Low-cost Bioreactor Research Articles

Use of spent yeasts from bioethanol production plant as low-cost nitrogen sources for ethanol fermentation from sweet sorghum stem juice in low-cost bioreactors

Two spent yeasts from an ethanol production plant, spent yeast after distillation (SY-AD) and spent yeast after fermentation (SY-AF), were used as low-cost nitrogen sources for ethanol fermentation from sweet sorghum stem juice (SSJ) by a commercial dry yeast (Saccharomyces cerevisiae) in air-locked flasks. SY-AF was the more effective nitrogen source for ethanol fermentation, giving ethanol concentration (PE) and ethanol productivity (QE) values of 95.22 g/L and 1.98 g/L·h, respectively. When SY-AF was disrupted by autolysis, and the spent yeast hydrolysate (SYH) obtained was used as a nitrogen supplement. It was found that ethanol production in terms of PE and QE values increased to 102.20 g/L and 2.83 g/L·h, respectively. When three bioreactors, a stirred-tank bioreactor (STR, a typical bioreactor), a column bioreactor with stirrer (CS-R, a tower bioreactor) and an external loop bioreactor (ELR, a low-cost bioreactor with no agitation), were used for ethanol production from the SSJ supplemented with SYH, the fermentation efficiencies of all bioreactors were not different. Appropriate aeration during fermentation (0.31 vvm for 12 h) in the three bioreactors could enhance the QE value, reaching 3.36 g/L·h. Both the CR-S and ELR could be successfully used for ethanol production from SSJ supplemented with SYH.

Enhanced Growth of Bacterial Cells in a Smart 3D Printed Bioreactor.

In the last decade, there has been a notable advancement in diverse bioreactor types catering to various applications. However, conventional bioreactors often exhibit bulkiness and high costs, making them less accessible to many researchers and laboratory facilities. In light of these challenges, this article aims to introduce and evaluate the development of a do-it-yourself (DIY) 3D printed smart bioreactor, offering a cost-effective and user-friendly solution for the proliferation of various bioentities, including bacteria and human organoids, among others. The customized bioreactor was fabricated under an ergonomic design and assembled with 3D printed mechanical parts combined with electronic components, under 3D printed housing. The 3D printed parts were designed using SOLIDWORKS® CAD Software (2022 SP2.0 Professional version) and fabricated via the fused filament fabrication (FFF) technique. All parts were 3D printed with acrylonitrile butadiene styrene (ABS) in order for the bioreactor to be used under sterile conditions. The printed low-cost bioreactor integrates Internet-of-things (IoT) functionalities, since it provides the operator with the ability to change its operational parameters (sampling frequency, rotor speed, and duty cycle) remotely, via a user-friendly developed mobile application and to save the user history locally on the device. Using this bioreactor, which is adjusted to a standard commercial 12-well plate, proof of concept of a successful operation of the bioreactor during a 2-day culture of Escherichia coli bacteria (Mach1 strain) is presented. This study paves the way for more in-depth investigation of bacterial and various biological-entity growth cultures, utilizing 3D printing technology to create customized low-cost bioreactors.

Capability of immobilized Clostridium beijerinckii for batch and repeated-batch butanol fermentation from sweet sorghum stem juice in various bioreactors

Mixing in bioreactors is important for butanol fermentations. The type of impeller (6-blade disc, 2-pitch blade and 3-pitch blade turbines) in a stirred-tank bioreactor (STR) did not affect butanol production by immobilized Clostridium beijerinckii TISTR 1461 on bamboo chopstick pieces. Using a spin-filter in the STR could reduce the fermentation time from 48 h to 36 h. Butanol production from sweet sorghum stem juice (butanol concentration, PB, ∼11 g/L and butanol productivity, QB, 0.30 g/L·h) by the immobilized cells in an internal loop gas-lift bioreactor using a carrier inside the draft tube were similar to those using an STR with a 6-blade disc turbine integrated with a spin-filter. Repeated-batch butanol fermentation in the internal loop gas-lift bioreactor could be operated for four cycles. However, high levels of butanol production could be maintained for only two cycles. Simultaneous immobilization and fermentation was a suitable process for repeated-batch fermentation by immobilized cells. An internal loop gas-lift bioreactor is a potential low-cost bioreactor for batch and repeated-batch butanol fermentation by the immobilized cells.

Mechanism of lactose assimilation in microalgae for the bioremediation of dairy processing side-streams and co-production of valuable food products

Abstract This study investigated the mechanism of lactose assimilation in Nannochloropsis oceanica for dairy-wastewater bioremediation and co-production of valuable feed/food ingredients in a circular dairy system (β-galactosidase and omega-3 polyunsaturated fatty acids). Mixotrophic cultivation was found to be mandatory for lactose assimilation in N. oceanica, with biomass production in mixotrophic cultures reaching a fourfold increase over that under heterotrophic conditions. Under mixotrophic conditions, the microalgae were able to produce β-galactosidase enzyme to hydrolyse lactose, with maximum extracellular secretion recorded on day 8 of growth cycle at 41.47 ± 0.33 U gbiomass−1. No increase in the concentration of glucose or galactose was observed in the medium, confirming the ability of microalgae to indiscriminately absorb the resultant monosaccharides derived from lactose breakdown. Population analysis revealed that microalgae cells were able to maintain dominance in the mixotrophic culture, with bacteria accounting for < 12% of biomass. On the other hand, under heterotrophic conditions, native bacteria took over the culture (occupying over 95% of total biomass). The bacteria, however, were also unable to effectively assimilate lactose, resulting in limited biomass increase and negligible production of extracellular β-galactosidase. Results from the study indicate that N. oceanica can be effectively applied for onsite dairy wastewater treatment under strict mixotrophic conditions. This is commercially disadvantageous as it rules out the possibility of deploying heterotrophic fermentation with low-cost bioreactors and smaller areal footprint.

Enhancing bioreactor arrays for automated measurements and reactive control with ReacSight

Small-scale, low-cost bioreactors provide exquisite control of environmental parameters of microbial cultures over long durations. Their use is gaining popularity in quantitative systems and synthetic biology. However, existing setups are limited in their measurement capabilities. Here, we present ReacSight, a strategy to enhance bioreactor arrays for automated measurements and reactive experiment control. ReacSight leverages low-cost pipetting robots for sample collection, handling and loading, and provides a flexible instrument control architecture. We showcase ReacSight capabilities on three applications in yeast. First, we demonstrate real-time optogenetic control of gene expression. Second, we explore the impact of nutrient scarcity on fitness and cellular stress using competition assays. Third, we perform dynamic control of the composition of a two-strain consortium. We combine custom or chi.bio reactors with automated cytometry. To further illustrate ReacSight’s genericity, we use it to enhance plate-readers with pipetting capabilities and perform repeated antibiotic treatments on a bacterial clinical isolate.

Citrus sudden death-associated virus as a new expression vector for rapid in planta production of heterologous proteins, chimeric virions, and virus-like particles

The more we understand the strategies used by viruses for protein expression, the more possibilities we have to exploit viruses as expression vectors for heterologous protein production. Advances in the development of virus-based expression systems have been possible due to generation of many virus infectious clones, especially those derived from plant viruses, which have the capability for rapid and high-level transient expression of proteins in plant cells, a robust and low-cost bioreactor. In this work, we generated new replicative virus expression vectors based on a previously constructed citrus sudden death-associated virus (CSDaV) infectious cDNA clone. These vectors were generated to express the reporter green fluorescent protein (GFP) in Nicotiana benthamiana leaves by taking advantage of the expression strategies used by CSDaV to produce its structural proteins. We show that higher amounts of GFP can be produced from a coat protein (CP)-independent CSDaV-based vector, compared to levels of GFP expressed from a widely used non-replicative vector (pEAQ series); or GFP can be produced in fusion with the major CSDaV CP (CPp21) to be incorporated into chimeric virions. However, GFP-recombinant CSDaV virions do not appear uniformly assembled, but more likely as mosaic particles. Cryo-electron microscopy analysis from this work revealed the structures of the wild-type and the GFP-recombinant CSDaV virions, but it was not able to reveal where exactly the GFP is displayed in the chimeric virions. We show though that the incorporation of GFP-CPp21 fusion protein into virions occurs solely due to its interaction with free/non-fused CPp21, independent of other viral proteins. Therefore, individual co-expression of GFP-CPp21 and CPp21 in the same plant cells leads to the production of chimeric virus-like particles (VLPs), while GFP-CPp21 fusion protein itself is not able to self-assemble into VLPs. The new CSDaV-based expression vectors may provide an alternative platform for use in molecular farming, either for production of heterologous proteins or as scaffold for heterologous protein display.

Effectiveness of Low-Cost Bioreactors Integrated with a Gas Stripping System for Butanol Fermentation from Sugarcane Molasses by Clostridium beijerinckii

The effectiveness of column bioreactors for butanol fermentation from sugarcane molasses by Clostridium beijerinckii TISTR 1461 was investigated. This fermentation was operated at an initial pH of 6.5 and temperature of 37 °C under anaerobic conditions. A 1-L bubble column bioreactor was used with various gas circulation rates ranging from 0.2 to 1.0 L/min. The highest butanol concentration (PB, 8.72 g/L), productivity (QB, 0.24 g/L∙h) and yield (YB/S, 0.21 g/g) were obtained with a gas circulation of 0.2 L/min. To improve butanol production efficiency, gas-lift column bioreactors with internal and external loops at 0.2 L/min of circulating gas were used. Higher PB (10.50–10.58 g/L), QB (0.29 g/L∙h) and YB/S (0.22–0.23 g/g) values were obtained in gas-lift column bioreactors. These values were similar to those using a more complex 2-L stirred-tank bioreactor (PB, 10.10 g/L; QB, 0.28 g/L h and YB/S, 0.22 g/g). Hence, gas-lift column bioreactors have potential for use as low-cost fermenters instead of stirred-tank bioreactors for butanol fermentation. When the gas-lift column bioreactor with an internal loop was coupled with a gas stripping system, it yielded an enhanced PB and sugar consumption of approximately 9% and 7%, respectively, compared to a system with no gas stripping.

Butanol Production by a Novel Efficient Method Using Mixed Cultures of Clostridium beijerinckii and Arthrobacter sp. in Stirred-Tank and Gas-Lift Bioreactors

Arthrobacter sp. BCC 72131, an obligate aerobic bacterium, was used to create anaerobic conditions, and Clostridium beijerinckii TISTR 1461 was used as a butanol producer in an acetone-butanol–ethanol (ABE) fermentation. Sweet sorghum juice (SSJ) medium containing 60 g/L of total sugar supplemented with 1.27 g/L of (NH4)2SO4 was used as a butanol production (BP) medium. Arthrobacter sp. was inoculated into the BP medium in 1-L screw-capped bottles. After 2, 4, 6 and 12 h of Arthrobacter sp. cultivation at 30 °C, C. beijerinckii was transferred into the BP medium to start butanol production at 37 °C. The results showed that C. beijerinckii inoculation after 6 h of Arthrobacter sp. cultivation gave the highest butanol titer (PB) at 12.56 g/L, with a butanol yield (YB/S) and volumetric butanol productivity (QB) of 0.34 g/g and 0.23 g/L·h, respectively. These values are approximately 10–27% higher than those of the control experiment using a single culture of C. beijerinckii TISTR 1461 and oxygen-free nitrogen (OFN) gas flushing to create anaerobic conditions. Field emission scanning electron microscopic (FE-SEM) images of Clostridium cells, as well as protein and free amino nitrogen concentrations in the broth during butanol fermentation were also studied to confirm the results. The butanol fermentation was then carried out in a 5.6-L stirred-tank and a 1.2-L low-cost gas-lift bioreactor by the mixed cultures using the optimal time of Clostridium inoculation. The PB, YB/S and QB values obtained were not significantly different from those in the 1-L screw-capped bottles. Hence, Arthrobacter sp. can be used as a novel method to create anaerobic conditions instead of a traditional method employing OFN gas flushing. Using mixed cultures of Arthrobacter sp. BCC 72131 and C. beijerinckii TISTR 1461 is a practical method to produce butanol on a large-scale, both in complex and low-cost bioreactors.

A Novel, Open-Source, Low-Cost Bioreactor for Load-Controlled Cyclic Loading of Tendon Explants.

A major risk factor for tendinopathy is tendon overuse (i.e., fatigue loading). Fatigue loading of tendon damages the extracellular matrix and induces tissue degeneration. However, the specific mechanisms linking tendon fatigue damage with tissue degeneration are unclear. While explant models of tendon fatigue loading have been used to address this knowledge gap, they predominantly employ bioreactors that apply cyclic displacements/strains rather than loads/stresses, which are more physiologically relevant. This is because of the technical complexity and cost of building a load-controlled bioreactor, which requires multiple motors, load cells, and computationally intensive feedback loops. Here, we present a novel, low-cost, load-controlled bioreactor that applies cyclic loading to multiple tendon explants by offloading weights from a single motorized stage. Using an optional load cell, we validated that the bioreactor can effectively provide load-controlled fatigue testing of mouse and rat tendon explants while maintaining tissue viability. Furthermore, all the design files, bill of materials, and operating software are available "open source"1 so that anyone can easily manufacture and use the bioreactor for their own research. Therefore, this novel load-controlled bioreactor will enable researchers to study the mechanisms driving fatigue-induced tendon degeneration in a more physiologically relevant and cost-effective manner.

An innovative jet loop-airlift bioreactor for simultaneous removal of carbon and nitrogen from soft drink industrial wastewater: Process performance and kinetic evaluation

Abstract Creating different redox conditions in different regions can be effective for nutrient removal in a single bioreactor. Jet loop-airlift bioreactors are presented as an innovative system that can provide regions with different oxidation–reduction potentials (ORP). In the present study, the performance of a novel jet loop-airlift bioreactor for the simultaneous removal of nutrients from soft drink wastewater (SDW) (COD:N:P of 100:16:1) was investigated. The anoxic and aerobic zones with settling tank could successfully be integrated in a compact high rate bioreactor under continuous aeration and feeding. Two independent variables, hydraulic retention time (HRT) (6–14 h) and air flow rate (2.5-4.5 l/min), were chosen at three levels for modeling, optimization, and trend analysis. According to experimental data, optimum conditions were achieved at HRT of 11 h and air flow rate of 4 l/min with TCOD and TN removal of 98% and 83.6%, respectively. Also, kinetic analysis of the soft drink wastewater treatment process in the jet loop-airlift bioreactor was performed and Y, K d , K s , K and μ max were found to be 0.2425 gVSS/gCOD, 0.038 d−1, 0.00786 g/l, 0.6321 d−1 and 0.165 d−1, respectively. In conclusion, the jet loop-airlift system can be described as an effective low-cost bioreactor for treating wastewater with high carbon and nitrogen.

Applications of novel bioreactor technology to enhance the viability and function of cultured cells and tissues.

As the field of tissue engineering continues to advance rapidly, so too does the complexity of cell culture techniques used to generate in vitro tissue constructs, with the overall aim of mimicking the in vivo microenvironment. This complexity typically comes at a cost with regards to the size of the equipment required and associated expenses. We have developed a small, low-cost bioreactor system which overcomes some of the issues of typical bioreactor systems while retaining a suitable scale for the formation of complex tissues. Herein, we have tested this system with three cell populations/tissues: the culture of hepatocellular carcinoma cells, where an improved structure and basic metabolic function is seen; the culture of human pluripotent stem cells, in which the cultures can form more heterogeneous tissues resembling the in vivo teratoma and ex vivo liver tissue slices, in which improved maintenance of cellular viability is seen over the 3 days tested. This system has the flexibility to be used for a variety of further uses and has the potential to provide a more accessible alternative to current bioreactor technologies.

Promoter engineering for microbial bio-alkane gas production

Successful industrial biotechnological solutions to biofuels and other chemicals production rely on effective competition with existing lower-cost natural sources and synthetic chemistry approaches enabled by adopting low-cost bioreactors and processes. This is achievable by mobilizing Halomonas as a next generation industrial chassis, which can be cultivated under non-sterile conditions. To increase the cost effectiveness of an existing sustainable low carbon bio-propane production strategy, we designed and screened a constitutive promoter library based on the known strong porin promoter from Halomonas. Comparative studies were performed between Escherichia coli and Halomonas using the reporter gene red fluorescent protein (RFP). Later studies with a fatty acid photodecarboxylase-RFP fusion protein demonstrated tuneable propane production in Halomonas and E. coli, with an ∼8-fold improvement in yield over comparable isopropyl-β-D-thiogalactoside-inducible systems. This novel set of promoters is a useful addition to the synthetic biology toolbox for future engineering of Halomonas to make chemicals and fuels.

A Cell Culture Platform for the Cultivation of Cryptosporidium parvum.

Cryptosporidium is a genus of ubiquitous unicellular parasites belonging to the phylum Apicomplexa. Cryptosporidium species are the second largest cause of childhood diarrhea and are associated with increased morbidity. Accompanying this is the low availability of treatment and lack of vaccines. The major barrier to developing effective treatment is the lack of reliable in vitro culture methods. Recently, our lab has successfully cultivated C. parvum in the esophageal cancer-derived cell line COLO-680N, and has been able to maintain infection for several weeks. The success of this cell line was assessed with a combination of various techniques including fluorescent microscopy and qPCR. In addition, to tackle the issue of long-term oocyst production in vitro, a simple, low-cost bioreactor system using the COLO-680N cell line was established, which produced infectious oocysts for 4 months. This chapter provides details on the methodologies used to culture, maintain, and assess Cryptosporidium infection and propagation in COLO-680N. © 2019 by John Wiley & Sons, Inc.

Chemical hydrodynamics of a downward microbubble flow for intensification of gas‐fed bioreactors

Bioreactors are of interest for value‐upgrading of stranded or waste industrial gases. Reactor intensification requires development of low cost bioreactors with fast gas–liquid mass transfer rate. Here we assess published reactor technology in comparison with a novel downward bubble flow created by a micro‐jet array. Compared to known technology, the advanced design achieves higher volumetric gas transfer efficiency (kLa per power density) and can operate at higher kLa. We measure the effect of four reactor heights (height‐to‐diameter ratios of 12, 9, 6, and 3) on the gas transfer coefficient kL, total interfacial area a, liquid residence time distribution, energy consumption, and turbulent hydrodynamics. Leading models for predicting kL and a are appraised with experimental data. The results show kL is governed by “entrance effects” due to Higbie penetration dominate at short distances below the micro‐jet array, while turbulence dominates at intermediate distances, and finally terminal rise velocity dominates at large distances. © 2017 American Institute of Chemical Engineers AIChE J, 64: 1399–1411, 2018

Effect of Rotation on Scaffold Motion and Cell Growth in Rotating Bioreactors.

Efficient use of different bioreactor designs to improve cell growth in three-dimensional scaffolds requires an understanding of their mechanism of action. To address this for rotating wall vessel bioreactors, fluid and scaffold motion were investigated experimentally at different rotation speeds and vessel fill volumes. Low cost bioreactors with single and dual axis rotation were developed to investigate the effect of these systems on human osteoblast proliferation in free floating and constrained collagen-glycosaminoglycan porous scaffolds. A range of scaffold motions (free fall, periodic oscillation, and orbital motion) were observed at the rotation speeds and vessel fluid/air ratios used, with 85% fluid fill and an outer vessel wall velocity of ∼14 mm s−1 producing a scaffold in a free fall state. The cell proliferation results showed that after 14 and 21 days of culture, this combination of fluid fill and speed of rotation produced significantly greater cell numbers in the scaffolds than when lower or higher rotation speeds (p < 0.002) or when the chamber was 60% or 100% full (p < 0.01). The fluid flow and scaffold motion experiments show that biaxial rotation would not improve the mass transfer of medium into the scaffold as the second axis of rotation can only transition the scaffold toward oscillatory or orbital motion and, hence, reduce mass transport to the scaffold. The cell culture results confirmed that there was no benefit to the second axis of rotation with no significant difference in cell proliferation either when the scaffolds were free floating or constrained (p > 0.05).

Untranslated regions of diverse plant viral RNAs vary greatly in translation enhancement efficiency

BackgroundWhole plants or plant cell cultures can serve as low cost bioreactors to produce massive amounts of a specific protein for pharmacological or industrial use. To maximize protein expression, translation of mRNA must be optimized. Many plant viral RNAs harbor extremely efficient translation enhancers. However, few of these different translation elements have been compared side-by-side. Thus, it is unclear which are the most efficient translation enhancers. Here, we compare the effects of untranslated regions (UTRs) containing translation elements from six plant viruses on translation in wheat germ extract and in monocotyledenous and dicotyledenous plant cells.ResultsThe highest expressing uncapped mRNAs contained viral UTRs harboring Barley yellow dwarf virus (BYDV)-like cap-independent translation elements (BTEs). The BYDV BTE conferred the most efficient translation of a luciferase reporter in wheat germ extract and oat protoplasts, while uncapped mRNA containing the BTE from Tobacco necrosis virus-D translated most efficiently in tobacco cells. Capped mRNA containing the Tobacco mosaic virus omega sequence was the most efficient mRNA in tobacco cells. UTRs from Satellite tobacco necrosis virus, Tomato bushy stunt virus, and Crucifer-infecting tobamovirus (crTMV) did not stimulate translation efficiently. mRNA with the crTMV 5′ UTR was unstable in tobacco protoplasts.ConclusionsBTEs confer the highest levels of translation of uncapped mRNAs in vitro and in vivo, while the capped omega sequence is most efficient in tobacco cells. These results provide a basis for understanding mechanisms of translation enhancement, and for maximizing protein synthesis in cell-free systems, transgenic plants, or in viral expression vectors.

The Expression of a Xylanase Targeted to ER-Protein Bodies Provides a Simple Strategy to Produce Active Insoluble Enzyme Polymers in Tobacco Plants

BackgroundXylanases deserve particular attention due to their potential application in the feed, pulp bleaching and paper industries. We have developed here an efficient system for the production of an active xylanase in tobacco plants fused to a proline-rich domain (Zera) of the maize storage protein γ-zein. Zera is a self-assembling domain able to form protein aggregates in vivo packed in newly formed endoplasmic reticulum-derived organelles known as protein bodies (PBs).Methodology/Principal FindingsTobacco leaves were transiently transformed with a binary vector containing the Zera-xylanase coding region, which was optimized for plant expression, under the control of the 35S CaMV promoter. The fusion protein was efficiently expressed and stored in dense PBs, resulting in yields of up to 9% of total protein. Zera-xylanase was post-translationally modified with high-mannose-type glycans. Xylanase fused to Zera was biologically active not only when solubilized from PBs but also in its insoluble form. The resistance of insoluble Zera-xylanase to trypsin digestion demonstrated that the correct folding of xylanase in PBs was not impaired by Zera oligomerization. The activity of insoluble Zera-xylanase was enhanced when substrate accessibility was facilitated by physical treatments such as ultrasound. Moreover, we found that the thermostability of the enzyme was improved when Zera was fused to the C-terminus of xylanase.Conclusion/SignificanceIn the present work we have successfully produced an active insoluble aggregate of xylanase fused to Zera in plants. Zera-xylanase chimeric protein accumulates within ER-derived protein bodies as active aggregates that can easily be recovered by a simple density-based downstream process. The production of insoluble active Zera-xylanase protein in tobacco outlines the potential of Zera as a fusion partner for producing enzymes of biotechnological relevance. Zera-PBs could thus become efficient and low-cost bioreactors for industrial purposes.

Characterisation of an attenuation system for the remediation of Mn(II) contaminated waters

Soluble manganese (Mn(II)) can be problematic in surface, ground waters and in domestic water supplies. Many mine drainage waters contain highly elevated concentrations of manganese (often > 100 mg l − 1 ). Current systems for remediating mine waters, although effective for other metals, are often inefficient at removing manganese. This paper describes the development of self-sustaining, low-cost bioreactors that can be used in situ for passive removal of manganese from contaminated water courses of pH > 4. A prototype bioreactor, set-up using Mn(IV)-coated pebbles from a fresh-water stream, was tested over one year and shown to be effective in removing Mn(II) from 10 to < 0.25 mg l − 1 . Managanese-oxidising microorganisms isolated from the bioreactor included two species of fungi and one bacterial species. The fungi were identified as belonging to the order Pleosporales (Ascomycetes), and one was related (98% 18S rRNA gene sequence identity) to a known Mn(II)-oxidising fungus. The bacterial isolate was closely related to the α-proteobacterium Bosea thiooxidans. T-RFLP analysis showed that one or both of the fungal isolates were the dominant eukaryotes in the bioreactor community, while the Bosea isolate did not appear to be numerically important in the bacterial community.

Development of a Novel Biological System for Removing Manganese from Contaminated Waters

Soluble manganese (Mn(II)) can cause nuisance and occasionally toxicity problems, both in surface and ground waters and in domestic water supplies. Many mine drainage waters contain highly elevated concentrations (often &gt;100 mg l-1). Current systems for remediating mine waters, although effective for other metals, are often inefficient at removing manganese. This paper describes the development of self-sustaining, low-cost bioreactors that can be used in situ for passive removal of manganese from mine waters and other contaminated water courses of pH &gt;4. A prototype bioreactor, set-up using Mn(IV)-coated pebbles from a fresh-water stream, was tested over one year and shown to be effective in removing Mn(II) from 10 to &lt;0.25 mg l-1. Two species of fungi and one bacterial species were isolated from this system as Mn(II)-oxidizers. The fungi were identified as belonging to the order Pleosporales (Ascomycetes), and one was related (98 % 18S rRNA gene sequence identity) to a known Mn(II)-oxidising fungus. The bacterial isolate was closely related to the α-proteobacterium, Bosea thiooxidans. T-RFLP analysis showed that one or both of the fungal isolates were the dominant eukaryotes in the bioreactor community.

Expression, purification and characterization of human GM-CSF using silkworm pupae (Bombyx mori) as a bioreactor

To date, many recombinant proteins have been expressed in Bombyx mori cells or silkworm larvae, apart from in pupae. Silkworm pupae may be more suitable for the expression of heterologous proteins as a bioreactor. If maintained at an appropriate temperature, silkworm pupae could be inoculated with recombinant baculovirus for the expression of a protein of interest. In this study, human granulocyte-macrophage colony-stimulating factor was successfully expressed in silkworm pupae using B. mori nucleopolyhedrovirus, purified and characterized with respect to its physico-chemical properties. The target protein expressed had an apparent molecular mass of 29 kDa and an isoelectric point of 5.1. The protein was purified using three chromatographic steps with a final recovery of 10.3%. Finally, approximately 3.5mg of the protein was obtained with a biological activity of up to 8.4 x 10(6) cfu mg(-1). The results of this study suggest that silkworm pupae represent a convenient and low-cost bioreactor for the expression of heterologous proteins.