Shake Cultures Research Articles

Enhanced cellulase producing mutants developed from heterokaryotic Aspergillus strain

A heterokaryon 28, derived through protoplast fusion between Aspergillus nidulans and Aspergillus tubingensis (Dal8), was subjected cyclic mutagenesis followed by selection on increasing levels of 2-deoxy glucose (2-DG) as selection marker. The derived deregulated cellulase hyper producing mutant '64', when compared to fusant 28, produced 9.83, 7.8, 3.2, 4.2 and 19.74 folds higher endoglucanase, β-glucosidase, cellobiohydrolase, FPase and xylanase, respectively, under shake cultures. The sequence analysis of PCR amplified β-glucosidase gene from wild and mutant showed nucleotide deletion/substitution. The mutants showed highly catalytic efficient β-glucosidase as evident from low Km and high Vmax values. The expression profiling through zymogram analysis also indicated towards over-expression of cellulases. The up/down regulated expressed proteins observed through SDS-PAGE were identified by Peptide mass fingerprinting The cellulase produced by mutants in conjunction with cellulase free xylanase derived from Thermomyces lanuginosus was used for efficient utilization of alkali treated rice straw for obtaining xylo-oligosaccharides and ethanol.

Optimized Zebrafish Apolipoprotein A-I Expression and Purification for Nabbs Assembly

G protein-coupled receptors (GPCRs) are heptahelical transmembrane proteins that are highly susceptible to their lipid environment. Reconstitution of GPCRs into detergent micelles or liposomes allows researchers to control the lipid environment. Yet, micelle and liposome preparations are in homogeneous and protein incorporation is difficult to control. To address these issues, nanoscale apolipoprotein bound bilayers (NABBs), soluble phospholipid bilayers stabilized by the helical protein apolipoprotein A-I derived from zebra fish (zap1), have been previously devised. Yet, our approach has been limited by the poor expression of zap1 in E. coli. [1] To increase expression of zap1, we designed a synthetic gene (zapN1) with a codon-optimized sequence encoding the same amino acid sequence as zap1. We transformed BL21-Gold (DE3) cells with the zapN1 gene and performed a double-screen of protein expression from single transformed colonies. We show that zapN1 expresses in a shaking culture at higher levels than the native gene and the membrane scaffold protein MSP1D1 from Stephen Sligar's group. We carefully parameterized the induction and temperature values, finding that induction at an OD600 of 0.8 with 1 mM IPTG and 3 hours incubation at 30 °C yields the highest protein yield per ml of culture. We also show that a batch purification procedure of zapN1 using the His60 Ni superflow resin yields highly pure zapN1. The optimized protocol is easy to implement and requires no extensive preparation with fermenter or FPLC purification. The exceptional purity and high yield of the procedure makes it attractive for single-molecule fluorescence assays, such as fluorescence (cross-) correlation spectroscopy and total internal reflection fluorescence microscopy, and structural studies. [1] AM Knepp, A Grunbeck, S Banerjee, TP Sakmar, T Huber (2011) Direct measurement of thermal stability of expressed CCR5 and stabilization by small molecule ligands. Biochemistry, 50, 502-511.

石油污染土壤中苯酚降解菌ad049的鉴定及降解特性研究

以苯酚为唯一碳源，采用富集培养方法，从陕北靖边油田污染土壤中分离获得1株苯酚高效降解菌（ad049），对菌株进行形态观察、生理生化检验及 16S rDNA 序列分析，确定该菌株为红球菌（<em>Rhodococcus</em>）。采用摇瓶振荡培养方法，研究了接种量、pH 值、温度和底物浓度对 ad049 生长量和苯酚降解率的影响，同时对该菌株脱氢酶和邻苯二酚双加氧酶活性进行了测定。结果表明，ad049 具有较强的苯酚降解能力；在苯酚浓度1000 mg/L，温度 35 ℃，pH 值8，接种量 5% 的培养条件下，反应24 h后，苯酚降解率达 99%以上，且整个降解过程符合零级动力学方程，速率常数<em>k</em>₀ =41.51，相关系数 <em>R</em>²=0.96。通过邻苯二酚双加氧酶活性的测定，推测出该菌株降解苯酚的途径可能是以邻苯二酚 1，2 双加氧酶为主要途径进行邻位开环，辅以邻苯二酚 2，3双加氧酶进行间位开环。;A bacterium strain, which prove to be highly effective in phenol degrading, was isolated from soil contaminated with oil in Jingbian oilfield of Northern Shaanxi by using enrichment culture method with phenol as a sole carbon source. The bacterium was named ad049 and identified as<em> Rhodococcus</em> on the basis of the morphological characterizations, physiological and biochemical identification, and alignment analysis of the 16S rDNA sequence. Effects of inoculation quantity of ad049, pH, temperature, and concentration of phenol as a substrate on bacterial growth and the efficiency of ad049 on phenol degradation were also investigated by shaker culture method, and dehydrogenase and catechol dioxygenase were determined as well. Results showed that the ad049 had a strong biodegradation capability for phenol. It degraded 99% of the phenol in a concentration of 1000 mg/L in 24 hours under the incubating condition of 30 ℃, pH 8, and 5% of ad049 inoculation. The degradation process could be described with zero order kinetics equation with the k₀ and <em>R</em>² values of 41.51and 0.96, respectively. The degradation of phenol by ad049 might be achieved largely by 1,2-dioxygenase for ortho ring opening of benzene, and partly by catechol 2,3-dioxygenase for meta-cleavage.

Small-scale slow glucose feed cultivation of Pichia pastoris without repression of AOX1 promoter: towards high throughput cultivations

Recombinant protein synthesis in Pichia pastoris is generally controlled by the strong methanol inducible AOX1 promoter which is repressed by glucose and glycerol. In shake flasks, commonly one or two methanol pulses are added per day for induction. Such pulse feeding procedure leads to carbon starvation phases, which may enhance proteolytic activities and, therefore, cause product losses. Starvation between the methanol pulses could be avoided with a continuous enzymatic feed of glucose from a glucose-based polymer. The amount of glucose was low enough to prevent AOX1 repression by glucose. Energy and carbon were continuously supplied for cell maintenance resulting in significantly increased cell densities and product activities, as shown here at the example of a fungal lipase expressed in P. pastoris. A threefold improvement in measured product activity was obtained by applying enzymatic glucose feed and a further improvement was achieved by applying a defined mixture of ammonium compounds. The strategy described here simplifies the general procedure in shaken cultures by allowing the direct continuation of the cultivation from glucose to the methanol-based production phase without a medium change. It is easily applicable to multiwell plates and thus beneficial for high throughput applications.

Production of rotavirus core-like particles in Sf9 cells using recombinase-mediated cassette exchange

A flexible Sf9 insect cell line was recently developed leveraging the recombinase-mediated cassette exchange (RMCE) technology, which competes with the popular baculovirus expression vector system (BEVS) in terms of speed to produce new proteins. Herein, the ability of this cell platform to produce complex proteins, such as rotavirus core-like particles, was evaluated. A gene construct coding for a VP2-GFP fusion protein was targeted to a pre-characterized high recombination efficiency locus flanked by flipase (Flp) recognition target sites and, after three weeks in selection, an isogenic cell population was obtained. Despite the lower cell specific productivities with respect to those obtained by baculovirus infection, the titers of VP2-GFP reached in shake flask batch cultures were comparable as a result of higher cell densities. To further improve the VP2-GFP levels from stable expression, analysis of exhausted medium was undertaken to design feeding strategies enabling higher cell densities as well as increased culture duration. The implementation of the best strategy allowed reaching 20 million cells per ml in bioreactor cultures; the integrity of the rotavirus core-like particles could be confirmed by electron microscopy. Overall, we show that this Sf9-Flp cell platform represents a valuable alternative to the BEVS for producing complex recombinant proteins, such as rotavirus core-like particles.

Evaluation of process parameters in shake flasks for mammalian cell culture

Shake flask cultivation is nowadays a routine technique during process development for mammalian cell lines. During shaken culture, changes in agitation velocity, shaking diameter or shake flask size affect the hydrodynamics in the shake flask. This might be reflected in the growth of the cultured cells. Process parameters such as power input, mixing time, fluid velocity etc. have been determined and described mathematically for shake flasks used for microbial cultivation, but only to some extend for mammalian cell culture. Especially the relationship between these parameters and growth characteristics of mammalian cells is still a relatively uncovered issue. In this work, process parameters like specific power input, mixing time, maximum fluid velocity and Reynolds number were determined for four different shake flasks (baffled and unbaffled) in a range of shaking velocities on a shaking machine. The specific growth rate (μmax) of the human industrial cell line AGE1.HN® (ProBioGen AG, Berlin, Germany) was compared to the respective process parameters.

Decolorization of oxygen-delignified bleaching effluent and biobleaching of oxygen-delignified kraft pulp by non-white-rot fungus Geotrichum candidum Dec 1

Decolorization of oxygen-delignified bleaching effluent (abbreviated as OBE) and biobleaching of oxygen-delignified kraft pulp (OKP) were conducted using a non-white rot fungus Geotrichum candidum Dec 1 (abbreviated as Dec 1) which has ability to decolorize various synthetic dyes and molasses. Dec 1 decolorized up to 77% of OBE for 6 days. In addition, Dec 1 increased the brightness of OKP from 47.8% to 51.2% and decreased the kappa value of OKP from 12.4 to 10.4 points during a 6-day incubation period at a 25% of pulp-concentration. At 2% pulp-concentration, the brightness of OKP increased by 13% and the kappa value of OKP decreased by 4 points only for a 3-day incubation period. When the decolorized OBE was used for bleaching of OKP, the brightness of OKP increased to 62.7% under the shaking culture to a 2% pulp-concentration using culture fluid of decolorized OBE. It was revealed that Dec 1 is a potential to apply for decolorization of wastewater and biobleaching of pulp in paper-mills.

Codon modification for the DNA sequence of a single-chain Fv antibody against clenbuterol and expression in Pichia pastoris

The expression efficiency was improved for the recombinant single-chain variable fragment (scFv) against clenbuterol (CBL) obtained from mouse and expressed in the methylotrophic yeast Pichia pastoris GS115, by redesigning and synthesizing the DNA sequence encoding for CBL-scFv based on the codon bias of P. pastoris. The codons encoding 124 amino acids were optimized, in which a total of 156 nucleotides were changed, and the G+C ratio was simultaneously decreased from 53 to 47.2 %. Under the optimized expression conditions, the yield of the recombinant CBL-scFv (41 kDa) antibodies was 0.223 g L⁻¹ in shake culture. Compared to the non-optimized control, the expression level of the optimized recombinant CBL-scFv based on preferred codons in P. pastoris demonstrated a 2.35-fold higher yield. Furthermore, the recombinant CBL-scFv was purified by Ni-NTA column chromatography, and the purity was 95 %. The purified CBL-scFv showed good CBL recognition by a competitive indirect enzyme-linked immunoassay. The average concentration required for 50 % inhibition of binding and the limit of detection for the assay were 5.82 and 0.77 ng mL⁻¹, respectively.

Enhanced production of Fumigaclavine C in liquid culture of Aspergillus fumigatus under a two-stage process

Fumigaclavine C (FC) produced by Aspergillus fumigatus is a conidiation associated ergot alkaloid with strong anti-inflammatory activity. However, its wide application has been severely limited by low FC production from submerged culture. In this work, a novel two-stage culture process by combining shake culture with static culture was proposed to enhance the production of FC. After the process optimization, the FC production reached 62.7 mg/L, which was significantly higher than ever report. For scaling up this new culture process, the gas-liquid interfacial area per unit volume (Agas-liq) was identified as the key factor. The results showed that in a combined stirred-static bioreactor system, a maximum FC production (58.97 mg/L) was obtained at an Agas-liq value of 1.30 cm(2)/mL. These results demonstrated that two-stage culture is an efficient strategy to enhance FC production and the information obtained will be useful to production of this powerful bioactive compound on a large scale.

Biodegradation of the textile dye Mordant Black 17 (Calcon) by Moraxella osloensis isolated from textile effluent-contaminated site

The bacterium with dye degrading ability was isolated from effluent disposal sites of textile industries, Tirupur and was identified as Moraxella osloensis based on the biochemical and morphological characterization as well as 16S rRNA sequencing. This organism was found to decolorize 87 % of Mordant Black 17 at 100 mg l⁻¹ under shake culture condition compared to 92 % under stationary culture condition. Maximum degradation of the dye by M. osloensis was achieved when the mineral salt medium was supplemented with 0.5 % glucose and 0.1 % ammonium nitrate at 35 °C. Degradation of dye was found to follow first order kinetics with the k value of 0.06282 h⁻¹ and a R² value of 0.955. Analyses for the identification of intermediate compounds confirmed the presence of naphthalene, naphthol, naphthoquinone, salicylic acid and catechol. Based on this finding a probable pathway for the degradation of Mordant Black 17 by M. osloensis has been proposed.

Hfq and ArcA Are Involved in the Stationary Phase-Dependent Activation of Salmonella Pathogenicity Island 1 (SPI1) Under Shaking Culture Conditions

In Salmonella enterica serovar Typhimurium, many genes encoded within Salmonella pathogenicity island 1 (SPI1) are required to induce intestinal/diarrheal disease. In this study, we compared the expression of four SPI1 genes (hilA, invF, prgH, and sipC) under shaking and standing culture conditions and found that the expression of these genes was highest during the transition from the exponential to stationary phase under shaking conditions. To identify regulators associated with the stationary phase-dependent activation of SPI1, the effects of selected regulatory genes, including relA/spoT (ppGpp), luxS, ihfB, hfq, and arcA, on the expression of hilA and invF were compared under shaking conditions. Mutations in the hfq and arcA genes caused a reduction in hilA and invF expression (more than 2-fold) in the early stationary phase only, whereas the lack of ppGpp and IHF decreased hilA and invF gene expression during the entire stationary phase. We also found that hfq and arcA mutations caused a reduction of hilD expression upon entry into the stationary phase under shaking culture conditions. Taken together, these results suggest that Hfq and ArcA regulate the hilD promoter, causing an accumulation of HilD, which can trigger a stationary phase-dependent activation of SPI1 genes under shaking culture conditions.

Fast identification of reliable hosts for targeted cell line development from a limited‐genome screening using combined φC31 integrase and CRE‐Lox technologies

The use of targeted integration (TI) in cell line development (CLD) usually introduces one copy of a recombinant gene into a predetermined transcriptionally active locus. This reduces the heterogeneity typically associated with traditional random integration (RI) CLD with regards to varied productivity and instability, resulting from diverse chromosomal influences, varied copy numbers, and repeat-induced rearrangement. As such, TI CLD offers the hope of a predictable and consistent CLD process for establishing stable clones. However, given the low copy number, cell lines established from a TI CLD process tend to exhibit low productivity. Here, we describe our nonviral-based approach for quickly establishing and identifying TI hosts from a limited genome screening. Importantly, the TI hosts identified are consistent and reliable in supporting the production of diverse antibodies regardless of antibody subclass (IgG1 vs. IgG4) or prior traditional CLD performance (relatively easy vs. difficult to express antibodies). Moreover, an approximately twofold increase in titer can be achieved by using a CRE recombinase-mediated cassette exchange (RMCE) strategy with an exchange vector carrying two units of the antibody gene. Two RMCE hosts that were established were able to produce up to ∼ 1.7 and 2 g/L of antibodies in nonoptimized fed-batch shake flask production cultures with chemically defined media. Potentially, this strategy may be applied to the production of bispecific antibodies with a fast turnaround time.

Optimization and monitoring of water soluble substrate for synthesis of mannosylerythritol lipids by Pseudozyma antarctica (ATCC 32657)

Biosurfactants are amphipathic molecules, consisting of hydrophobic and hydrophilic domains, which can partition at the interfaces between different fluid phases, such as oil/water or water/air interfaces. Mannosylerithritol lipids (MELs) are a type of glycolipid. The yeast Candida (Pseudozyma) antarctica (ATCC 32657) secretes an extracellular MEL, with biosurfactant properties, when grown on a water soluble and insoluble substrate, such as glycerine or oils. Value added MEL was biologically synthesized, by using two different types of honey as natural and newer water soluble carbon source. Those types of honey as a carbon source were optimized at different concentration of total carbohydrate present in the honey. Further optimized substrate concentration of honey was monitored for 15 days, for studies of MEL yield and cell growth concentration per day on shake flask batch culture. Surfactant properties, such as surface tension, interfacial tension, foaming, emulsification and wetting of MEL, were observed. Produced MEL was confirmed by thin layer chromatography (TLC), Fourier Transform Infrared (FTIR) spectra, and 1H Nuclear Magnetic Resonance (NMR) techniques.

Generation and Characterization of Adeno-Associated Virus Producer Cell Lines for Research and Preclinical Vector Production

Adeno-associated virus (AAV) producer cell lines represent an effective method for large-scale production of AAV vectors. We set out to evaluate and characterize the use of an abbreviated protocol to generate "masterwells" (MWs; a nonclonal cell population) as a platform for research and preclinical vector production. In this system, a single plasmid containing three components, the vector sequence, the AAV rep, and cap genes, and a selectable marker gene is stably transfected into HeLaS3 cells. Producer cell lines generating an AAV2 vector expressing a secreted form of human placental alkaline phosphatase (SEAP) have been created. Several MWs showed vector yields in the 5×10(4) to 2×10(5) DNase-resistant particles/cell range, and the productivity was stable over >60 population doublings. Integrated plasmid copy number in three high-producing MWs ranged from approximately 12 to 50; copies were arranged in a head-to-tail configuration. Upon infection with adenovirus, rep/cap copy number was amplified approximately 100-fold and high yield appeared to be dependent on the extent of amplification. Rep/cap gene expression and vector packaging both reached a peak at 48 hr postinfection. AAV2-SEAP vector was produced in 1-liter shaker culture and purified for assessment of vector quality and potency. The data showed that the majority of the capsids from the MWs contained vector DNA (≥70%) and that purified vector was free of replication-competent AAV. In vitro and in vivo analyses demonstrated that potency of the producer cell-derived vector was comparable to vector generated via the standard transfection method.

In Vitro Tuberization of Chlorophytum Borivilianum Sant &amp; Fern (Safed Musli) As Influenced by Sucrose, CCC and Culture Systems

This study focuses on the establishment of in vitro tuberization of Chlorophytum borivilianum using solid and liquid culture systems. A high in vitro tuberization rate on solid and stationary liquid Murashige and Skoog media was observed in the presence of 60 g l⁻¹ sucrose with 950, 1,265 and 1,580 µM 2-chloroethyl-trimethylammonium chloride (CCC). Application of a higher sucrose concentration of 90 g l⁻¹ showed a negative interaction with CCC on in vitro tuber number and days to in vitro tuber induction. For economic feasibility, 950 µM CCC with 60 g l⁻¹ sucrose was chosen as the best combination for in vitro tuberization in both solid and stationary liquid media. For optimization of in vitro tuber production,a comparison between solid, stationary liquid and shake liquid culture was carried out. Liquid culture with shaking at 80 r.p.m. resulted in a >2.5-fold increase in in vitro tuber production compared with solid culture.

Production and optimization of poly-γ-glutamic acid by Bacillus subtilis BL53 isolated from the Amazonian environment

The aims of this research were to screen and characterize a new microbial source of γ-PGA, to optimize aspects of culture conditions and medium composition using central composite design and response surface methodologies. The influence of bioreactor stirring rates on the production of γ-PGA was also investigated and the oxygen volumetric mass transfer coefficients (k La) were established. The most productive strain was identified by 16S rDNA analysis as Bacillus subtilis, and its γ-PGA production in rotatory shaker was threefold increased under optimized conditions (37°C, pH 6.9, and 1.22mM Zn(2+)), compared to conventional medium. In bioreactor, the γ-PGA production was further increased, reaching 17gl(-1), 70% higher than shaker cultures. γ-PGA production showed high dependency on oxygen transfer. At k La of 210h(-1), the cultivation time could be reduced to 48h, about 50% of the time required for operations at k La 55h(-1).

Chlorobenzoic acid degradation by Lentinus (Panus) tigrinus: In vivo and in vitro mechanistic study-evidence for P-450 involvement in the transformation

Aim of this work was to investigate the ability of Lentinus (Panus) tigrinus to degrade and detoxify a chlorobenzoate (CBA) mixture composed of mono-, di- and tri-chlorinated isomers. The degradation process was investigated as a function of both the growing medium (i.e. low N Kirk's and malt extract-glucose medium) and cultivation conditions (i.e. stationary and shaken cultures). The majority of CBAs were quantitatively degraded within the early 15 d from spiking with the notable exception of the double ortho-chlorinated compounds, 2,6-di-, 2,3,6-tri- and 2,4,6-tri-CBA. Analysis of the degradation intermediates indicated the occurrence of side chain reduction, hydroxylation and methylation reactions. Although CBAs stimulated laccase production, in vitro experiments with a purified L. tigrinus laccase isoenzyme demonstrated its inability to participate in the initial attack on CBAs even in the presence of redox mediators; similar results were found with a Mn-peroxidase isoenzyme. Conversely, prompt degradation was observed upon 1h incubation of CBAs with a purified microsomal fraction containing cytochrome P-450 monooxygenase. The nature of some reaction products (i.e. hydroxylated derivatives), the dependency of the reaction on NADPH and its susceptibility to either CO or piperonyl butoxide inhibition confirmed the involvement of L. tigrinus cytochrome P-450 in the early steps of CBA degradation.

Systems-level understanding of how Propionibacterium acidipropionici respond to propionic acid stress at the microenvironment levels: Mechanism and application

In previous work, three evolved Propionibacterium acidipropionici mutants with higher tolerant capacity of propionic acid (PA) were obtained by genome shuffling. Here, we attempted to unravel the acid-tolerant mechanism of P. acidipropionici by comparing the physiological changes between P. acidipropionici and three mutants. The parameters used for comparison included intracellular pH (pHi), NAD+/NADH ratio, H+-ATPase activity, and the intracellular amino acids concentrations. It was indicated that the acid tolerance of P. acidipropionici was systematically regulated. Specifically, low pHi promoted the P. acidipropionici to biosynthesize more H+-ATPase to pump the protons out of the cells, and as a result, the NAD+/NADH ratio increased due to the decreased protons concentration. The increased arginine, aspartic acid, and glutamic acid concentrations helped to resist the acidic environment by consuming more H+ and generating more ATP and NH3. Based on what was analyzed above, 20mM arginine and aspartic acid were added during the shaker culture of P. acidipropionici, and the maximal PA titer reached 14.38g/L, which was increased by 39.9% compared with the control.

A new bioreactor for paclitaxel production based on foam separation

ABSTRACTWe report here the development of a new bioreactor (bioreactor with foam separation, BFS) for efficient production and recovery of paclitaxel that circumvents paclitaxel's inhibitory effects on growth through a feedback mechanism. The BFS captures paclitaxel through its hydrophobic interaction with the foam generated by aerated culture medium. The BFS comprises co‐axial 0.65 dm3 outer (150 mm height and 75 mm diameter) and 0.15 dm3 inner vessels (90 mm height and 50 mm diameter) equipped with a ball‐type sparger (5–10 µm pore size, 10 mm diameter) and a wire‐framed draft tube covered by a water‐permeable cellulose bag (150 × 40 mm, 100 µm maximum pore size). The BFS was tested by adding paclitaxel alone to the medium of 0.12 dm3 in the inner vessel and collecting it in the foam that overflowed into the outer vessel. Paclitaxel was separated with first‐order kinetics and was optimally recovered using an aeration rate of 0.05 dm3 min−1 (0.21 vvm). Next, a Taxus cuspidata callus was cultured using the optimal aeration rate. A conventional shaking culture (110 rpm) served as the control using a 0.2 dm3 Erlenmeyer flask (0.12 dm3 medium). Paclitaxel was maintained at less than 0.02 g m−3 in the inner vessel of the BFS. Consequently, there was no feedback inhibition, and the paclitaxel production increased by a factor of five in the BFS compared with the control culture where there was feedback inhibition by the high concentration of paclitaxel. © 2013 Curtin University of Technology and John Wiley &amp; Sons, Ltd.

Consortia-mediated bioprocessing of cellulose to ethanol with a symbiotic Clostridium phytofermentans/yeast co-culture

BackgroundLignocellulosic ethanol is a viable alternative to petroleum-based fuels with the added benefit of potentially lower greenhouse gas emissions. Consolidated bioprocessing (simultaneous enzyme production, hydrolysis and fermentation; CBP) is thought to be a low-cost processing scheme for lignocellulosic ethanol production. However, no single organism has been developed which is capable of high productivity, yield and titer ethanol production directly from lignocellulose. Consortia of cellulolytic and ethanologenic organisms could be an attractive alternate to the typical single organism approaches but implementation of consortia has a number of challenges (e.g., control, stability, productivity).ResultsEthanol is produced from α-cellulose using a consortium of C. phytofermentans and yeast that is maintained by controlled oxygen transport. Both Saccharomyces cerevisiae cdt-1 and Candida molischiana “protect” C. phytofermentans from introduced oxygen in return for soluble sugars released by C. phytofermentans hydrolysis. Only co-cultures were able to degrade filter paper when mono- and co-cultures were incubated at 30°C under semi-aerobic conditions. Using controlled oxygen delivery by diffusion through neoprene tubing at a calculated rate of approximately 8 μmol/L hour, we demonstrate establishment of the symbiotic relationship between C. phytofermentans and S. cerevisiae cdt-1 and maintenance of populations of 105 to 106 CFU/mL for 50 days. Comparable symbiotic population dynamics were observed in scaled up 500 mL bioreactors as those in 50 mL shake cultures. The conversion of α-cellulose to ethanol was shown to improve with additional cellulase indicating a limitation in hydrolysis rate. A co-culture of C. phytofermentans and S. cerevisiae cdt-1 with added endoglucanase produced approximately 22 g/L ethanol from 100 g/L α-cellulose compared to C. phytofermentans and S. cerevisiae cdt-1 mono-cultures which produced approximately 6 and 9 g/L, respectively.ConclusionThis work represents a significant step toward developing consortia-based bioprocessing systems for lignocellulosic biofuels production which utilize scalable, environmentally-mediated symbiosis mechanisms to provide consortium stability.