400 calibrated years before present (cal yr BP). Correction of the AIO ages of the diatomaceous ooze by only subtracting the marine reservoir effect (MRE) of 1,300 years indicated deposition of the diatom-rich sediments between 14,100 and 11,900 cal yr BP. Most of these ages are consistent with age constraints between 13.0 and 8.0 ka BP for the diatom-rich unit, which we obtained by correlating the relative palaeomagnetic intensity (RPI) records of three of the sediment cores with global and regional reference curves for palaeomagnetic intensity. As a third dating technique we applied conventional 53 radiocarbon dating of the AIO included in acid-cleaned diatom hard parts that were extracted from the diatomaceous ooze. This method yielded uncorrected 14C ages of only 5,111±38 and 5,106±38 yr BP, respectively. We reject these young ages, because they are likely to be overprinted by the adsorption of modern atmospheric carbon dioxide onto the surfaces of the extracted diatom hard parts prior to sample graphitisation and combustion for 14C dating. The deposition of the diatom-rich unit in the western Amundsen Sea suggests deglaciation of the inner shelf before ca. 13 ka BP. The deposition of diatomaceous oozes on other parts of the Antarctic shelf around the same time, however, seems to be coincidental rather than directly related.

The effect of a reduction of nitrogen and phosphorus to eutrophic and mesotrophic conditions towards silver nanoparticle (AgNP) on freshwater green algae species

We investigated the sensitivity of algae towards silver nanoparticles with OECD test medium and lower nutrient concentrations under standard test conditions to improve comparability and to exclude any other confounding factor aside nutrient levels. Two unicellular freshwater microalgae Desmodesmus subspicatus and Raphidocelis subcapitata were chosen due to their status as standard test organisms for the algae growth inhibition test and the response to changes in nutrient supply was compared. The original medium was used as the reference (standard). For the other four media, the amount of either nitrogen or phosphorus in the medium was lowered from half (50%) to one-fourth (25 %) of that of the OECD guideline, resulting in the following media: 50% N, 25% N, 50% P and 25% P medium. As test substance, the OECD reference material NM-300K was used. For this reason, the characterization of AgNP was done using DLS and Absorption spectra (UV/vis). Actual silver concentrations and ionic silver concentrations were measured at the highest test concentration used (100 µg Ag L-1) in R. subcapitata treatments only to reduce the number of samples. All tests were run according to the OECD guideline 201 with sterilized 50 mL cell culture flask. Each medium was tested using the test conditions for culturing with 3 replicates. Test concentrations for both algae species were 0, 25, 50 and 100 µg Ag L-1 for OECD, 50% P and 25% P while for both N reductions, the silver concentrations were 0, 10, 25 and 100 µg Ag L-1. Samples for determining the algal density were taken at every 24 h.

Core scans and lithologic analysis of Subglacial Lake Whillans sediment cores

The hydrologic system beneath the Antarctic Ice Sheet is thought to influence both the dynamics and distribution of fast flowing ice streams, which discharge most of the ice lost by the ice sheet. Despite considerable interest in understanding this subglacial network and its affect on ice flow, in situ observations from the ice sheet bed are exceedingly rare. Here we describe the first sediment cores recovered from an active subglacial lake. The lake, known as Subglacial Lake Whillans, is part of a broader, dynamic hydrologic network beneath the Whillans Ice Stream in West Antarctica. Even though "floods" pass through the lake, the lake floor shows no evidence of erosion or deposition by flowing water. By inference, these floods must have insufficient energy to erode or transport significant volumes of sediment coarser than silt. Consequently, water flow beneath the region is probably incapable of incising continuous channels into the bed and instead follows preexisting subglacial topography and surface slope. Sediment on the lake floor consists of till deposited during intermittent grounding of the ice stream following flood events. The fabrics within the till are weaker than those thought to develop in thick deforming beds suggesting subglacial sediment fluxes across the ice plain are currently low and unlikely to have a large stabilizing effect on the ice stream's grounding zone.

Production of Polyhydroxyalkanoates by Pseudomonas mendocina using vegetable oils and their characterisation

Synthesis of Polyhydroxyalkanoates (PHAs) by Pseudomonas mendocina, using different vegetable oils such as, coconut oil, groundnut oil, corn oil and olive oil, as the sole carbon source was investigated for the first time. The PHA yield obtained was compared with that obtained during the production of PHAs using sodium octanoate as the sole carbon source. The fermentation profiles at shaken flask and bioreactor levels revealed that vegetable oils supported the growth of Pseudomonas mendocina and PHA accumulation in this organism. Moreover, when vegetable oil (coconut oil) was used as the sole carbon source, fermentation profiles showed better growth and polymer production as compared to conditions when sodium octanoate was used as the carbon source. In addition, comparison of PHA accumulation at shaken flask and fermenter level confirmed the higher PHA yield at shaken flask level production. The highest cell mass found using sodium octanoate was 1.8 g/L, whereas cell mass as high as 5.1 g/L was observed when coconut oil was used as the feedstock at flask level production. Moreover, the maximum PHA yield of 60.5% dry cell weight (dcw) was achieved at shaken flask level using coconut oil as compared to the PHA yield of 35.1% dcw obtained using sodium octanoate as the sole carbon source. Characterisations of the chemical, physical, mechanical, surface and biocompatibility properties of the polymers produced have been carried out by performing different analyses as described in the second chapter of this study. Chemical analysis using GC and FTIR investigations showed medium chain length (MCL) PHA production in all conditions. GC-MS analysis revealed a unique terpolymer production, containing 3-hydroxyoctanoic acid, 3-hydroxydecanoic acid and 3-hydroxydodecanoic acid when coconut oil, groundnut oil, olive oil, and corn oil were used as the carbon source. Whereas production of the homopolymer containing 3-hydroxyoctanoic acid was observed when sodium octanoate was used as the carbon source. MCL-PHAs produced in this study using sodium octanoate, coconut oil, and olive oil exhibited melting transitions, indicating that each of the PHA was crystalline or semi-crystalline polymer. In contrast, the thermal properties of PHAs produced from groundnut and corn oils showed no melting transition, indicating that they were completely amorphous or semi-crystalline, which was also confirmed by the X-Ray Diffraction (XRD) results obtained in this study. Mechanical analysis of the polymers produced showed higher stiffness of the polymer produced from coconut oil than the polymer from sodium octanoate. Surface characterisation of the polymers using Scanning Electron Microscopy (SEM) revealed a rough surface topography and surface contact angle measurement revealed their hydrophobic nature. Moreover, to investigate the potential applicability of the produced polymers as the scaffold materials for dental pulp regeneration, multipotent human Mesenchymal stem cells (hMSCs) were cultured onto the polymer films. Results indicated that these polymers are not cytotoxic towards the hMSCs and could support their attachment and proliferation. Highest cell growth was observed on the polymer samples produced from corn oil, followed by the polymer produced using coconut oil. In conclusion, this work established, for the first time, that vegetable oils are a good economical source of carbon for production of MCL-PHA copolymers effectively by Pseudomonas mendocina. Moreover, biocompatibility studies suggest that the produced polymers may have potential for dental tissue engineering application.

Stock assessment of whaler and hammerhead sharks (Carcharhinidae and Sphyrnidae) in Queensland

This stock assessment provides detailed results for the most common sharks encountered by Queensland commercial fishers. These sharks come from the whaler (Carcharhinidae) and hammerhead (Sphyrnidae) families and comprise sharpnose sharks (Rhizoprionodon taylori and R. oligolinx), the milk shark (R. acutus), the creek whaler (Carcharhinus fitzroyensis), the hardnose shark (C. macloti), the spot-tail shark (C. sorrah), the Australian blacktip shark (C. tilstoni), the common blacktip shark (C. limbatus), the spinner shark (C. brevipinna), bull and pigeye sharks (C. leucas and C. amboinensis), the winghead shark (Eusphyra blochii), the scalloped hammerhead (Sphyrna lewini) and the great hammerhead (S. mokarran). Reef sharks were excluded because fishery observer data indicated that they were largely spatially segregated from sharks caught in the inshore net fisheries. The three common species of reef sharks in Queensland, which are all whaler sharks, are the grey reef shark Carcharhinus amblyrhynchos, the blacktip reef shark C. melanopterus and the whitetip reef shark Triaenodon obesus.

Ability of Gordonia alkanivorans strain 1B for high added value carotenoids production

Currently, carotenoids are valuable bioactive molecules for several industries, such as chemical, pharmaceutical, food and cosmetics, due to their multiple benefits as natural colorants, antioxidants and vitamin precursors. Hence, the increasing interest on these high added-value products has led to the search of alternatives, more cost-effective and with better yields, towards their industrial production. Indeed, microbial metabolism offers a promising option for carotenoids production. Herein it is shown the potential of the dibenzothiophene desulfurizing bacterium Gordonia alkanivorans strain 1B as a high carotenoid-producer microorganism. The novel carotenoids, produced under different culture conditions, were extracted with DMSO and then further analyzed both through spectrophotometry and HPLC. When grown in glucose-sulfate-light, strain 1B was able of achieving 2015 g carotenoids per g DCW in shake-flask assays, with about 60% corresponding to lutein, canthaxanthin and astaxanthin. Further optimization studies open a new focus of research aiming to get a hyper pigment-producer strain that may be applied towards different industrial sectors.

Assessment of a data-limited, multi-species shark fishery in the Great Barrier Reef Marine Park and south-east Queensland

The status of five species of commercially exploited sharks within the Great Barrier Reef Marine Park (GBRMP) and south-east Queensland was assessed using a data-limited approach. Annual harvest rate, U, estimated empirically from tagging between 2011 and 2013, was compared with an analytically-derived proxy for optimal equilibrium harvest rate, UMSY Lim. Median estimates of U for three principal retained species, Australian blacktip shark, Carcharhinus tilstoni, spot-tail shark, Carcharhinus sorrah, and spinner shark, Carcharhinus brevipinna, were 0.10, 0.06 and 0.07 year-1, respectively. Median U for two retained, non-target species, pigeye shark, Carcharhinus amboinensis and Australian sharpnose shark, Rhizoprionodon taylori, were 0.27 and 0.01 year-1, respectively. For all species except the Australian blacktip the median ratio of U/UMSY Lim was <1. The high vulnerability of this species to fishing combined with life history characteristics meant UMSY Lim was low (0.04-0.07 year-1) and that U/UMSY Lim was likely to be > 1. Harvest of the Australian blacktip shark above UMSY could place this species at a greater risk of localised depletion in parts of the GBRMP. Results of the study indicated that much higher catches, and presumably higher U, during the early 2000s were likely unsustainable. The unexpectedly high level of U on the pigeye shark indicated that output-based management controls may not have been effective in reducing harvest levels on all species, particularly those caught incidentally by other fishing sectors including the recreational sector. © 2016 Elsevier B.V.

Comparing the expression of human DNA topoisomerase I in KM71H and X33 strains of Pichia pastoris

Background: Human is an essential cellular enzyme that is found in all human cells. As this enzyme is upregulated in cancer cells exceedingly, it is used as a target for cancer chemotherapeutic drug development. As such, producing the in-house enzyme for the purpose to speed up the search for more cost-effective and target specific hTopoI inhibitors is warranted. This study aims to compare the optimised conditions for the expression of hTopoI in KM71H (MutS) and X33 (Mut+) strains of Pichia pastoris P. pastoris transfected with an hTopoI recombinant vector was used for the optimization of a higher level of hTopoI expression. Results: In the process, fed-batch cultivation parameters that influence the expression of hTopoI, such as culture temperature, methanol induction and feeding strategy, were optimised in the transfected KM71H and X33 P. pastoris strains in a shake flask system. The cell density and total protein concentration (protein level) of transfected P. pastoris were compared to determine the optimum culture conditions for each transfected P. pastoris strain. A higher hTopoI level was observed in the transfected KM71H culture supernatant (2.26 ng/mL) when the culture was incubated in the optimum conditions. Conclusions: This study demonstrated that MutS strain (KM71H) expressed and secreted a higher level of hTopoI heterologous protein in the presence of methanol compared to the Mut+ strain; X33 (0.75 ng/mL). However, other aspects of optimization, such as pH, should also be considered in the future, to obtain the optimum expression level of hTopoI in P. pastoris.

Application of an online-biomass sensor in an optical multisensory platform prototype for growth monitoring of biotechnical relevant microorganism and cell lines in single-use shake flasks

In the context of this work we evaluated a multisensory, noninvasive prototype platform for shake flask cultivations by monitoring three basic parameters (pH, pO2 and biomass). The focus lies on the evaluation of the biomass sensor based on backward light scattering. The application spectrum was expanded to four new organisms in addition to E. coli K12 and S. cerevisiae [1]. It could be shown that the sensor is appropriate for a wide range of standard microorganisms, e.g., L. zeae, K. pastoris, A. niger and CHO-K1. The biomass sensor signal could successfully be correlated and calibrated with well-known measurement methods like OD600, cell dry weight (CDW) and cell concentration. Logarithmic and Bleasdale-Nelder derived functions were adequate for data fitting. Measurements at low cell concentrations proved to be critical in terms of a high signal to noise ratio, but the integration of a custom made light shade in the shake flask improved these measurements significantly. This sensor based measurement method has a high potential to initiate a new generation of online bioprocess monitoring. Metabolic studies will particularly benefit from the multisensory data acquisition. The sensor is already used in labscale experiments for shake flask cultivations.

Quantificação de Fases Cristalinas de Incrustações em Colunas de Produção de Petróleo pelo Método Rietveld

The scale is defined as chemical compounds from inorganic nature, initially soluble in salt solutions, which may precipitate accumulate in columns of production and surface equipment. This work aimd to quantify the crystalline phases of scale through the Rietveld method. The study was conducted in scale derived from columns production wells in development and recipients of pigs. After collecting samples of scale were performed the procedure for separations of inorganic and organic phase and preparation to be analyzed at the X-ray Laboratory. The XRD and XRF techniques were used to monitor whether identifying and quantifying crystalline phases present in the deposits. The SEM technique was used to visualize the morphology of the scales and assess their homogeneity after the milling process. XRD measurements were performed with and without milling and with or without the accessory spinner. For quantify crystalline phases the program DBWStools was used. The procedure for conducting the first refinement was instrumental in setting parameters, then the structural parameters of the phases in the sample and finally the parameters of the function profile used. In the diffraction patterns of samples of scale observed that the best measures were those that passed through the mill and used the accessory spinner. Through the results, it was noted that the quantitative analysis for samples of scale is feasible when need to monitor a particular crystalline phase in a well, pipeline or oil field. Routinely, the quantification of phases by the Rietveld method is hardwork because in many scale was very difficult to identify the crystalline phases present

Induction of Microalgal Lipids for Biodiesel Production in Tandem with Sequestration of High Carbon Dioxide Concentration

There is no doubt that sufficient energy supply is indispensable for the fulfillment of our fossil fuel crises in a stainable fashion. There have been many attempts in deriving biodiesel fuel from different bioenergy crops including corn, canola, soybean, palm, sugar cane and vegetable oil. However, there are some significant challenges, including depleting feedstock supplies, land use change impacts and food use competition, which lead to high prices and inability to completely displace fossil fuel [1-2]. In recent years, use of microalgae as an alternative biodiesel feedstock has gained renewed interest as these fuels are becoming increasingly economically viable, renewable, and carbon-neutral energy sources. One reason for this renewed interest derives from its promising growth giving it the ability to meet global transport fuel demand constraints with fewer energy supplies without compromising the global food supply. In this study, Chlorella protothecoides microalgae were cultivated under different conditions to produce high-yield biomass with high lipid content which would be converted into biodiesel fuel in tandem with the mitigation of high carbon dioxide concentration. The effects of CO2 using atmospheric and 15% CO2 concentration and light intensity of 35 and 140 µmol m-2s-1 on the microalgae growth and lipid induction were studied. The approach used was to culture microalgal Chlorella protothecoides with inoculation of 1×105 cells/ml in a 250-ml Erlenmeyer flask, irradiated with cool white fluorescent light at ambient temperature. Using these conditions we were able to determine the most suitable operating conditions for cultivating the green microalgae to produce high biomass and lipids. Nile red dye was used as a hydrophobic fluorescent probe to detect the induced intracellular lipids. Also, gas chromatograph mass spectroscopy was used to determine the CO2 concentrations in each culture flask using the closed continuous loop system. The goal was to study how the 15% CO2 concentration was being used up by the microalgae during cultivation. The results show that the condition of high light intensity of 140 µmol m-2s-1 with 15% CO2 concentration obtain high cell concentration of 7 x 105 cells mL-1 after culturing Chlorella protothecoides for 9 to 10 day in both open and closed systems respectively. Higher lipid content was estimated as indicated by fluorescence intensity with 1.3 to 2.5 times CO2 reduction emitted by power plants. The particle size of Chlorella protothecoides increased as well due to induction of lipid accumulation by the cells when culture under these condition (140 µmol m-2s-1 with 15% CO2 concentration).

SIMULATING LARGE DEFORMATION OF INTRANEURAL GANGLION CYST USING FINITE ELEMENT METHOD

Intraneural Ganglion Cyst is disorder observed in the nerve injury, it is still unknown and very difficult to predict its propagation in the human body so many times it is referred as an unsolved history. The treatments for this disorder are to remove the cystic substance from the nerve by a surgery. However these treatments may result in neuropathic pain and recurrence of the cyst. The articular theory proposed by Spinner et al., (Spinner et al. 2003) considers the neurological deficit in Common Peroneal Nerve (CPN) branch of the sciatic nerve and adds that in addition to the treatment, ligation of articular branch results into foolproof eradication of the deficit. Mechanical modeling of the affected nerve cross section will reinforce the articular theory (Spinner et al. 2003). As the cyst propagates, it compresses the neighboring fascicles and the nerve cross section appears like a signet ring. Hence, in order to mechanically model the affected nerve cross section; computational methods capable of modeling excessively large deformations are required. Traditional FEM produces distorted elements while modeling such deformations, resulting into inaccuracies and premature termination of the analysis. The methods described in research report have the capability to simulate large deformation. The results obtained from this research shows significant deformation as compared to the deformation observed in the conventional finite element models. The report elaborates the neurological deficit followed by detail explanation of the Smoothed Particle Hydrodynamic approach. Finally, the results show the large deformation in stages and also the successful implementation of the SPH method for the large deformation of the biological organ like the Intra-neural ganglion cyst.

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.