Is heterogeneity in large-scale bioreactors a real problem in recombinant protein synthesis by Pichia pastoris?

Culture medium heterogeneity is inherent in industrial bioreactors. The loss of mixing efficiency in a large-scale bioreactor yields to the formation of concentration gradients. Consequently, cells face oscillatory culture conditions that may deeply affect their metabolism. Herein, cell response to transient perturbations, namely high methanol concentration combined with hypoxia, has been investigated using a two stirred-tank reactor compartiments (STR-STR) scale-down system and a Pichia pastoris strain expressing the gene encoding enhanced green fluorescent protein (eGFP) under the control of the alcohol oxidase 1 (AOX1) promoter. Cell residence times under transient stressing conditions were calculated based on the typical hydraulic circulation times of bioreactors of tens and hundreds cubic metres. A significant increase in methanol and oxygen uptake rates was observed as the cell residence time was increased. Stressful culture conditions impaired biomass formation and triggered cell flocculation. More importantly, both expression levels of genes under the control of pAOX1 promoter and eGFP specific fluorescence were higher in those oscillatory culture conditions, suggesting that those a priori unfavourable culture conditions in fact benefit to recombinant protein productivity. Flocculent cells were also identified as the most productive as compared to ovoid cells. KEY POINTS: • Transient hypoxia and high methanol trigger high level of recombinant protein synthesis • In Pichia pastoris, pAOX1 induction is higher in flocculent cells • Medium heterogeneity leads to morphological diversification.

Synthesis of Secretory Proteins in Yarrowia lipolytica: Effect of Combined Stress Factors and Metabolic Load.

While overproduction of recombinant secretory proteins (rs-Prots) triggers multiple changes in the physiology of the producer cell, exposure to suboptimal growth conditions may further increase that biological response. The environmental conditions may modulate the efficiency of both the rs-Prot gene transcription and translation but also the polypeptide folding. Insights into responses elicited by different environmental stresses on the rs-Prots synthesis and host yeast physiology might contribute to a better understanding of fundamental biology processes, thus providing some clues to further optimise bioprocesses. Herein, a series of batch cultivations of Yarrowia lipolytica strains differentially metabolically burdened by the rs-Prots overproduction have been conducted. Combinations of different stress factors, namely pH (3/7) and oxygen availability (kLa 28/110 h-1), have been considered for their impact on cell growth and morphology, substrate consumption, metabolic activity, genes expression, and secretion of the rs-Prots. Amongst others, our data demonstrate that a highly metabolically burdened cell has a higher demand for the carbon source, although presenting a compromised cell growth. Moreover, the observed decrease in rs-Prot production under adverse environmental conditions rather results from the emergence of a less-producing cell subpopulation than from the decrease of the synthetic capacity of the whole cell population.

Recombinant protein production in Pichia pastoris: from transcriptionally redesigned strains to bioprocess optimization and metabolic modelling.

Pichia pastoris is one of the most widely used host for the production of recombinant proteins. Expression systems that rely mostly on promoters from genes encoding alcohol oxidase 1 or glyceraldehyde-3-phosphate dehydrogenase have been developed together with related bioreactor operation strategies based on carbon sources such as methanol, glycerol, or glucose. Although, these processes are relatively efficient and easy to use, there have been notable improvements over the last twenty years to better control gene expression from these promoters and their engineered variants. Methanol-free and more efficient protein production platforms have been developed by engineering promoters and transcription factors. The production window of P. pastoris has been also extended by using alternative feedstocks including ethanol, lactic acid, mannitol, sorbitol, sucrose, xylose, gluconate, formate or rhamnose. Herein, the specific aspects that are emerging as key parameters for recombinant protein synthesis are discussed. For this purpose, a holistic approach has been considered to scrutinize protein production processes from strain design to bioprocess optimization, particularly focusing on promoter engineering, transcriptional circuitry redesign. This review also considers the optimization of bioprocess based on alternative carbon sources and derived co-feeding strategies. Optimization strategies for recombinant protein synthesis through metabolic modelling are also discussed.

What makes Komagataella phaffii non-conventional?

The important industrial protein production host Komagataella phaffii (syn Pichia pastoris) is classified as a non-conventional yeast. But what exactly makes K. phaffii non-conventional? In this review, we set out to address the main differences to the 'conventional' yeast Saccharomyces cerevisiae, but also pinpoint differences to other non-conventional yeasts used in biotechnology. Apart from its methylotrophic lifestyle, K. phaffii is a Crabtree-negative yeast species. But even within the methylotrophs, K. phaffii possesses distinct regulatory features such as glycerol-repression of the methanol-utilization pathway or the lack of nitrate assimilation. Rewiring of the transcriptional networks regulating carbon (and nitrogen) source utilization clearly contributes to our understanding of genetic events occurring during evolution of yeast species. The mechanisms of mating-type switching and the triggers of morphogenic phenotypes represent further examples for how K. phaffii is distinguished from the model yeast S. cerevisiae. With respect to heterologous protein production, K. phaffii features high secretory capacity but secretes only low amounts of endogenous proteins. Different to S. cerevisiae, the Golgi apparatus of K. phaffii is stacked like in mammals. While it is tempting to speculate that Golgi architecture is correlated to the high secretion levels or the different N-glycan structures observed in K. phaffii, there is recent evidence against this. We conclude that K. phaffii is a yeast with unique features that has a lot of potential to explore both fundamental research questions and industrial applications.

Bioproducts generation from carboxylate platforms by the non-conventional yeast Yarrowia lipolytica.

In recent years, there has been a growing interest in the use of renewable sources for bio-based production aiming at developing sustainable and feasible approaches towards a circular economy. Among these renewable sources, organic wastes (OWs) can be anaerobically digested to generate carboxylates like volatile fatty acids (VFAs), lactic acid, and longer-chain fatty acids that are regarded as novel building blocks for the synthesis of value-added compounds by yeasts. This review discusses on the processes that can be used to create valuable molecules from OW-derived VFAs; the pathways employed by the oleaginous yeast Yarrowia lipolytica to directly metabolize such molecules; and the relationship between OW composition, anaerobic digestion, and VFA profiles. The review also summarizes the current knowledge about VFA toxicity, the pathways by which VFAs are metabolized and the metabolic engineering strategies that can be employed in Y. lipolytica to produce value-added biobased compounds from VFAs.

Improvement of glutathione production by a metabolically engineered Yarrowia lipolytica strain using a small-scale optimization approach.

OBJECTIVE: In this study, we aimed to maximize glutathione (GSH) production by a metabolically engineered Yarrowia lipolytica strain using a small-scale optimization approach. RESULTS: A three levels four factorial Box-Behnken Design was used to assess the effect of pH, inulin extract, yeast extract and ammonium sulfate concentrations on cell growth and to generate a mathematical model which predict optimal conditions to maximize biomass production and thus GSH titer. The obtained results revealed that only yeast and inulin extract concentrations significantly affect biomass production. Based on the generated model, a medium composed of 10 g/L of yeast extract and 10 g/L of inulin extract from Jerusalem artichoke was used to conduct batch cultures in 2 L bioreactor. After 48 h of culture, the biomass and the glutathione titer increased by 55% (5.8 gDCW/L) and 61% (1011.4 mg/L), respectively, as compared to non-optimized conditions. CONCLUSION: From the obtained results, it could be observed that the model established from small scale culture (i.e. 2 mL) is able to predict performance at larger scale (i.e. 2 L bioreactor, two orders of magnitude scale-up). Moreover, the results highlight the ability of the optimized process to ensure high titer of glutathione using a low-cost carbon source.

Waste soybean frying oil for the production, extraction, and characterization of cell-wall-associated lipases from Yarrowia lipolytica.

The lipolytic yeast Yarrowia lipolytica produces cell-wall-associated lipases, namely Lip7p and Lip8p, that could have interesting properties as catalyst either in free (released lipase fraction-RLF) or cell-associated (cell-bound lipase fraction-CBLF) forms. Herein, a mixture of waste soybean frying oil, yeast extract and bactopeptone was found to favor the enzyme production. Best parameters for lipase activation and release from the cell wall by means of acoustic wave treatment were defined as: 26 W/cm2 for 1 min for CBLF and 52 W/cm2 for 2 min for RLF. Optimal pH and temperature values for lipase activity together with storage conditions were similar for both the free enzyme and cell-associated one: pH 7.0; T = 37 °C; and > 70% residual activity for 60 days at 4, - 4 °C and for 15 days at 30 °C.

Biocompatibility and Cytotoxicity of Gold Nanoparticles: Recent Advances in Methodologies and Regulations.

Recent advances in the synthesis of metal nanoparticles (MeNPs), and more specifically gold nanoparticles (AuNPs), have led to tremendous expansion of their potential applications in different fields, ranging from healthcare research to microelectronics and food packaging. The properties of functionalised MeNPs can be fine-tuned depending on their final application, and subsequently, these properties can strongly modulate their biological effects. In this review, we will firstly focus on the impact of MeNP characteristics (particularly of gold nanoparticles, AuNPs) such as shape, size, and aggregation on their biological activities. Moreover, we will detail different in vitro and in vivo assays to be performed when cytotoxicity and biocompatibility must be assessed. Due to the complex nature of nanomaterials, conflicting studies have led to different views on their safety, and it is clear that the definition of a standard biosafety label for AuNPs is difficult. In fact, AuNPs' biocompatibility is strongly affected by the nanoparticles' intrinsic characteristics, biological target, and methodology employed to evaluate their toxicity. In the last part of this review, the current legislation and requirements established by regulatory authorities, defining the main guidelines and standards to characterise new nanomaterials, will also be discussed, as this aspect has not been reviewed recently. It is clear that the lack of well-established safety regulations based on reliable, robust, and universal methodologies has hampered the development of MeNP applications in the healthcare field. Henceforth, the international community must make an effort to adopt specific and standard protocols for characterisation of these products.

Effect of brine concentration on physico-chemical characteristics, texture, rheological properties and proteolysis level of cheeses produced by an optimized wild cardoon rennet.

The main objective of this study was to test the efficiency of a wild cardoon (Cynara cardunculus L.) rennet, previously optimized by response surface methodology, in cheese making process; then to select the best brine concentration, leading to excellent cheese quality. Results showed that the optimized C. cardunculus rennet and chymosin produced curds with similar properties (yield, colour, texture, viscoelasticity), suggesting that this coagulant could replace successfully calf rennet. After brining at different salt concentrations (5, 7, 10 and 15%), we concluded that the use of 15% of salt in brine was an efficient way to reduce considerably the proteolysis level in C. cardunculus cheeses, stored for 28 d at 4 °C. At this salt level, the highest hardness, gumminess, viscoelasticity and yield of soft cheeses were also recorded. In conclusion, the satisfactory findings could open new opportunities to produce industrially the optimized C. cardunculus rennet and its cheeses in the Mediterranean area.

Impact of oxygen availability on heterologous geneexpression and polypeptide secretion dynamics in Yarrowia lipolytica-based protein production platforms.

Industrially relevant traits of Yarrowia lipolytica, like high growth rate, capacity to grow at high cell density or to synthesize biomolecules with high productivities, strongly rely on sufficient oxygen provision. Although the impact of oxygen availability (OA) on the physiology of Y. lipolytica has been already studied, its influence on recombinant protein (rProt) synthesis and secretion has been largely neglected to date. With the aim to fill this gap, a fluorescent reporter protein (yellow fluorescent protein [YFP]) was used herein as a proxy to follow simultaneously rProt synthesis and secretion in Y. lipolytica under different OAs. This study covers the analysis of the reporter gene expression through reverse transcription quantitative polymerase chain reaction, polypeptide synthesis and its retention-to-secretion ratio using flow cytometry and fluorymetry during shake flasks and bioreactor cultivations under different OA. The results gathered demonstrate that OA has a dramatic impact on the kinetics of intracellular and extracellular YFP accumulation. Higher rProt production and secretion were favoured under high OA, and were largely related to OA and not to cell growth. Our observations also suggest the existence of some upper limit of secretory protein accumulation inside the cells above which massive secretion is initiated. Moreover, at low OA, the first bottleneck in rProt synthesis occurs as early as at transcription level, which could results from a lower availability of transcriptional machinery elements. Finally, using flow cytometry and bioreactor cultivations, we highlighted that ovoid cells are generally more efficient in terms of rProt synthesis.

In Yarrowia lipolytica erythritol catabolism ends with erythrose phosphate.

In response to osmotic stress, the yeast Yarrowia lipolytica produces erythritol, a four-carbon sugar alcohol, from erythrose-P, an intermediate of the pentose phosphate pathway. Under non-stressing conditions (isotonic environment), the produced erythritol is subsequently recycled into erythrose-P that can feed the pentose phosphate pathway. Herein, gene YALI0F01584g was characterized as involved in the erythritol catabolic pathway. Several experimental evidences suggested that it encodes an erythrulose-1P isomerase that converts erythrulose-1P into erythrulose-4P. On the basis of our previous reports and results gathered in this study with genetically modified strains, including ΔYALI0F01584g and ΔYALI0F01628g disrupted mutants, the entire erythritol catabolic pathway has been characterized.

Downregulation by organic nitrogen of AOX1 promoter used for controlled expression of foreign genes in the yeast Pichia pastoris.

Pichia pastoris is a well-established cell factory for recombinant protein synthesis. Various optimization strategies of processes based on AOX1 promoter have been investigated, including methanol co-feeding with glycerol or sorbitol during the induction stage. Compared with carbon sources, comparatively little research has been devoted to the effects of nitrogen sources. Several reports have described the benefits of adding casamino acids (CA) to the recombinant protein production medium, however, without considering its effects at the gene expression level. Using enhanced green fluorescent protein as a reporter protein, monitored using flow cytometry, CA was shown to downregulate AOX1 promoter induction. Despite higher growth rates, cultures containing CA exhibited slower transition to the induced state, whereas metabolite analysis revealed that methanol consumption was reduced in the presence of CA compared with its absence. The repressive effect of CA was further confirmed by analysing the synthesis of extracellular recombinant Candida antarctica lipase under control of the AOX1 promoter. These findings highlight nitrogen source selection as an important consideration for AOX1-based protein production.

Screening various Yarrowia lipolytica strains for citric acid production.

Citric acid (CA) productivity by Yarrowia lipolytica dependents on strain type, carbon source, carbon to nitrogen (C/N) molar ratio as well as physicochemical conditions (pH, temperature, oxygen transfer rate, etc.). In the current study, 10 different Y. lipolytica strains were first challenged in shake-flask culture for CA production in a glucose-based media under nitrogen-limited conditions. For the most promising one, strain K57, CA productivity was monitored during culture in batch bioreactor for three initial C/N molar ratio (167, 367, and 567 Cmol/Nmol). The highest CA yield (0.77 g/g glucose), titre (72.3 g/L CA), and productivity (0.04 g/g.h) were found for C/N ratio of 367. However, the highest growth rate was obtained for an initial C/N ratio of 167. From these results, Y. lipolytica strain K57 could be considered to produce CA at higher titre on glucose-based medium in batch bioreactor than others Y. lipolytica strain reported in the literature.

Expression of recombinant enhanced green fluorescent protein provides insight into foreign gene-expression differences between Mut+ and MutS strains of Pichia pastoris.

Pichia pastoris is a very popular yeast for recombinant protein production, mainly due to the strong, methanol-inducible PAOX1 promoter. Methanol induction however poses several drawbacks. One approach to improve processes is to use MutS strains with reduced methanol catabolic ability. Various reports claim that MutS allows higher recombinant protein production levels than Mut+ but scarcely elaborate on reasons for differences. In this study, enhanced green fluorescent protein was used as a PAOX1 -driven reporter for the investigation of expression differences between Mut+ and MutS strains. Mut+ exhibited higher responses to methanol, with faster growth (0.07 vs. 0.01 hr-1 ) and higher consumption of methanol (2.25 vs. 1.81 mmol/gDCW .hr) and oxygen (2.2 vs. 0.66 mmol/gDCW .hr) than MutS. Mut+ yielded more biomass than MutS (2.3 vs. 1.3 gDCW /L), and carbon dioxide analysis of bioreactor off-gas suggested that considerable amounts of methanol were consumed by Mut+ via the dissimilatory pathway. In contrast, it was demonstrated that the MutS population switched to an induced state more rapidly than Mut+. In addition, MutS exhibited 3.4-fold higher fluorescence levels per cell (77,509 vs. 23,783 SFU) indicative of higher recombinant protein production. The findings were verified by similar results obtained during the expression of a lipase. Based on the differences in response to methanol versus recombinant protein production, it was proposed that higher energy availability occurs in MutS for recombinant protein synthesis, contrary to Mut+ that uses the energy to maintain high levels of methanol catabolic pathways and biomass production.

Green pyomelanin-mediated synthesis of gold nanoparticles: modelling and design, physico-chemical and biological characteristics.

BACKGROUND: Synthesis of nanoparticles (NPs) and their incorporation in materials are amongst the most studied topics in chemistry, physics and material science. Gold NPs have applications in medicine due to their antibacterial and anticancer activities, in biomedical imaging and diagnostic test. Despite chemical synthesis of NPs are well characterized and controlled, they rely on the utilization of harsh chemical conditions and organic solvent and generate toxic residues. Therefore, greener and more sustainable alternative methods for NPs synthesis have been developed recently. These methods use microorganisms, mainly yeast or yeast cell extract. NPs synthesis with culture supernatants are most of the time the preferred method since it facilitates the purification scheme for the recovery of the NPs. Extraction of NPs, formed within the cells or cell-wall, is laborious, time-consuming and are not cost effective. The bioactivities of NPs, namely antimicrobial and anticancer, are known to be related to NPs shape, size and size distribution. RESULTS: Herein, we reported on the green synthesis of gold nanoparticles (AuNPs) mediated by pyomelanin purified from the yeast Yarrowia lipolytica. A three levels four factorial Box-Behnken Design (BBD) was used to evaluate the influence of temperature, pH, gold salt and pyomelanin concentration on the nanoparticle size distribution. Based on the BBD, a quadratic model was established and was applied to predict the experimental parameters that yield to AuNPs with specific size. The synthesized nanoparticles with median size value of 104 nm were of nanocrystalline structure, mostly polygonal or spherical. They exhibited a high colloidal stability with zeta potential of - 28.96 mV and a moderate polydispersity index of 0.267. The absence of cytotoxicity of the AuNPs was investigated on two mammalian cell lines, namely mouse fibroblasts (NIH3T3) and human osteosarcoma cells (U2OS). Cell viability was only reduced at AuNPs concentration higher than 160 µg/mL. Moreover, they did not affect on the cell morphology. CONCLUSION: Our results indicate that different process parameters affect significantly nanoparticles size however with the mathematical model it is possible to define the size of AuNPs. Moreover, this melanin-based gold nanoparticles showed neither cytotoxicity effect nor altered cell morphology.

Efficient expression vectors and host strain for the production of recombinant proteins by Yarrowia lipolytica in process conditions.

BACKGROUND: The oleaginous yeast Yarrowia lipolytica is increasingly used as an alternative cell factory for the production of recombinant proteins. Recently, regulated promoters from genes EYK1 and EYD1, encoding an erythrulose kinase and an erythritol dehydrogenase, respectively, have been identified and characterized in this yeast. Hybrid promoters up-regulated by polyols such as erythritol and erythrulose have been developed based on tandem copies of upstream activating sequences from EYK1 (UAS1EYK1) and XPR2 (encoding extracellular protease, UAS1XPR2) promoters. RESULTS: The strength of native (pEYD1) and engineered promoters (pEYK1-3AB and pHU8EYK) was compared using the extracellular lipase CalB from Candida antarctica as a model protein and a novel dedicated host strain. This latter is engineered in polyol metabolism and allows targeted chromosomal integration. In process conditions, engineered promoters pEYK1-3AB and pHU8EYK yielded 2.8 and 2.5-fold higher protein productivity, respectively, as compared to the reference pTEF promoter. We also demonstrated the possibility of multicopy integration in the newly developed host strain. In batch bioreactor, the CalB multi-copy strain RIY406 led to a 1.6 fold increased lipase productivity (45,125 U mL-1) within 24 h as compared to the mono-copy strain. CONCLUSIONS: The expression system described herein appears promising for recombinant extracellular protein production in Y. lipolytica.

Erythritol production by yeasts: a snapshot of current knowledge.

Erythritol is a four-carbon sugar alcohol produced by microorganisms as an osmoprotectant. It could be used as a natural sweetener in the pharmaceutical and food industries. Here, a snapshot of current knowledge on erythritol metabolism and synthesis, optimization of its production and more precise process and producer strain improvement is presented.

Gene of the transcriptional activator MET4 is involved in regulation of glutathione biosynthesis in the methylotrophic yeast Ogataea (Hansenula) polymorpha.

Glutathione is the most abundant cellular thiol and the low molecular weight peptide present in cells. The methylotrophic yeast Ogataea (Hansenula) polymorpha is considered as a promising cell factory for the synthesis of glutathione. In this study, a competitive O. polymorpha glutathione producer was constructed by overexpression of the GSH2 gene, encoding γ-glutamylcysteine synthetase, the first enzyme involved in glutathione biosynthesis, and the MET4 gene coding for central regulator of sulfur metabolism. Overexpression of MET4 gene in the background of overexpressed GSH2 gene resulted in 5-fold increased glutathione production during shake flask cultivation as compared to the wild-type strain, reaching 2167 mg L-1. During bioreactor cultivation, glutathione accumulation by obtained recombinant strain was 5-fold increased relative to that by the parental strain with overexpressed only GSH2 gene, on the first 25 h of batch cultivation in mineral medium. Obtained results suggest involvement of Met4 transcriptional activator in regulation of GSH synthesis in the methylotrophic yeast O. polymorpha.

Identification, characterization of two NADPH-dependent erythrose reductases in the yeast Yarrowia lipolytica and improvement of erythritol productivity using metabolic engineering.

BACKGROUND: Erythritol is a four-carbon sugar alcohol with sweetening properties that is used by the agro-food industry as a food additive. In the yeast Yarrowia lipolytica, the last step of erythritol synthesis involves the reduction of erythrose by specific erythrose reductase(s). In the earlier report, an erythrose reductase gene (YALI0F18590g) from erythritol-producing yeast Y. lipolytica MK1 was identified (Janek et al. in Microb Cell Fact 16:118, 2017). However, deletion of the gene in Y. lipolytica MK1 only resulted in some lower erythritol production but the erythritol synthesis process was still maintained, indicating that other erythrose reductase gene(s) might exist in the genome of Y. lipolytica. RESULTS: In this study, we have isolated genes g141.t1 (YALI0D07634g) and g3023.t1 (YALI0C13508g) encoding two novel erythrose reductases (ER). The biochemical characterization of the purified enzymes showed that they have a strong affinity for erythrose. Deletion of the two ER genes plus g801.t1 (YALI0F18590g) did not prevent erythritol synthesis, suggesting that other ER or ER-like enzymes remain to be discovered in this yeast. Overexpression of the newly isolated two genes (ER10 or ER25) led to an average 14.7% higher erythritol yield and 31.2% higher productivity compared to the wild-type strain. Finally, engineering NADPH cofactor metabolism by overexpression of genes ZWF1 and GND1 encoding glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase, respectively, allowed a 23.5% higher erythritol yield and 50% higher productivity compared to the wild-type strain. The best of our constructed strains produced an erythritol titer of 190 g/L in baffled flasks using glucose as main carbon source. CONCLUSIONS: Our results highlight that in the Y. lipolytica genome several genes encode enzymes able to reduce erythrose into erythritol. The catalytic properties of these enzymes and their cofactor dependency are different from that of already known erythrose reductase of Y. lipolytica. Constitutive expression of the newly isolated genes and engineering of NADPH cofactor metabolism led to an increase in erythritol titer. Development of fermentation strategies will allow further improvement of this productivity in the future.

Integrating metabolic modeling and population heterogeneity analysis into optimizing recombinant protein production by Komagataella (Pichia) pastoris.

The methylotrophic yeast Komagataella (Pichia) pastoris has become one of the most utilized cell factories for the production of recombinant proteins over the last three decades. This success story is linked to its specific physiological traits, i.e., the ability to grow at high cell density in inexpensive culture medium and to secrete proteins at high yield. Exploiting methanol metabolism is at the core of most P. pastoris-based processes but comes with its own challenges. Co-feeding cultures with glycerol/sorbitol and methanol is a promising approach, which can benefit from improved understanding and prediction of metabolic response. The development of profitable processes relies on the construction and selection of efficient producing strains from less efficient ones but also depends on the ability to master the bioreactor process itself. More specifically, how a bioreactor processes could be monitored and controlled to obtain high yield of production. In this review, new perspectives are detailed regarding a multi-faceted approach to recombinant protein production processes by P. pastoris; including gaining improved understanding of the metabolic pathways involved, accounting for variations in transcriptional and translational efficiency at the single cell level and efficient monitoring and control of methanol levels at the bioreactor level.