Bioreactor Research Articles

Chemical reaction influence on nanofluid flow in a porous layer: Stability analysis

An influence of exothermic chemical reaction on the natural thermo-solutal convection in a horizontal channel filled with sparsely packed permeable nanofluid is investigated. It is assumed that the fluid viscosity is different from the effective viscosity. The Brinkman approach is engaged for the porous material, while the nanofluid approach features the Buongiorno model. To figure out the stability of the linear terms, normal mode analysis is opted. Galerkin technique is selected to work out the nonlinear terms. The Rayleigh number and its relevant wave numbers are assessed for all dimensionless parameters and exposed in the form of graphs. It is found that there is a critical value of Frank–Kamenetskii number at which the system is most unstable. It is found that increasing the viscosity ratio delays the onset of convection. With exothermic chemical reactions, the fluid in the porous medium is more prone to instability as compared to the case in which chemical reactions are absent. A critical value of Frank–Kamenetskii number is also identified at which the system is most unstable, and this is shown to be independent of both porous media parameter and the viscosity ratio. Applications of the study arise in nano-doped geothermal energy extraction, chemical and bio reactors and other engineering systems.

Multiobjective Optimization of a Fed-Batch Bienzymatic Reactor for Mannitol Production

Enzymatic reactions can successfully replace complex chemical syntheses using milder reaction conditions and generating less waste. The developed model-based numerical analysis turned out to be a beneficial tool to determine the optimal operating policies of complex multienzymatic reactors. As proved, for such cases, the determination of a Fed-Batch Reactor (FBR) optimal operating policy results in a difficult multiobjective optimization problem. Exemplification is made for the bienzymatic reduction of D-fructose to mannitol by using MDH (mannitol dehydrogenase) and nicotinamide adenine dinucleotide (NADH) cofactor with the in situ continuous regeneration of NADH at the expense of formate degradation in the presence of FDH (formate dehydrogenase). For such a coupled system, the model-based engineering evaluations must account for multiple competing (opposable) optimization objectives. Among the multiple novelty elements: i) an optimally operated FBR with a tightly controlled variable feeding (of the time stepwise type) during the batch can lead to higher performance; ii) the optimally operated FBR reported better performance compared to an optimally single or cyclic BR, or to optimally serial batch-to-batch reactors (SeqBR), when considering a multiobjective optimization; iii) the concomitant variable feeding with substrate, enzymes, and cofactor during the FBR “time-arcs” is an option seldom approached in the literature but which is proved here, leading to consistent economic benefits.

Kinetic Investigations of the Electrochemically Assisted Leaching of Metals for the Recycling of Photovoltaics

To describe and predict the leaching of Ag, Cu and Sn from waste photovoltaic modules with an electrochemical-assisted process kinetic investigations were performed. In this process, peroxydisulfate is generated from sulfuric acid to oxidize metals. It was found that under the reaction conditions peroxymonosulfate is formed as well and has a major contribution to the leaching process. For Ag, autocatalytic decomposition of the leaching reagents is determined to be a limiting step while for Sn passivation influences the process. The leaching is modeled for three different reaction types, a batch reaction, the reaction in a static H-cell with continuous generation of S₂O₈2− and a fed-batch reaction with an electrochemical flow cell for the production of peroxydisulfate.

Enzymatic synthesis of geranyl acetate in batch and fed-batch reactors and evaluation of its larvicidal activity against Rhipicephalus (Boophilus) microplus

Geraniol esters, such as geranyl acetate, are known to have antimicrobial and insect-repellent action, but their acaricidal activity has been little explored. This study aimed to investigate the solvent-free synthesis of geranyl acetate in batch (BSTR) and fed-batch (FBSTR) stirred tank reactors using the immobilized lipases NS 88011 and Novozym 435 and evaluate its larvicidal activity against the cattle tick Rhipicephalus (Boophilus) microplus. A central composite design (CCD) was used to maximize the synthesis of geranyl acetate in a BSTR. The independent variables were temperature reaction (40–60 °C), acetic acid/geraniol molar ratio (1:1–1:3), and enzyme loading (1–5% w/w in relation to the substrates). Novozym 435 yielded the best results, achieving 99% of geranyl acetate conversion using 1:3 acetic acid/geraniol molar ratio, 60 °C and 5% (w/w) of enzyme loading after 1 h of reaction in a BSTR. In the FBSTR system, a high content of geranyl acetate was obtained (about 80%) in 4 h of reaction using a 1:1 acetic acid/geraniol molar ratio, 60 °C and 5% (w/w) Novozym 435. Geranyl acetate showed potential as an acaricide, with an LC90 of 6.25 mg/mL and 100% mortality at a concentration of 12.5 mg/mL.

Life cycle cost analysis of wastewater treatment technologies

With the ever-increasing population, volumes of wastewater treatment are a major concern in our country. The Activated Sludge Process (ASP), Biological Filtration and Oxygenated Reactor (BIOFOR), Upflow Anaerobic Sludge Blanket (UASB), and Moving Bed Bio Reactor (MBBR) are all monetarily investigated in the present study using the Life Cycle Cost Assessment (LCCA) tool. In this study, life cycle costing is done using the present value method, which involves discounting the costs for a 20-year economic life. The costs of treating wastewater per million litres per day (MLD) of wastewater treatment technology are obtained from the literature. Moreover, this study takes into account the capital, annual operation, energy, salvage, and replacement costs to compare the life cycle costs of ASP, UASB, BIOFOR, and MBBR to make the best guess of an economical technology. The LCCA demonstrates that the MBBR has the highest costs of treatment, resulting in the highest Life Cycle Cost (LCC). BIOFOR has the largest energy requirement making LCC the second-highest among the technologies. In India, ASP is one of the most widely used technologies, whose LCC is the third most advanced of the four technologies. Because of its lower energy and operating costs, UASB has the lowest LCC.

Fed-batch anaerobic digestion of raw and pretreated hazelnut skin over long-term operation

This study provided important insights on the anaerobic digestion (AD) of hazelnut skin (HS) by operating a fed-batch AD reactor over 240 days and focusing on several factors impacting the process in the long term. An efficient reactor configuration was proposed to increase the substrate load while reducing the solid retention time during the fed-batch AD of HS. Raw HS produced maximally 19.29 mL CH4/g VSadd/d. Polyphenols accumulated in the reactor and the use of NaOH to adjust the pH likely inhibited AD. Maceration and methanol-organosolv pretreatments were, thus, used to remove polyphenols from HS (i.e. 82 and 97%, respectively) and improve HS biodegradation. Additionally, organosolv pretreatment removed 9% of the lignin. The organosolv-pretreated HS showed an increment in methane potential of 21%, while macerated HS produced less methane than the raw substrate, probably due to the loss of non-structural sugars during maceration.

Customized Woven Carbon Fiber Electrodes for Bioelectrochemical Systems—A Study of Structural Parameters

Commercial carbon fiber (CF) fabrics are popular electrode materials for bioelectrochemical systems (BES), but are usually not optimized for the specific application. This study investigates BES-relevant material characteristics on fabric level, such as weave types and weave parameters. The two contrasting weave types plain and leno weave were characterized with respect to their envisaged application types: 1) BES with mainly advective flow regimes and 2) stirred systems, which could benefit from fluid flow through a fabric electrode. Experiments with batch and continuously fed pure cultures of Geobacter sulfurreducens PCA and Shewanella oneidensis MR-1 reveal that µm-scale electrode topologies are of limited use for the thick biofilms of G. sulfurreducens, but can boost S. oneidensis’ current generation especially in batch and fed-batch reactors. For advective flow regimes, deeper layers of biofilm inside microporous electrodes are often mass transport limited, even with thin biofilms of S. oneidensis. Therefore, low porosity plain weave electrodes for advective flow operation as in wastewater treating BES should be thin and flat. A trade-off between maximized current density and electrode material utilization exists, which is optimized exemplarily for an advective flow operation. For stirred BES of biotechnological applications, a flow-through of electrolyte is desired. For this, leno weave fabrics with pores at cm-scale are produced from 100% CF for the first time. In a preliminary evaluation, they outperform plain weave fabrics. Mass transfer investigations in stirred BES demonstrate that the large pores enable efficient electrode utilization at lower power input in terms of stirring speed.

Design and Optimization of a Penicillin Fed-Batch Reactor Based on a Deep Learning Fault Detection and Diagnostic Model

Design and Optimization of a Penicillin Fed-Batch Reactor Based on a Deep Learning Fault Detection and Diagnostic Model

Heterogeneity of CH4-derived carbon induced by O2:CH4 mediates the bacterial community assembly processes

The mechanism by which O2:CH4 controls microbial community assembly in the process of aerobic methane oxidation coupled to denitrification (AMED) remains largely uncharacterized, which hinders the design of engineering microbiomes for the AMED. In this study, the changes in the bacterial community in fed-batch serum bottle reactors under different O2:CH4 ratios were systematically characterized. The ratios of CH4 consumption to the amount of nitrate removal in the treatment with O2:CH4 = 1.5:1, O2:CH4 = 0.5:1, and O2:CH4 = 0.25:1 were 13.1 ± 3.4, 4.7 ± 1.1, and 5.9 ± 3.0 mol-CH4 mol−1-NO3−, respectively. The α-diversity of the bacterial community increased as O2:CH4 decreased. Significantly different selection patterns were found for the high and low O2:CH4 ratios. The coherence process dominated the selection at high O2:CH4 ratios, while the diversification process played a role when O2:CH4 was low. Differences were also observed in the composition of CH4-derived carbon between treatments with O2:CH4 = 1.5:1 and O2:CH4 = 0.5:1. Compared with the treatments with O2:CH4 = 1.5:1, the concentrations of methanol, formaldehyde, acetate, and ethanol in the treatment with O2:CH4 = 0.5:1 were significantly higher, while the concentration of formate was significantly lower. The heterogeneity of CH4-derived carbon induced by O2:CH4 was likely to be responsible for the differences in the selection patterns. Our findings bridge the gaps between the observations of bacterial community perturbations and ecological community assembly theories, highlighting the potential of the bottom-up design approach to improve the nitrate removal rate of the AME-D.

Sustainable development of environment and energy aspects of methane fermentation on family farms in Poland

The goal of these study was to present results of investigation concerning possibilities of utilization of harmful wastes in countryside area to produce ecological energy. Biogas production can be important from the point of view of environment protection especially in case of overproduction of animal wastes. Production and utilization of energy from agriculture residues gives a great chance for diversification and grows of income for family farms. Besides energetic and environment gains, we can obtain very valuable fertilizer, which is easy absorbed by plants in field crop production. The experimental study was conducted to investigate the effect of mixing process on the parameters of methane fermentation process. Temperature inside fermentation chamber, pH of fermented material, redox potential and carbon to nitrogen ratio (C:N) were investigated. Utilize wastes from pig and poultry houses were used for the study. Digestion in a chamber was provided at constant temperature of 37 °C. After adding fresh substrate to the digester, the temperature of the raw material decreased by 1,0-1.5 °C depending on the location in the tank. Also, it was observed that biogas production decreased. The mixing process had a positive effect on the homogeneity of the material throughout the digester volume. The best results for biogas production were obtained when the pH value was 7.0. Research results obtained from tested biogas installation show, that from two bio reactors at total capacity of 410 m3 we can get electrical energy at cost of 34,52 € MWh-1 and thermal energy at cost of 62,54 €×MWh-1. While the cost of producing electricity in a professional power plant based on lignite was 76.23 €MWh-1. The energy produced was used for the operational activities of the farm.

From sucrose to fructo-oligosaccharides: Production and purification of fructo-oligosaccharides by an integrated enzymatic catalysis and membrane separation process

Fructo-oligosaccharides (FOS) are a group of linear fructose oligomers with numerous health benefits, which can be synthesized by the transfructosylation of sucrose using fructosyltransferases (FTases). In this study, it was found that the selected FTases showed both hydrolytic and transfructosylating activities. For FOS production, the hydrolytic activities of FTases became significant when the reaction time was above 1 h. Thus, FTases needed to be inactivated or removed from the reactor after achieving the highest FOS conversion. We therefore developed an integrated ultrafiltration-diafiltration- concentration process for FTases reuse and FOS purification. Because of the 100% rejection of FTases activity and a higher average flux, a PES-10 ultrafiltration membrane was selected for FTases recovery, where the cake layer formation was the main membrane fouling mechanism. Then, a fed-batch reaction process was developed for FOS production, where the hydrolytic behavior of FTases was negligible, and the FTases dosage was reduced by 75% compared to single batch operation (four batches). Moreover, the FOS production was comparable, and 95.5% FTases was recovered by the PES10 membrane after the four fed-batches processes. Subsequently, a NF5 nanofiltration membrane was selected to further separate FOS from the FTases-free FOS solution by a constant volume diafiltration process under 45 °C, and the monosaccharides in the reaction mixture (glucose and fructose) were completely removed, increasing the FOS purity from 55.8% to 92.3%. Finally, the FOS and sucrose in the diafiltration permeate was recovered by a DL nanofiltration membrane under 60 °C. The outcome of this work offers a complete technical route for FOS production using sucrose as substrate.

Performance evaluation of textile wastewater treatment techniques using sustainability index: An integrated fuzzy approach of assessment

The Textile Wastewater Treatment Technique (TWWTT) plays a prominent role in reducing effluent contaminants and thus keeping the environment cleaner. To select appropriate technology, evaluating the unified performance of TWWTTs towards social, technical, economic, and environmental sustainability parameters is of utmost importance. Though there are TWWTTs available, no ready framework exists that can help decision-makers choose the appropriate technology based on their requirement. The study proposes a novel and systematic decision-making framework and a comprehensive mathematical model for judiciously selecting the TWWTT. It integrates fuzzy Delphi and hybrid Fuzzy Analytical Hierarchy Process (FAHP) approach of Multi-Criteria Decision Making (MCDM). A total of 38 sub-indicators of sustainability are identified from past studies and expert opinions. Fuzzy Delphi is applied to identify the essential sub-indicators of sustainability, and hybrid FAHP is used to rank the sustainability dimensions, key sub-indicators, and alternatives. Twenty-eight sub-indicators are rounded down from the initial 38. The results from hybrid FAHP indicate that the technical dimension of sustainability is of paramount importance while selecting the TWWTTs followed by the economic dimension. The key sub-indicators for the selection of TWWTTs that scored higher than the others in technical, economic, social, and environmental aspects respectively are as under (i) color removal efficiency, COD removal efficiency, and quantity of sludge generation; (ii) cost of construction, operation, and maintenance; (iii) awareness within textile industries, public safety; (iv) effluent suitability for reuse and space requirements. The five TWWTTs, namely, Activated Sludge Process (ASP), Membrane Biological Reactor (MBR), Electrochemical Coagulation (EC), Mixed Bed Bio Reactor (MBBR), and the Rotating Biological Contactors (RBC), are compared using the estimated entropy weights also called as sustainability indices. MBR has scored the highest sustainability index value, and ASP has the least value. MBR, EC and MBBR have higher sustainability indices proving them better sustainable alternatives than ASP and RBC. The MBR permeate quality is good enough to be reused in the textile industry without any further treatment. This will reduce the effluent quantity and groundwater demand leading to cleaner production of textile. The study will help the decision-makers in the overall assessment of the sustainability of TWWTTs prior to selection.

Integration of transepithelial electrical resistance (TEER) measurement in the corneal bioreactor

AbstractPurposeThe corneal bioreactor (BR) patented by our laboratory allows to restore a physiological epithelium of porcine or human corneas. The measurement of the transepithelial electrical resistance (TEER), correlated with the presence of tight junctions, is a robust parameter for assessing the integrity of the corneal epithelium.AimTo integrate the measurement of TEER with BR and to evaluate its relevance on porcine corneas.MethodsTwo Ag/AgCl electrodes (WPI, Saratosa, FL) were placed, one in an endothelial chamber filled with Corneamax storage medium (Eurobio, Les Ulis, France), the other in an epithelial chamber and under 3 conditions: immersed in the conservation medium; immersed in the conservation medium while isolating the cornea from the sclera thanks to an applicator cone usually used for iontophoresis (Opia Technologies, Paris, France); in direct contact with the epithelium with interposition of Gel‐larmes (Théa, Clermont‐Ferrand, France). The electric current frequencies tested ranged from 50 Hz to 1 MHz. Three fresh pig corneas (with and without epithelium) were placed in BR and the TEER measured in the 3 conditions, with and without epithelium. Controls: Fresh pig sclera and Corneamax alone in BR.ResultsResults are expressed as median. TEER of controls: 302 Ω (sclera) and 266 Ω (Corneamax alone) TEER of corneas without epithelium: 321 Ω, in the 3 experimental conditions, thus close to the control. TEER of corneas with epithelium: 397 Ω with electrode immersed in the medium, thus close to the sclera (397 Ω). In the other 2 conditions, the TEER was higher, with a greater variability, depending on the current frequency: from 1456 Ω to 143 Ω with the applicator cone and from 2335 Ω to 242 Ω with the Gel‐Larmes.ConclusionsThe sclera placed in BR is a conductive tissue. It is possible to determine the TEER of a cornea ex vivo in the BR, if the cornea is isolated from the sclera and the epithelium present on the cornea is healthy.

Optimal control of renewable biohydrogen production: A switched system approach*

Biohydrogen produced from microorganisms such as cyanobacteria is a promising low cost, sustainable and environmentally friendly energy source. Recent studies have shown that high biohydrogen yield can be obtained from Cyanothece sp. ATCC 51142 in a fed-batch reactor. This system has been accurately described with a modified Droop model that can be used for optimization studies. Searching for the optimal operating conditions and the switching time from batch to fed-batch operation, such that the biohydrogen production is maximized, leads to a challenging singular optimal control problem. In this study, a novel reformulation based on the theory of switched systems and time-scaling transformation is proposed to address the switching of the operating modes and the optimal control structure. Solutions are found by solving an embedded optimal control problem that can be solved efficiently as a nonlinear programming problem. No mesh refinement is required to capture the switching times. Smooth optimal control profiles and clear switching structures that maximize the biohydrogen yield were found for two types of control parametrization.

Use of the corneal bioreactor to evaluate the anti‐edema effect and the ulcer healing rate between two commercial hyperosmotic eye drops: ODM5 and Muro 128

AbstractPurposeThe corneal bioreactor (BR) in our laboratory, with an intraocular pressure (IOP) of 21 mmHg and the renewal of the culture medium, allows to maintain the homeostasis of the 3 layers of the cornea. We showed that an IOP reduced to 10 mmHg allowed to simulate a corneal edema (760 ± 57 µm) with a healthy epithelium.AimTo compare 2 hyperosmolar eye drops in the evolution of corneal deturgescence and epithelial ulcer healing: ODM5 composed mainly of NaCl 5% and hyaluronic acid (Horus Pharma, Saint‐Laurent‐du‐Var, France) and Muro 128 composed mainly of NaCl 5% and various excipients (Bausch+Lomb, Bridgewater, NJ)MethodsBeforehand, 3 pairs of human corneas were preserved for 2 weeks in the BR filled with CorneaMax (Eurobio, France), with a pressure maintained at 10 mmHg. A central epithelial ulcer of 6.64 ± 0.04 mm in diameter was made (D0). Each cornea received a drop of Muro 128 or ODM5 four times a day. Corneal central volume measurement (Casia 1, Tomey) was performed daily at the beginning and end of the day. Ulcer healing was monitored daily by fluorescein labelling and quantified by image analysis (Fiji) taken with a macroscope. At D14, the corneas were removed and fixed for immunostaining of E‐Cad (cell architecture), Zo‐1 (tight junctions), MUC16 (mucins), K3K12 (cell maturity) and for histology (Saffron Eosin Hemathein).ResultsMean daily volume loss was comparable: 2.43 ± 1.55 mm3 with ODM5 vs. 2.18 ± 1.71 mm3 with Muro 128 (p = 0.698 3). Corneal ulcers in all 3 pairs healed at D4, D5, D5 with ODM5 vs. D5, D13, and 1 cornea never healed with Muro 128. In IHC and histology, the epithelium was of better quality with ODM5.ConclusionsThe efficacy of the 2 eye drops is comparable. Epithelial preservation is partly better with ODM5, possibly related to the presence of hyaluronic acid.

Modeling and Simulation of Upstream and Downstream Processes for Monoclonal Antibody Production

We present a numerical case study for modeling and simulation of upstream and downstream processes for monoclonal antibody (mAb) production. We apply a systematic and intuitive modeling methodology for an existing upstream process and downstream process. The resulting models are based on differential mass balances and kinetic expressions for the reactions and adsorption. Mass balances for the fedbatch reactor yield a model consisting of five ordinary differential equations (ODEs). The downstream process is conducted batchwise in a chromatographic column for capture of mAbs. Mass balances of the chromatographic column yield a system of partial differential equations (PDEs). The chromatographic model applies the nonlinear shrinking core adsorption isotherm model for transition between the mobile phase and the stationary phase. We apply a high-order spectral nodal continuous Galerkin scheme for spatial discretization of the chromatographic column, which result in a semi-discrete ODE formulation. The resulting simulation model, coupling the upstream and downstream processes in batchwise mAb production, can be used as a benchmark for numerical estimation, control and optimization studies.

Bioprocess intensification for acetone-butanol-ethanol fermentation from brewer's spent grain: Fed-batch strategies coupled with in-situ gas stripping

A fed-batch ABE fermentation process coupled with in-situ gas-stripping to mitigate butanol toxicity was investigated. Two feeding strategies were compared: pulses of sugars and continuous feeding of the liquid released in the dilute acid pretreatment of brewer's spent grain. The concentrations of butanol (13.2 g L-1 total and 50 g L-1 average in the condensates for glucose pulse feeding) were higher than those obtained under batch conditions, showing that in-situ gas-stripping can relieve butanol toxicity. The continuous feeding of the fed-batch reactor produced similar butanol concentrations (10.2 g L-1 total and 65 g L-1 in the condensates), more stable concentrations of solvents in the condensates and enhanced monosaccharides uptake (99.1%) in comparison with the pulse feeding strategy. The efficient utilization of the enzymatic and pretreatment hydrolysates in the same fed-batch reactor is an integrated approach that could reduce capital and operating costs. The mathematical model proposed showed good performance to predict concentrations in the fermentation broth.

TWO-STAGE ANAEROBIC DIGESTION OF ORGANIC WASTES: A REVIEW

Anaerobic digestion is a biotechnological process, in which microorganisms degrade the complex organic matter to simpler components under anaerobic conditions to produce biogas and fertilizer. This process has many environmental benefits, such as green energy production, organic waste treatment, environmental protection and greenhouse gas emissions reduction. It has long been known that the two main communities of microorganisms (acidogenic and methanogenic) taking part in the anaerobic digestion differ in many aspects e.g. their optimal conditions for cell growth and development. Therefore, the anaerobic digestion in a single bioreactor (BR) (single-phase process) required selected optimal conditions, taking into account the slow-growing methanogens at the expense of fast-growing acidogens, which affects the efficiency of the whole process. This has led in recent years to development of two-stage anaerobic digestion, in which the processes are divided into a cascade of two separate BRs. It is known that this separation of the processes into two consecutive BRs leads to significantly higher energy yields for the two-phase system (H2 + CH4), compared to the traditional single-stage CH4 production process. The present paper aims to review the literature in the field of two-stage anaerobic digestion of organic wastes.

EXTREMUM SEEKING CONTROL OF TWO-STAGE ANAEROBIC DIGESTION SYSTEM: A MINI REVIEW

Anaerobic digestion (AD) is a biotechnological process, in which microorganisms degrade organic matter under anaerobic conditions to produce biogas. It has long been known that the two main species (acidogenic and methanogenic) in the community of microorganisms in AD differ in many aspects and the optimal conditions for their growth and development are different. Therefore, in AD in a single bioreactor (BR) (single-phase process), the optimal conditions are selected taking into account the slow-growing methanogens at the expense of fast-growing acidogens, which affects the efficiency of the whole process. This has led in recent years to the development of two-stage AD (TSAD), in which processes are divided into a cascade of two separate BRs. It is known that this division of the processes into two consecutive BRs leads to significantly higher energy yields for the two-phase system (H2 + CH4), compared to the traditional single-stage CH4 production process. In our previous studies different mathematical models of the TSAD have been developed. It was shown that in both BRs the input-output characteristics have a clear maximum, which allows the yields to increase significantly if operations are provided around the maximum points. However, in order to maintain the sustainability of the biogas plants work, it is necessary to introduce automatic control with sophisticated extremum seeking control (ESC) algorithms. This paper presents the pioneering research on ESC of AD process with production of hydrogen and methane. This research has been realized by the Department of Biotechnology at The Stephan Angeloff Institute of Microbiology (SAIM) and the French-Chinese Laboratory on Automatic Control and Signal Processing (LaFCAS), in collaboration with the Laboratory of Signals and Systems (L2S) at the French National Center of Scientific Research CNRS.

Adaptation of anammox bacteria to low temperature via gradual acclimation and cold shocks: Distinctions in protein expression, membrane composition and activities

Anammox bacteria enable efficient removal of nitrogen from sewage in processes involving partial nitritation and anammox (PN/A) or nitrification, partial denitrification, and anammox (N-PdN/A). In mild climates, anammox bacteria must be adapted to ≤15 °C, typically by gradual temperature decrease; however, this takes months or years. To reduce the time necessary for the adaptation, an unconventional method of ‘cold shocks’ is promising, involving hours-long exposure of anammox biomass to extremely low temperatures. We compared the efficacies of gradual temperature decrease and cold shocks to increase the metabolic activity of anammox (fed batch reactor, planktonic “Ca. Kuenenia”). We assessed the cold shock mechanism on the level of protein expression (quantitative shot-gun proteomics, LCHRMS/MS) and the structure of membrane lipids (UPLCHRMS/MS). The shocked culture was more active (0.66±0.06 vs 0.48±0.06 kg-N/kg-VSS/d) and maintained the relative content of N-respiration proteins at levels consistent levels with the initial state, whereas the content of these proteins decreased in gradually acclimated culture. Cold shocks also induced a more efficient expression of potential cold shock proteins (e.g. ppiD, UspA, pqqC), while putative cold shock proteins CspB and TypA were upregulated in both cultures. Ladderane lipids characteristic for anammox evolved to a similar end-point in both cultures; this confirms their role in anammox bacteria adaptation to cold and indicates a three-pronged adaptation mechanism (ladderane alkyl length, introduction of shorter non-ladderane alkyls, polar headgroup). Overall, we show the outstanding potential of cold shocks for low-temperature adaptation of anammox bacteria and provide yet unreported detailed mechanisms of anammox adaptation to low temperatures.