Growth In Chemostat Research Articles

Substrate and nutrient manipulation during continuous cultivation of extremophilic algae, Galdieria spp. RTK 37.1, substantially impacts biomass productivity and composition.

The extremophilic nature and metabolic flexibility of Galdieria spp. highlights their potential for biotechnological application. However, limited research into continuous cultivation of Galdieria spp. has slowed progress towards the commercialization of these algae. The objective of this research was to investigate biomass productivity and growth yields during continuous photoautotrophic, mixotrophic and heterotrophic cultivation of Galdieria sp. RTK371; a strain recently isolated from within the Taupō Volcanic Zone in Aotearoa-New Zealand. Results indicate Galdieria sp. RTK371 grows optimally at pH 2.5 under warm white LED illumination. Photosynthetic O2 production was dependent on lighting intensity with a maximal value of (133.5 ± 12.1 nmol O2 mgbiomass -1 h-1) achieved under 100 μmol m-2 s-1 illumination. O2 production rates slowed significantly to 42 ± 1 and <0.01 nmol O2 mgbiomass -1 h-1 during mixotrophic and heterotrophic growth regimes respectively. Stable, long-term chemostat growth of Galdieria sp. RTK371 was achieved during photoautotrophic, mixotrophic and heterotrophic growth regimes. During periods of ammonium limitation, Galdieria sp. RTK371 increased its intracellular carbohydrate content (up to 37% w/w). In contrast, biomass grown in ammonium excess was composed of up to 65% protein (w/w). Results from this study demonstrate that the growth of Galdieria sp. RTK371 can be manipulated during continuous cultivation to obtain desired biomass and product yields over long cultivation periods.

Adaptive laboratory evolution of Clostridium autoethanogenum to metabolize CO2 and H2 enhances growth rates in chemostat and unravels proteome and metabolome alterations.

Gas fermentation of CO2 and H2 is an attractive means to sustainably produce fuels and chemicals. Clostridium autoethanogenum is a model organism for industrial CO to ethanol and presents an opportunity for CO2-to-ethanol processes. As we have previously characterized its CO2/H2 chemostat growth, here we use adaptive laboratory evolution (ALE) with the aim of improving growth with CO2/H2. Seven ALE lineages were generated, all with improved specific growth rates. ALE conducted in the presence of 2% CO along with CO2/H2 generated Evolved lineage D, which showed the highest ethanol titres amongst all the ALE lineages during the fermentation of CO2/H2. Chemostat comparison against the parental strain shows no change in acetate or ethanol production, while Evolved D could achieve a higher maximum dilution rate. Multi-omics analyses at steady state revealed that Evolved D has widespread proteome and intracellular metabolome changes. However, the uptake and production rates and titres remain unaltered until investigating their maximum dilution rate. Yet, we provide numerous insights into CO2/H2 metabolism via these multi-omics data and link these results to mutations, suggesting novel targets for metabolic engineering in this bacterium.

Template switching between the leading and lagging strands at replication forks generates inverted copy number variants through hairpin-capped extrachromosomal DNA.

Inherited and germ-line de novo copy number variants (CNVs) are increasingly found to be correlated with human developmental and cancerous phenotypes. Several models for template switching during replication have been proposed to explain the generation of these gross chromosomal rearrangements. We proposed a model of template switching (ODIRA-origin dependent inverted repeat amplification) in which simultaneous ligation of the leading and lagging strands at diverging replication forks could generate segmental inverted triplications through an extrachromosomal inverted circular intermediate. Here, we created a genetic assay using split-ura3 cassettes to trap the proposed inverted intermediate. However, instead of recovering circular inverted intermediates, we found inverted linear chromosomal fragments ending in native telomeres-suggesting that a template switch had occurred at the centromere-proximal fork of a replication bubble. As telomeric inverted hairpin fragments can also be created through double strand breaks we tested whether replication errors or repair of double stranded DNA breaks were the most likely initiating event. The results from CRISPR/Cas9 cleavage experiments and growth in the replication inhibitor hydroxyurea indicate that it is a replication error, not a double stranded break that creates the inverted junctions. Since inverted amplicons of the SUL1 gene occur during long-term growth in sulfate-limited chemostats, we sequenced evolved populations to look for evidence of linear intermediates formed by an error in replication. All of the data are compatible with a two-step version of the ODIRA model in which sequential template switching at short inverted repeats between the leading and lagging strands at a replication fork, followed by integration via homologous recombination, generates inverted interstitial triplications.

Simulating Bacterial Chemostat Growth as an Introduction to Rate Modeling with Calculus

Simulating Bacterial Chemostat Growth as an Introduction to Rate Modeling with Calculus

Microbial maintenance energy quantified and modeled with microcalorimetry

Refining the energetic costs of cellular maintenance is essential for predicting microbial growth and survival in the environment. Here, we evaluate a simple batch culture method to quantify energy partitioning between growth and maintenance using microcalorimetry and thermodynamic modeling. The constants derived from the batch culture system were comparable to those that have been reported from meta-analyses of data derived from chemostat studies. The model accurately predicted temperature-dependent biomass yield and the upper temperature limit of growth for Desulfovibrio alaskensis G20, suggesting the method may have broad application. An Arrhenius temperature dependence for the specific energy consumption rate, inferred from substrate consumption and heat evolution, was observed over the entire viable temperature range. By combining this relationship for specific energy consumption rates and observed specific growth rates, the model describes an increase in nongrowth associated maintenance at higher temperatures and the corresponding decrease in energy available for growth. This analytical and thermodynamic formulation suggests that simply monitoring heat evolution in batch culture could be a useful complement to the recognized limitations of estimating maintenance using extrapolation to zero growth in chemostats.

An Experimental Approach to Assessing the Roles of Magnesium, Calcium, and Carbonate Ratios in Marine Carbonates

Marine biomineralization is a globally important biological and geochemical process. Understanding the mechanisms controlling the precipitation of calcium carbonate [CaCO3] within the calcifying fluid of marine organisms, such as corals, crustose coralline algae, and foraminifera, presents one of the most elusive, yet relevant areas of biomineralization research, due to the often-impenetrable ability to measure the process in situ. The precipitation of CaCO3 is assumed to be largely controlled by the saturation state [Ω] of the extracellular calcifying fluid. In this study, we mimicked the typical pH and Ω known for the calcifying fluid in corals, while varying the magnesium, calcium, and carbonate concentrations in six chemo-static growth experiments, thereby mimicking various dissolved inorganic carbon concentration mechanisms and ionic movement into the extracellular calcifying fluid. Reduced mineralization and varied CaCO3 morphologies highlight the inhibiting effect of magnesium regardless of pH and Ω and suggests the importance of strong magnesium removal or calcium concentration mechanisms. In respect to ocean acidification studies, this could allow an explanation for why specific marine calcifiers respond differently to lower saturation states.

Enhanced production of heterologous proteins by a synthetic microbial community: Conditions and trade-offs.

Synthetic microbial consortia have been increasingly utilized in biotechnology and experimental evidence shows that suitably engineered consortia can outperform individual species in the synthesis of valuable products. Despite significant achievements, though, a quantitative understanding of the conditions that make this possible, and of the trade-offs due to the concurrent growth of multiple species, is still limited. In this work, we contribute to filling this gap by the investigation of a known prototypical synthetic consortium. A first E. coli strain, producing a heterologous protein, is sided by a second E. coli strain engineered to scavenge toxic byproducts, thus favoring the growth of the producer at the expense of diverting part of the resources to the growth of the cleaner. The simplicity of the consortium is ideal to perform an in depth-analysis and draw conclusions of more general interest. We develop a coarse-grained mathematical model that quantitatively accounts for literature data from different key growth phenotypes. Based on this, assuming growth in chemostat, we first investigate the conditions enabling stable coexistence of both strains and the effect of the metabolic load due to heterologous protein production. In these conditions, we establish when and to what extent the consortium outperforms the producer alone in terms of productivity. Finally, we show in chemostat as well as in a fed-batch scenario that gain in productivity comes at the price of a reduced yield, reflecting at the level of the consortium resource allocation trade-offs that are well-known for individual species.

Evolving Populations in Biofilms Contain More Persistent Plasmids.

Bacterial plasmids substantially contribute to the rapid spread of antibiotic resistance, which is a crisis in healthcare today. Coevolution of plasmids and their hosts promotes this spread of resistance by ameliorating the cost of plasmid carriage. However, our knowledge of plasmid–bacteria coevolution is solely based on studies done in well-mixed liquid cultures, even though biofilms represent the main way of bacterial life on Earth and are responsible for most infections. The spatial structure and the heterogeneity provided by biofilms are known to lead to increased genetic diversity as compared with well-mixed liquids. Therefore, we expect that growth in this complex environment could affect the evolutionary trajectories of plasmid–host dyads. We experimentally evolved Shewanella oneidensis MR-1 with plasmid pBP136Gm in biofilms and chemostats and sequenced the genomes of clones and populations. Biofilm populations not only maintained a higher diversity of mutations than chemostat populations but contained a few clones with markedly more persistent plasmids that evolved via multiple distinct trajectories. These included the acquisition of a putative toxin–antitoxin transposon by the plasmid and chromosomal mutations. Some of these genetic changes resulted in loss of plasmid transferability or decrease in plasmid cost. Growth in chemostats led to a higher proportion of variants with decreased plasmid persistence, a phenomenon not detected in biofilms. We suggest that the presence of more stable plasmid–host dyads in biofilms reflects higher genetic diversity and possibly unknown selection pressures. Overall, this study underscores the importance of the mode of growth in the evolution of antibiotic-resistant bacteria.

Optimal proteome allocation strategies for phototrophic growth in a light-limited chemostat

BackgroundCyanobacteria and other phototrophic microorganisms allow to couple the light-driven assimilation of atmospheric text {CO}_{2} directly to the synthesis of carbon-based products, and are therefore attractive platforms for microbial cell factories. While most current engineering efforts are performed using small-scale laboratory cultivation, the economic viability of phototrophic cultivation also crucially depends on photobioreactor design and culture parameters, such as the maximal areal and volumetric productivities. Based on recent insights into the cyanobacterial cell physiology and the resulting computational models of cyanobacterial growth, the aim of this study is to investigate the limits of cyanobacterial productivity in continuous culture with light as the limiting nutrient.ResultsWe integrate a coarse-grained model of cyanobacterial growth into a light-limited chemostat and its heterogeneous light gradient induced by self-shading of cells. We show that phototrophic growth in the light-limited chemostat can be described using the concept of an average light intensity. Different from previous models based on phenomenological growth equations, our model provides a mechanistic link between intracellular protein allocation, population growth and the resulting reactor productivity. Our computational framework thereby provides a novel approach to investigate and predict the maximal productivity of phototrophic cultivation, and identifies optimal proteome allocation strategies for developing maximally productive strains.ConclusionsOur results have implications for efficient phototrophic cultivation and the design of maximally productive phototrophic cell factories. The model predicts that the use of dense cultures in well-mixed photobioreactors with short light-paths acts as an effective light dilution mechanism and alleviates the detrimental effects of photoinhibition even under very high light intensities. We recover the well-known trade-offs between a reduced light-harvesting apparatus and increased population density. Our results are discussed in the context of recent experimental efforts to increase the yield of phototrophic cultivation.

On Adaptive Estimation of Bacterial Growth in the Competitive Chemostat

In this paper, the problem of state estimation of a bioreactor containing a single substrate and several competing species is studied. This scenario is well-known as a competition model, in which multiple species compete for the same nutrient. The goal is to estimate concentrations of the substrate and all species, considering the concentration of total biomass as measurement. To address such a challenge, the estimation scheme relies in the coupling of two estimation techniques: an asymptotic observer, which takes advantage of the model structure being independent of its kinetics, and a finite-time parameter estimation technique, which also drops the usual requirement of persistence of excitation.

The impact of growth rate and environmental factors on mutation rates and spectra in Escherichia coli.

Genetic variation in bacterial populations is remarkably sensitive to environmental influences, including simple, nutritional differences. Not only the rate but also the kind of mutational changes is biased by the nutritional state of bacteria. Here we investigate the mutational consequences of a universal variable for free-living bacteria, namely the growth rate. By controlling growth in chemostats, the rate and mix of mutations was investigated for populations of Escherichia coli subject to different specific growth rates. Both aerobic and anaerobic cultures were compared with see if growth rate is a factor in the commonest respiratory conditions for E. coli. We find mutation rates are raised markedly with decreasing growth rate. Base pair substitutions and 1-bp insertions and deletions increase with reduced growth rate, but less so in anaerobic cultures. Insertion sequence movements are particularly sensitive to growth rate, with IS2 being optimal at intermediate growth rates whereas IS1 and IS150 movements are highest at the slowest tested growth rate. A comprehensive comparison of growth rate effects, as well as six other environmental factors, provides the most complete picture yet of the range of mutational signatures in bacterial genetic variation.

Guanine Limitation Results in CodY-Dependent and -Independent Alteration of Staphylococcus aureus Physiology and Gene Expression.

In Staphylococcus aureus, the global transcriptional regulator CodY modulates the expression of hundreds of genes in response to the availability of GTP and the branched-chain amino acids isoleucine, leucine, and valine (ILV). CodY DNA-binding activity is high when GTP and ILV are abundant. When GTP and ILV are limited, CodY's affinity for DNA drops, altering expression of CodY-regulated targets. In this work, we investigated the impact of guanine nucleotides (GNs) on S. aureus physiology and CodY activity by constructing a guaA null mutant (ΔguaA strain). De novo biosynthesis of guanine monophosphate is abolished due to the guaA mutation; thus, the mutant cells require exogenous guanosine for growth. We also found that CodY activity was reduced when we knocked out guaA, activating the Agr two-component system and increasing secreted protease activity. Notably, in a rich, complex medium, we detected an increase in alternative sigma factor B activity in the ΔguaA mutant, which results in a 5-fold increase in production of the antioxidant pigment staphyloxanthin. Under biologically relevant flow conditions, ΔguaA cells failed to form robust biofilms when limited for guanine or guanosine. Transcriptome sequencing (RNA-Seq) analysis of the S. aureus transcriptome during growth in guanosine-limited chemostats revealed substantial CodY-dependent and -independent alterations of gene expression profiles. Importantly, these changes increase production of proteases and δ-toxin, suggesting that S. aureus exhibits a more invasive lifestyle when limited for guanosine. Further, gene products upregulated under GN limitation, including those necessary for lipoic acid biosynthesis and sugar transport, may prove to be useful drug targets for treating Gram-positive infections.IMPORTANCEStaphylococcus aureus infections impose a serious economic burden on health care facilities and patients because of the emergence of strains resistant to last-line antibiotics. Understanding the physiological processes governing fitness and virulence of S. aureus in response to environmental cues is critical for developing efficient diagnostics and treatments. De novo purine biosynthesis is essential for both fitness and virulence in S. aureus since inhibiting production cripples S. aureus's ability to cause infection. Here, we corroborate these findings and show that blocking guanine nucleotide synthesis severely affects S. aureus fitness by altering metabolic and virulence gene expression. Characterizing pathways and gene products upregulated in response to guanine limitation can aid in the development of novel adjuvant strategies to combat S. aureus infections.

Prodigiosin pigment of Serratia marcescens is associated with increased biomass production.

Serratia marcescens is a gram-negative, facultatively-anaerobic bacterium and opportunistic pathogen which produces the red pigment prodigiosin. We employed both batch culture and chemostat growth methods to investigate prodigiosin function in the producing organism. Pigmentation correlated with an increased rate of ATP production during population lag phase. Results with a lacZ transcriptional fusion to the prodigiosin (pig) biosynthetic operon revealed that operon transcription is activated by low cellular levels of ATP at high cell density. Furthermore, these data enabled estimation of the ATP per cell minimum value at which the operon is induced. Pigmented cells were found to accumulate ATP more rapidly and to multiply more quickly than non-pigmented cells during the high density growth phase. Finally, results with both batch and chemostat culture revealed that pigmented cells grow to approximately twice the biomass yield as non-pigmented S. marcescens bacteria. Prodigiosin production may, therefore, provide a growth advantage at ambient temperatures.

Experimental Evolution Reveals Favored Adaptive Routes to Cell Aggregation in Yeast.

Yeast flocculation is a community-building cell aggregation trait that is an important mechanism of stress resistance and a useful phenotype for brewers; however, it is also a nuisance in many industrial processes, in clinical settings, and in the laboratory. Chemostat-based evolution experiments are impaired by inadvertent selection for aggregation, which we observe in 35% of populations. These populations provide a testing ground for understanding the breadth of genetic mechanisms Saccharomyces cerevisiae uses to flocculate, and which of those mechanisms provide the biggest adaptive advantages. In this study, we employed experimental evolution as a tool to ask whether one or many routes to flocculation are favored, and to engineer a strain with reduced flocculation potential. Using a combination of whole genome sequencing and bulk segregant analysis, we identified causal mutations in 23 independent clones that had evolved cell aggregation during hundreds of generations of chemostat growth. In 12 of those clones, we identified a transposable element insertion in the promoter region of known flocculation gene FLO1, and, in an additional five clones, we recovered loss-of-function mutations in transcriptional repressor TUP1, which regulates FLO1 and other related genes. Other causal mutations were found in genes that have not been previously connected to flocculation. Evolving a flo1 deletion strain revealed that this single deletion reduces flocculation occurrences to 3%, and demonstrated the efficacy of using experimental evolution as a tool to identify and eliminate the primary adaptive routes for undesirable traits.

Programmable, Pneumatically Actuated Microfluidic Device with an Integrated Nanochannel Array To Track Development of Individual Bacteria

We describe a microfluidic device with an integrated nanochannel array to trap individual bacteria and monitor growth and reproduction of lineages over multiple generations. Our poly(dimethylsiloxane) device comprises a pneumatically actuated nanochannel array that includes 1280 channels with widths from 600 to 1000 nm to actively trap diverse bacteria. Integrated pumps and valves perform on-chip fluid and cell manipulations that provide dynamic control of cell loading and nutrient flow, permitting chemostatic growth for extended periods of time (typically 12 to 20 h). Nanochannels confine bacterial growth to a single dimension, facilitating high-resolution, time-lapse imaging and tracking of individual cells. We use the device to monitor the growth of single bacterial cells that undergo symmetric (Bacillus subtilis) and asymmetric (Caulobacter crescentus) division and reconstruct their lineages to correlate growth measurements through time and among related cells. Furthermore, we monitor the motility state of single B. subtilis cells across multiple generations by the expression of a fluorescent reporter protein and observe that the state of the epigenetic switch is correlated over five generations. Our device allows imaging of cellular lineages with high spatiotemporal resolution to facilitate the analysis of biological processes spanning multiple generations.

Solids Retention Time Dependent Phototrophic Growth and Population Changes in Chemostat Cultivation Using Wastewater

There has been renewed interest in using algae for wastewater polishing and treatment in recent years. Because solids retention time (SRT) is a key design and operating parameter in bioreactor operation, this research determined the effect of SRT on phototrophic growth and microbial population dynamics in continuous-flow chemostat systems. There was a unique feature of phototrophic growth that differed from chemotrophic growth in chemostat. It was found that the phototrophic biomass concentration increased proportionally as SRT increased from 3 to 9 days. Regardless of the change in SRT, a step function model was successfully applied with the predicted phototrophic production rate of 4.5 ± 0.9 g/m(2)·d at the light intensity of 68.5 μmol/m(2)·s. Even though the continuous-flow systems were initially seeded with a 1:1 mixture of green algae and cyanobacteria, Chlorella vulgaris always dominated (98%) in the continuous-flow chemostat systems under steady-state conditions.

Mycobacterium tuberculosis Is Resistant to Isoniazid at a Slow Growth Rate by Single Nucleotide Polymorphisms in katG Codon Ser315.

An important aim for improving TB treatment is to shorten the period of antibiotic therapy without increasing relapse rates or encouraging the development of antibiotic-resistant strains. In any M. tuberculosis population there is a proportion of bacteria that are drug-tolerant; this might be because of pre-existing populations of slow growing/non replicating bacteria that are protected from antibiotic action due to the expression of a phenotype that limits drug activity. We addressed this question by observing populations of either slow growing (constant 69.3h mean generation time) or fast growing bacilli (constant 23.1h mean generation time) in their response to the effects of isoniazid exposure, using controlled and defined growth in chemostats. Phenotypic differences were detected between the populations at the two growth rates including expression of efflux mechanisms and the involvement of antisense RNA/small RNA in the regulation of a drug-tolerant phenotype, which has not been explored previously for M. tuberculosis. Genotypic analyses showed that slow growing bacilli develop resistance to isoniazid through mutations specifically in katG codon Ser315 which are present in approximately 50–90% of all isoniazid-resistant clinical isolates. The fast growing bacilli persisted as a mixed population with katG mutations distributed throughout the gene. Mutations in katG codon Ser315 appear to have a fitness cost in vitro and particularly in fast growing cultures. Our results suggest a requirement for functional katG-encoded catalase-peroxide in the slow growers but not the fast-growing bacteria, which may explain why katG codon Ser315 mutations are favoured in the slow growing cultures.

Growth retardation of Escherichia coli by artificial increase of intracellular ATP.

Overexpression of phosphoenolpyruvate carboxykinase (PCK) was reported to cause the harboring of higher intracellular ATP concentration in Escherichia coli, accompanied with a slower growth rate. For systematic determination of the relationship between the artificial increase of ATP and growth retardation, PCKWT enzyme was directly evolved in vitro and further overexpressed. The evolved PCK67 showed a 60% greater catalytic efficiency than that of PCKWT. Consequently, the PCK67-overexpressing E. coli showed the highest ATP concentration at the log phase of 1.45 μmol/gcell, with the slowest growth rate of 0.66 h(-1), while the PCKWT-overexpressing cells displayed 1.00 μmol/gcell ATP concentration with the growth rate of 0.84 h(-1) and the control had 0.28 μmol/gcell with 1.03 h(-1). To find a plausible reason, PCK-overexpressing cells in a steady state during chemostat growth were applied to monitor intracellular reactive oxygen species (ROS). Higher amount of intracellular ROS were observed as the ATP levels increased. To confirm the hypothesis of slower growth rate without perturbation of the carbon flux by PCK-overexpression, phototrophic Gloeobacter rhodopsin (GR) was expressed. The GR-expressing strain under illumination harbored 81% more ATP concentration along with 82% higher ROS, with a 54% slower maximum growth rate than the control, while both the GR-expressing strain under dark and dicarboxylate transporter (a control membrane protein)-expressing strain showed a lower ATP and increased ROS, and slower growth rate. Regardless of carbon flux changes, the artificial ATP increase was related to the ROS increase and it was reciprocally correlated to the maximum growth rate. To verify that the accumulated intracellular ROS were responsible for the growth retardation, glutathione was added to the medium to reduce the ROS. As a result, the growth retardation was restored by the addition of 0.1 mM glutathione. Anaerobic culture even enabled the artificial ATP-increased E. coli to grow faster than control. Collectively, it was concluded that artificial ATP increases inhibit the growth of E. coli due to the overproduction of ROS.

Physiological, biomass elemental composition and proteomic analyses of Escherichia coli ammonium-limited chemostat growth, and comparison with iron- and glucose-limited chemostat growth.

Escherichia coli physiological, biomass elemental composition and proteome acclimations to ammonium-limited chemostat growth were measured at four levels of nutrient scarcity controlled via chemostat dilution rate. These data were compared with published iron- and glucose-limited growth data collected from the same strain and at the same dilution rates to quantify general and nutrient-specific responses. Severe nutrient scarcity resulted in an overflow metabolism with differing organic byproduct profiles based on limiting nutrient and dilution rate. Ammonium-limited cultures secreted up to 35% of the metabolized glucose carbon as organic byproducts with acetate representing the largest fraction; in comparison, iron-limited cultures secreted up to 70 % of the metabolized glucose carbon as lactate, and glucose-limited cultures secreted up to 4% of the metabolized glucose carbon as formate. Biomass elemental composition differed with nutrient limitation; biomass from ammonium-limited cultures had a lower nitrogen content than biomass from either iron- or glucose-limited cultures. Proteomic analysis of central metabolism enzymes revealed that ammonium- and iron-limited cultures had a lower abundance of key tricarboxylic acid (TCA) cycle enzymes and higher abundance of key glycolysis enzymes compared with glucose-limited cultures. The overall results are largely consistent with cellular economics concepts, including metabolic tradeoff theory where the limiting nutrient is invested into essential pathways such as glycolysis instead of higher ATP-yielding, but non-essential, pathways such as the TCA cycle. The data provide a detailed insight into ecologically competitive metabolic strategies selected by evolution, templates for controlling metabolism for bioprocesses and a comprehensive dataset for validating in silico representations of metabolism.

Investigating the physiological response of Pichia (Komagataella) pastoris GS115 to the heterologous expression of misfolded proteins using chemostat cultures.

Pichia pastoris is widely used as a host system for heterologous protein expression in both academia and industry. Production is typically accomplished by a fed-batch induction process that is known to have negative impacts on cell physiology that impose limits on both protein yields and quality. We have analysed recombinant protein production in chemostat cultures to understand the physiological responses associated with methanol-induced production of two human lysozyme variants with different degrees of misfolding by P. pastoris. Confounding variables associated with nutrient stress or growth-rate are minimised during steady-state growth in chemostats. Comparison of transcriptome-level data obtained during the non-inducing and inducing steady states identified changes in expression of only about 1 % of the genome during production of either an amyloidogenic human lysozyme variant prone to intracellular aggregation (I56T) or a misfolded but secretable variant (T70N), indicating near-complete acclimation to their production. A marked, but temporary, stress response involving both the unfolded protein response (UPR) and ER-associated degradation pathway was observed during the transient between steady states, particularly following induction of the T70N variant synthesis, and was accompanied by changes in expression of around 50 antisense transcripts. The results suggest that optimal heterologous protein production could best be achieved by a continuous process that minimises the number of methanol-induced transients experienced by the cultures. The processing of HAC1 mRNA required for the UPR was found to be constitutive in the culture conditions used, even in the absence of recombinant protein induction.Electronic supplementary materialThe online version of this article (doi:10.1007/s00253-013-5186-1) contains supplementary material, which is available to authorized users.