In Vitro Culture of Cryptosporidium parvum Using Hollow Fiber Bioreactor: Applications for Simultaneous Pharmacokinetic and Pharmacodynamic Evaluation of Test Compounds.

Hollow fiber technology is a powerful tool for the culture of difficult-to-grow cells. Cryptosporidium parvum has a multistage sexual and asexual life cycle that has proved difficult to culture by conventional in vitro culture methods. Here, we describe a method utilizing a hollow fiber bioreactor for the continuous in vitro growth of C. parvum that produces sexual and asexual stages. The method enables the evaluation of potential therapeutic compounds under conditions that mirror the dynamic conditions found in the gut facilitating preliminary pharmacokinetic and pharmacodynamic data to be obtained.

Response of Anaerobic Protozoa to Oxygen Tension in Anaerobic System

The effect of oxygen on anaerobic protozoa was studied in anaerobic batch reactors inoculated with sludge and protozoa cultures. Among the protozoa genera, Metopus, Brachonella, Plagiopyla, Trepomonas, and Vanella were more sensitive to oxygen compared to other genera. Protozoa genera Menoidium, Rhynchomonas, Cyclidium, Spathidium, and Amoeba were found to survive under aerobic conditions, and the growth rate was slightly higher or similar to anaerobic condition. O2 tension resulted in the loss of free and endosymbiotic methanogens in anaerobic system, while methanogens were observed inside the protozoan cysts. Survival of anaerobic protozoa declined considerably when the O2 tension exceeded 1% atm. sat. and showed chemosensory behavior in response to O2 exposure. Superoxide dismutase activity was detected in survived protozoa cells under O2 tension. Facultative anaerobic protozoa with SOD activity can provide a mechanism to overcome possible occurrence of oxygen toxicity in the treatment of wastewater in anaerobic reactor

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Response of Anaerobic Protozoa to Oxygen Tension in Anaerobic System.

The effect of oxygen on anaerobic protozoa was studied in anaerobic batch reactors inoculated with sludge and protozoa cultures. Among the protozoa genera, Metopus, Brachonella, Plagiopyla, Trepomonas, and Vanella were more sensitive to oxygen compared to other genera. Protozoa genera Menoidium, Rhynchomonas, Cyclidium, Spathidium, and Amoeba were found to survive under aerobic conditions, and the growth rate was slightly higher or similar to anaerobic condition. O2 tension resulted in the loss of free and endosymbiotic methanogens in anaerobic system, while methanogens were observed inside the protozoan cysts. Survival of anaerobic protozoa declined considerably when the O2 tension exceeded 1% atm. sat. and showed chemosensory behavior in response to O2 exposure. Superoxide dismutase activity was detected in survived protozoa cells under O2 tension. Facultative anaerobic protozoa with SOD activity can provide a mechanism to overcome possible occurrence of oxygen toxicity in the treatment of wastewater in anaerobic reactor.

Large-scale cultivation of Leishmania infantum promastigotes in stirred bioreactor.

BACKGROUND & OBJECTIVES: Bioreactors are practical tools that are used for economical, time-conserving and large-scale production of biomass from cell cultivation. They provide optimal environmental conditions such as pH and temperature required for obtaining maximum amounts of biomass. However, there is no evidence in the literature on the large-scale cultivation of Leishmania infantum parasites in the bioreactor. Therefore, the present study was undertaken to develop a new approach for obtaining L. infantum biomass by using pH and temperature controllable stirred bioreactor and to compare parasitic growth kinetics with classical method within erlenmeyers. METHODS: In order to obtain parasite biomass, a newly developed pH and temperature controlled stirred bioreactor was used and its efficacy was compared with a graduated classical scale-up method. Growth kinetics of parasites within erlenmeyers and bioreactors were determined by evaluating promastigote numbers using haemocytometer. The graduated scale enlargement of culture was followed by T25 flask, T75 flask, and 1 L erlenmeyer, respectively. RESULTS: Obtained results showed a 10-fold increase in the number of promastigotes within the conventional culture performed in 700 ml medium, while parasite numbers increased approximately 15 times due to initial inoculation amounts in the bioreactor culture performed in the 3.5 l medium. Thus, there was 7.5 times more biomass collection in bioreactor compared to classical method. INTERPRETATION & CONCLUSION: It is postulated that constant culture pH and temperature in the bioreactor extends cultivation time. This could lead to significant increase in parasite numbers. Hence, pH and temperature controllable bioreactors provided acquisition of sufficient amounts of biomass in contrast to classical methods. Therefore, this type of bioreactors may substitute classical culture methods in the production of antigenic molecules for vaccine development.

A bioreactor system for the manufacture of a genetically modified Plasmodium falciparum blood stage malaria cell bank for use in a clinical trial.

BACKGROUND: Although the use of induced blood stage malaria infection has proven to be a valuable tool for testing the efficacy of vaccines and drugs against Plasmodium falciparum, a limiting factor has been the availability of Good Manufacturing Practice (GMP)-compliant defined P. falciparum strains for in vivo use. The aim of this study was to develop a cost-effective method for the large-scale production of P. falciparum cell banks suitable for use in clinical trials. METHODS: Genetically-attenuated parasites (GAP) were produced by targeted deletion of the gene encoding the knob associated histidine rich protein (kahrp) from P. falciparum strain 3D7. A GAP master cell bank (MCB) was manufactured by culturing parasites in an FDA approved single use, closed system sterile plastic bioreactor. All components used to manufacture the MCB were screened to comply with standards appropriate for in vivo use. The cryopreserved MCB was subjected to extensive testing to ensure GMP compliance for a phase 1 investigational product. RESULTS: Two hundred vials of the GAP MCB were successfully manufactured. At harvest, the GAP MCB had a parasitaemia of 6.3%, with 96% of parasites at ring stage. Testing confirmed that all release criteria were met (sterility, absence of viral contaminants and endotoxins, parasite viability following cryopreservation, identity and anti-malarial drug sensitivity of parasites). CONCLUSION: Large-scale in vitro culture of P. falciparum parasites using a wave bioreactor can be achieved under GMP-compliant conditions. This provides a cost-effective methodology for the production of malaria parasites suitable for administration in clinical trials.

Specialized proteomic responses and an ancient photoprotection mechanism sustain marine green algal growth during phosphate limitation.

Marine algae perform approximately half of global carbon fixation, but their growth is often limited by the availability of phosphate or other nutrients1,2. As oceans warm, the area of phosphate-limited surface waters is predicted to increase, resulting in ocean desertification3,4. Understanding the responses of key eukaryotic phytoplankton to nutrient limitation is therefore critical5,6. We used advanced photo-bioreactors to investigate how the widespread marine green alga Micromonas commoda grows under transitions from replete nutrients to chronic phosphate limitation and subsequent relief, analysing photosystem changes and broad cellular responses using proteomics, transcriptomics and biophysical measurements. We find that physiological and protein expression responses previously attributed to stress are critical to supporting stable exponential growth when phosphate is limiting. Unexpectedly, the abundance of most proteins involved in light harvesting does not change, but an ancient light-harvesting-related protein, LHCSR, is induced and dissipates damaging excess absorbed light as heat throughout phosphate limitation. Concurrently, a suite of uncharacterized proteins with narrow phylogenetic distributions increase multifold. Notably, of the proteins that exhibit significant changes, 70% are not differentially expressed at the mRNA transcript level, highlighting the importance of post-transcriptional processes in microbial eukaryotes. Nevertheless, transcript-protein pairs with concordant changes were identified that will enable more robust interpretation of eukaryotic phytoplankton responses in the field from metatranscriptomic studies. Our results show that P-limited Micromonas responds quickly to a fresh pulse of phosphate by rapidly increasing replication, and that the protein network associated with this ability is composed of both conserved and phylogenetically recent proteome systems that promote dynamic phosphate homeostasis. That an ancient mechanism for mitigating light stress is central to sustaining growth during extended phosphate limitation highlights the possibility of interactive effects arising from combined stressors under ocean change, which could reduce the efficacy of algal strategies for optimizing marine photosynthesis.

The components of shear stress affecting insect cells used with the baculovirus expression vector system.

Insect-based expression platforms such as the baculovirus expression vector system (BEVS) are widely used for the laboratory- and industrial-scale production of recombinant proteins. Thereby, major drawbacks to gain high-quality proteins are the lytic infection cycle and the shear sensitivity of infected insect cells due to turbulence and aeration. Smaller bubbles were formerly assumed to be more harmful than larger ones, but we found that cell damage is also dependent on the concentration of protective agents such as Pluronic®. At the appropriate concentration, Pluronic forms a layer around air bubbles and hinders the attachment of cells, thus limiting the damage. In this context, we used microaeration to vary bubble sizes and confirmed that size is not the most important factor, but the total gas surface area in the reactor is. If the surface area exceeds a certain threshold, the concentration of Pluronic is no longer sufficient for cell protection. To investigate the significance of shear forces, a second study was carried out in which infected insect cells were cultivated in a hollow fiber module to protect them from shear forces. Both model studies revealed important aspects of the design and scale-up of BEVS processes for the production of recombinant proteins.

Prokaryote species richness is positively correlated with eukaryote abundance in wastewater treatment biofilms.

Biological treatment represents a key step in nutrient removal from wastewater. Until now these process has mainly been considered prokaryotic, with the interactions between prokaryotes and eukaryotes not being properly explored. We therefore investigated the co-occurrence of eukaryotes and prokaryotes in biological nitrogen removal biofilms. We found that biofilms in the nitrifying reactor contained the highest diversity and abundance of both prokaryotes and eukaryotes, with nearly three times higher prokaryote species richness than for the denitrifying reactor. The positive associations between eukaryote abundance and prokaryote diversity could potentially be explained by mutualism - and/or predator/prey interactions. Further mechanistic insight, however, is needed to determine the main diversifying mechanisms. In summary, eukaryote and prokaryote interactions seem to play a fundamental yet underexplored role in biological wastewater treatment. SIGNIFICANCE AND IMPACT OF THE STUDY: Eukaryote and prokaryote interactions may play an important role in wastewater treatment. This study found that prokaryote species richness was nearly three times higher in the aerobe nitrification than in an anaerobe denitrification reactor, coinciding with the highest level of eukaryotes. This knowledge can be important in process control, and potentially in the development of novel approaches based on nitrate accumulating denitrifying eukaryotes.

Putrescine: Essential factor for in vitro proliferation of Babesia bovis.

This study reports the effect of putrescine addition, either alone or in combination with insulin, transferrin and selenite (ITS), to serum-free Advanced DMEM/F12 (A-DMEM/F12) medium, on the in vitro culture of Babesia bovis and using a perfusion bioreactor to improve efficiency of the process. A B. bovis strain previously adapted to proliferate in serum-free medium (Bbovis-SF) was evaluated using eight increasing concentrations of putrescine. The percentage of parasitized erythrocytes (PPE) obtained from cultures supplemented with 0.101 mg/L was 6.23% compared with 2.3% for control cultures with M199 with Earle's salts (M199) and 40% serum. The combination of putrescine (0.101 mg/L) and a mixture of ITS (2000, 1100, and 1.34 mg/L, respectively) (Pu-ITS), in A-DMEM/F12 culture medium without serum yielded a maximum PPE of 17.26% compared to 2.58% in the control medium. This new formulation of culture medium, together with the use of a hollow-fiber perfusion bioreactor system (HFPBS), caused a substantial increase in the proliferation of B. bovis, yielding a maximum cumulative PPE of 118.8% after five days, compared to 58.6% in cultures treated with control medium M199 and 40% serum. We concluded that the addition of the ITS mixture and putrescine to the culture medium stimulated the proliferation of B. bovis in vitro. This new medium formulation, used in a HFPBS culture system, can be an effective, automated-prone system that can induce massive proliferation of B. bovis for use as a source of parasite antigens and immunogens.

Eukaryotic Community Shift in Response to Organic Loading Rate of an Aerobic Trickling Filter (Down-Flow Hanging Sponge Reactor) Treating Domestic Sewage.

In this study, changes in eukaryotic community structure and water quality were investigated in an aerobic trickling filter (down-flow hanging sponge, DHS) treating domestic sewage under different organic loading rates (OLRs). The OLR clearly influenced both sponge pore water quality and relative flagellates and ciliates (free-swimming, carnivorous, crawling, and stalked protozoa) abundances in the retained sludge. Immediately after the OLR was increased from 1.05 to 1.97 kg chemical oxygen demand (COD) m-3 day-1, COD and NH4+-N treatment efficiencies both deteriorated, and relative flagellates and ciliates abundances then increased from 2-8 % to 51-65 % total cells in the middle-bottom part of the DHS reactor. In a continuous operation at a stable OLR (2.01 kg COD m-3 day-1), effluent water quality improved, and relative flagellates and ciliates abundances decreased to 15-46 % total cells in the middle-bottom part of the DHS reactor. This result may indicate that flagellates and ciliates preferentially graze on dispersed bacteria, thus, stabilizing effluent water quality. Additionally, to investigate eukaryotic community structure, clone libraries based on the 18S ribosomal ribonucleic acid (rRNA) gene of the retained sludge were constructed. The predominant group was Nucletmycea phylotypes, representing approximately 29-56 % total clones. Furthermore, a large proportion of the clones had <97 % sequence identity in the NCBI database. This result indicates that phylogenetically unknown eukaryotes were present in the DHS reactor. These results provide insights into eukaryotic community shift in the DHS reactor treating domestic sewage.

Effect of high levels of the rotifer Lecane inermis on the ciliate community in laboratory-scale sequencing batch bioreactors (SBRs).

Due to its ability to feed on filamentous bacteria, the rotifer Lecane inermis has already been recognized as a potential control agent of activated sludge bulking, which is usually caused by the excessive growth of filamentous microorganisms. However, their effectiveness depends, in part, on their abundance. We studied the influence of high densities of L. inermis on the protozoan community in activated sludge from a wastewater treatment plant (WWTP) in 4 laboratory-scale sequencing batch bioreactors (SBRs). Two treatments and two controls were subjected to nutrient removal system in process similar to that used in a WWTP. The experiment lasted 9 days and was repeated in 24-h cycles, including phases of agitation with feeding, aeration and agitation and sedimentation with decantation at the end of the cycle. In total, 32 taxa were identified, among which 25 were ciliated protozoa, 4 were amoebae, 2 were flagellates, and one was a nematode. Rotifers were then introduced to 2 bioreactors at a final concentration of 500ind.mL(-1), and the taxonomic composition and abundance of the activated sludge microfauna were assessed 2, 5 and 8 days thereafter. The mean density of ciliates on the first day of experiment was 12,610ind.mL(-1) and diminished to 4868±432ind.mL-±432ind.mL(-1) in the control and 5496±638ind.mL(-1) in the rotifer-treated group on the last day. Thus, even extremely high densities of artificially introduced rotifers did not negatively affect the protozoan community. On the contrary, the protozoan community was more diverse in the treatment group than in the control.

Assessing the fate of Ascaris suum ova during mesophilic anaerobic digestion.

There is limited knowledge about the survival of geohelminths, which are soil-transmitted human pathogens, in mesophilic anaerobic digestion processes. This study examined the fate of embryonated and unembryonated Ascaris suum ova in six laboratory-scale mesophilic (35 °C) anaerobic digesters processing swine manure to identify their survival strategies and investigate potential mechanisms to enhance their destruction. There was no significant difference in inactivation of Ascaris suum ova in digesters operated at different solids residence times (SRT) or feeding frequencies. Ova exposed to an anaerobic environment became dormant, or remained unembryonated throughout their residence in the reactors. Approximately 65% of ova were able to retain their viability for up to 16 days, after which the rate of inactivation increased until nearly all ova were nonviable by day 24. In contrast, ova exposed to aerobic conditions did not become dormant and progressed through several developmental stages until day 16, after which nearly all ova were observed to be nonviable. In addition, only 35% of fully developed ova exposed to the anaerobic environment retained their viability by day 16 compared to 65% for dormant ova. Results suggest that some ova are physically destroyed during digestion and ova can be inactivated faster if their development cycle is aerobically triggered before entering the anaerobic digestion process. Results also suggest that transfer of resource recovery technologies such as mesophilic anaerobic digestion to developing world settings must account for local climatic and health conditions so mutually beneficial outcomes can be attained.

NaCS-PDMDAAC immobilized cultivation of recombinant Dictyostelium discoideum for soluble human Fas ligand production.

Dictyostelium discoideum is a promising eukaryotic host for the expression of heterologous proteins requiring post-translational modifications. However, the dilute nature of D. discoideum cell culture limits applications for high value proteins production. D. discoideum cells, entrapped in sodium cellulose sulfate/poly-dimethyl-diallyl-ammonium chloride (NaCS-PDMDAAC) capsules were used for biosynthesis of the heterologous protein, soluble human Fas ligand (hFasL). Semi-continuous cultivations with capsules recycling were carried out in shake flasks. Also, a scaled-up cultivation of immobilized D. discoideum for hFasL production in a customized vitreous airlift bioreactor was conducted. The results show that NaCS-PDMDAAC capsules have desirable biophysical properties including biocompatibility with the D. discoideum cells and good mechanical stability throughout the duration of cultivation. A maximum cell density of 2.02 × 10(7) cells mL(-1) (equivalent to a maximum cell density of 2.22 × 10(8) cells mL(-1) in capsules) and a hFasL concentration of 130.40 µg L(-1) (equivalent to a hFasL concentration of 1434.40 µg L(-1) in capsules) were obtained in shake flask cultivation with capsules recycling. Also, a maximum cell density of 1.72 × 10(7) cells mL(-1) (equivalent to a maximum cell density of 1.89 × 10(8) cells mL(-1) in capsules) and a hFasL concentration of 106.10 µg L(-1) (equivalent to a hFasL concentration of 1167.10 µg L(-1) in capsules) were obtained after ∼170 h cultivation in the airlift bioreactor (with a working volume of 200 mL in a 315 mL bioreactor). As the article presents a premier work in the application of NaCS-PDMDAAC immobilized D. discoideum cells for the production of hFasL, more work is required to further optimize the system to generate higher cell densities and hFasL titers for large-scale applications.

Quantitative analysis of ammonia-oxidizing bacteria in a combined system of MBR and worm reactors treating synthetic wastewater.

The Static Sequencing Batch Worm Reactor (SSBWR) followed by the MBR (S-MBR) is one of the advanced excess sludge treatments. In this paper, the control MBR (C-MBR) and the SSBWR-MBR were operated in parallel to study the changes of NH3-N removal and ammonia oxidizing bacteria (AOB). The results showed that the capacity of NH3-N removal of the S-MBR was improved by the worm reactors along with the operation. The S-MBR was favorable because it selected for the higher activity of the ammonia oxidization and better cells appearance of the sludge. The five species (Nitrosomonas, Betaproteobacteria, Clostridium, Dechloromonas and Bacteria) were found to be significantly correlate with the ammonia oxidization functions and performance of NH3-N removal in the C-MBR and S-MBR. The Nitrosomonas, Betaproteobacteria and Dechloromonas remained and eventually enriched in the S-MBR played a primary role in the NH3-N removal of the S-MBR.

Fouling potential evaluation of soluble microbial products (SMP) with different membrane surfaces in a hybrid membrane bioreactor using worm reactor for sludge reduction.

The fouling characteristics of soluble microbial products (SMP) in the membrane bioreactor coupled with Static Sequencing Batch Worm Reactor (SSBWR-MBR) were tested with different types of membranes. It was noted that the flux decrements of S-SMP (SMP in SSBWR-MBR) with cellulose acetate (CA), polyvinylidene fluoride (PVDF) and polyether sulfones (PES) membranes were respectively 6.7%, 8.5% and 9.5% lower compared to those of C-SMP (SMP in Control-MBR) with corresponding membranes. However, for both the filtration of the C-SMP and S-SMP, the CA membrane exhibited the fastest diminishing rate of flux among the three types of membranes. The surface morphology analysis showed that the CA membrane exhibited more but smaller protuberances compared to the PVDF and PES. The second minimums surrounding each protruding asperity on CA membrane were more than those on the PVDF and PES membranes, enhancing the attachment of SMP onto the membrane surface.

Microfauna communities as performance indicators for an A/O Shortcut Biological Nitrogen Removal moving-bed biofilm reactor.

The microfauna communities present in the mixed liquor and biofilm of an Anoxic/Oxic Shortcut Biofilm Nitrogen Removal moving-bed biofilm process were characterised in order to optimise process control through the use of bioindicators. The system operated at high ammonium concentrations, with an average of 588 ± 220 mg N-NH4(+) L(-1) in the influent, 161 ± 80 mg L(-1) in the anoxic reactor and 74 ± 71.2 mg L(-1) in the aerobic reactor. Up to 20 different taxa were identified, including ciliates (4), flagellates (11), amoebae (4) and nematodes (1). Compared to conventional wastewater treatment processes (WWTPs), this process can be defined as a flagellates-predominant system with a low diversity of ciliates. Flagellates were mainly dominant in the mixed liquor, demonstrating high tolerance to ammonium and the capacity for survival over a long time under anoxic conditions. The data obtained provide interesting values of maximum and minimum tolerance ranges to ammonium, nitrates and nitrites for the ciliate species Cyclidium glaucoma, Colpoda ecaudata, Vorticella microstoma-complex and Epistylis cf. rotans. The last of these was the only ciliate species that presented a constant and abundant population, almost exclusively in the aerobic biofilm. Epistylis cf. rotans dynamics showed a high negative correlation with ammonium variations and a positive correlation with ammonium removal efficiency. Hence, the results indicate that Epistylis cf. rotans is a good bioindicator of the nitrification process in this system. The study of protozoan communities in unexplored WWTPs sheds light on species ecology and their role under conditions that have been little studied in WWTPs, and could offer new biological management tools.

H2-driven cofactor regeneration with NAD(P)⁺-reducing hydrogenases.

A large number of industrially relevant enzymes depend upon nicotinamide cofactors, which are too expensive to be added in stoichiometric amounts. Existing NAD(P)H-recycling systems suffer from low activity, or the generation of side products. H2-driven cofactor regeneration has the advantage of 100% atom efficiency and the use of H2 as a cheap reducing agent, in a world where sustainable energy carriers are increasingly attractive. The state of development of H2-driven cofactor-recycling systems and examples of their integration with enzyme reactions are summarized in this article. The O2-tolerant NAD⁺-reducing hydrogenase from Ralstonia eutropha is a particularly attractive candidate for this approach, and we therefore discuss its catalytic properties that are relevant for technical applications.

Impact of worm predation on pseudo-steady-state of the circulating fluidized bed biofilm reactor.

This paper studies integrated simultaneous carbon and nitrogen removal as well as worm predation, in a circulating fluidized bed biofilm reactor (CFBBR) operated with an anoxic-aerobic bioparticle recirculation. A lab-scale CFBBR with a 8.5-liter reaction zone comprising 2L anoxic and 6.5L aerobic compartments was designed to evaluate the aquatic Oligochaete worm effect. Long-term (200 days) performance showed that stable and high-rate chemical oxygen demand (COD) with sodium acetate as the carbon source and total nitrogen (NH(4)Cl as nitrogen source) conversions were achieved simultaneously, with low sludge production of 0.082 g VSS (volatile suspended solids) g COD(-1) at pseudo-steady-state. Worm predation, which causes considerable sludge reduction of the bioparticle process, was studied. The results proved that the worm predation has a significant impact on the pseudo-steady-state performance of the CFBBR, decreasing biomass yield, decreasing oxygen concentration and increasing expanded bed height.

Activated sludge rheology: a critical review on data collection and modelling.

Rheological behaviour is an important fluid property that severely impacts its flow behaviour and many aspects related to this. In the case of activated sludge, the apparent viscosity has an influence on e.g. pumping, hydrodynamics, mass transfer rates, sludge-water separation (settling and filtration). It therefore is an important property related to process performance, including process economics. To account for this, rheological behaviour is being included in process design, necessitating its measurement. However, measurements and corresponding protocols in literature are quite diverse, leading to varying results and conclusions. In this paper, a vast amount of papers are critically reviewed with respect to this and important flaws are highlighted with respect to rheometer choice, rheometer settings and measurement protocol. The obtained rheograms from experimental efforts have frequently been used to build viscosity models. However, this is not that straightforward and a lot of errors can be detected with respect to good modelling practice, including fair model selection criteria, qualitative parameter estimations and proper model validation. These important steps are however recurrently violated, severely affecting the model reliability and predictive power. This is illustrated with several examples. In conclusion, dedicated research is required to improve the rheological measurements and the models derived from them. At this moment, there is no guidance with respect to proper rheological measurements. Moreover, the rheological models are not very trustworthy and remain very "black box". More insight in the physical background needs to be gained. A model-based approach with dedicated experimental data collection is the key to address this.