Volatile fatty acid (VFA) rich streams from fermentation of organic residuals and wastewater are suitable feedstocks for mixed microbial culture (MMC) Polyhydroxyalkanoate (PHA) production. However, many such streams have low total VFA concentration (1–10 gCOD/L). PHA accumulation requires a flow-through bioprocess if the VFAs are not concentrated. A flow through bioprocess must balance goals of productivity (highest possible influent flow rates) with goals of substrate utilization efficiency (lowest possible effluent VFA concentration). Towards these goals, dynamics of upshift and downshift respiration kinetics for laboratory and pilot scale MMCs were evaluated. Monod kinetics described a hysteresis between the upshift and downshift responses. Substrate concentrations necessary to stimulate a given substrate uptake rate were significantly higher than the concentrations necessary to sustain the attained substrate uptake rate. A benefit of this hysteresis was explored in Monte Carlo based PHA accumulation bioprocess numerical simulations. Simulations illustrated for a potential to establish continuous flow-through PHA production bioprocesses even at a low (1 gCOD/L) influent total VFA concentration. Process biomass recirculation into an engineered higher substrate concentration mixing zone, due to the constant influent substrate flow, enabled to drive the process to maximal possible PHA production rates without sacrificing substrate utilization efficiency. Polyhydroxyalkanoates (PHAs) are a family of semi-crystalline polyesters that are naturally accumulated by many species of bacteria, for carbon and energy intermediate storage . AccumulLimited types and amounts of PHAs are available commercially today , with suMMCs can be made to accumulate PHA with VFA rich feedstocks. Effluents of suspended solids separated acidogenic fermentation liquors from wastewater and sludges, dominated by (C2 to C5) VFAs, are typically found to result in co-polymer blends of poly . Other tPilot scale projects have repeatedly demonstrated technical feasibility for commercial scale PHA production based on direct accumulation of surplus biomass treating wastewater or using biomass produced with selection pressures specifically for MMC PHA production . NotwithPure culture methods for PHA production classically rely on fed-batch fermentation processes with concentrated feedstocks. A mass of organic substrate is to be delivered to a microbial biomass to achieve as high a titre of PHA containing bacteria as possible, in the shortest possible time, and within the limits of the available bioprocess volume . Batch sMMC PHA production methods must similarly strive to achieve as high a productivity as possible in mass polymer produced per unit volume and time. Acidogenic fermentation effluents can be sourced from commonly available regional sources of municipal and/or industrial organic wastewater and sludge. However, a challenge is that these typically available fermentation feedstocks do not often have high total VFA concentrations. VFA rich feedstocks from fermented organic waste streams from industrial and municipal sources may often, and in own direct experience with examples over 20 years, be in the low VFA concentration range between 0.5 to 10 g/L . Higher If VFAs are the product for transport and sale, then innovations in methods of VFA up concentration as part of recovery are required . VFA extWith dilute VFA-rich feedstocks it is anticipated that the feed volumes will be larger than the available bioprocess working volume. The feed volume must flow through the process, and it is required that the biomass is retained in the process by, for example, membranes  or graviIn previous research, it was observed that traditional Monod kinetics could not adequately describe the dynamics of response of a MMC biomass during fed-batch PHA accumulation . ConsequThe objective in the present work was to systematically evaluate and confirm a model for stimulation and attenuation of substrate uptake rates during PHA accumulation. Specifically, it was of interest to assess if a common model framework could be applied to two distinctly different MMCs enriched for PHA accumulation. The goal was to refine insight for conditions and principles of bioprocess engineering to maintain both high substrate utilization efficiency and optimal volumetric productivity during continuous feed mixed culture PHA production. The approach is intended to be flexible and to work well in cases for PHA production with low VFA concentration feedstock at industrial scale.Dynamics of respiration for laboratory and pilot scale MMC enrichment cultures were characterized by oxygen and chemical oxygen demand (COD) mass balances as a function of initial substrate concentration. Acetic acid and acetate mixtures were used as the model substrate. The dynamic response of the biomass was translated into a process model that was applicable to both cultures. This process model was then applied by means of Monte Carlo numerical simulations to experiment with ideas of a constant volume, flow through, PHA production bioprocess design. The numerical simulations expressed how stimulation and maintenance mixing zones could, in principle, be applied for achieving high rates of PHA production while still maintaining low effluent substrate concentrations.A PHA-storing mixed microbial culture was produced in a 1.0 L working volume aerobic sequencing batch reactor (SBR) at room temperature (20 to 25 °C). A mechanical stirrer provided complete mixing of the culture medium without any settling, and oxygen was delivered through air pumps connected to an array of ceramic diffusors. The SBR was inoculated with activated sludge from the “Roma Nord”  full-scale municipal wastewater treatment plant (WWTP). At the time of the present study, the enrichment culture was established under steady state conditions for 3 months (12 h cycle and organic loading rate (OLR) of 8.5 gCOD/L/d). The biomass was characterized with a PHA accumulation potential in the range from 0.63 to 0.70 gPHA/gVSS. Full details of the SBR operations and performances are reported elsewhere .2HPO4 (334), KH2PO4 (259), MgSO4· 7H2O (100), CaCl2· 2H2O (50), thiourea (20), Na2EDTA (3), FeCl3· 6H2O (2), H3BO3 (0.3), CoCl2· 6H2O (0.2), ZnSO4· 7H2O (0.1), MnCl2· 4H2O (0.03), NaMoO4· 2H2O (0.03), NiCl2· 6H2O (0.02) and CuCl2· 2H2O (0.01). Stability of feast and famine selection conditions were routinely monitored from dissolved oxygen (DO) trends (WTW Cell Oxi 3310).The SBR cycle program sequence with timer controlled peristaltic pumps was as follows: (a) feast substrate feeding , (b) feast reaction phase (150 min), (c) surplus biomass (mixed liquor) withdrawal , (d) famine substrate feeding , (e) famine reaction phase (552 min). The 1-day sludge retention time (SRT) was equal to the hydraulic retention time (HRT) due to no applied settling phase. The feast substrate was an 85:15 mixture (COD basis) of acetic and propionic acids in a mineral medium at 10.12 gCOD/L. The substrate was trimmed to pH 6.0 with NaOH from a 3.0 M stock solution. The famine substrate comprised ammonium sulphate at 7.33 g/L, and with 80 mL addition per cycle, giving a nitrogen loading of 249 mgN/d. A net excess nitrogen loading was established per cycle given a C/N balance of 34 gCOD/gN. The mineral medium composition was (mg/L): KThe biomass source for replicate respiration response experiments in the present work was the withdrawn surplus biomass (c) after feast reaction (b) as described above. The 500 mL withdrawn mixed liquor was directed to an aerated vessel with the added nitrogen rich famine substrate that the wasted biomass would otherwise have experienced for the remaining cycle segments (d and e). Famine conditions were maintained for at least 8 h before the biomass was then used for replicate respiration experiments. Experiments were conducted at room temperature after the applied famine period and these experiments were completed within 26 h of the biomass withdrawal from the SBR. The famine condition for the biomass was confirmed from measured nitrogen uptake during famine, and consistent performance in PHA content cycling (Fourier transform infrared spectroscopy (FTIR) measurements). Famine biomass in the present work refers to the biomass after famine conditions were applied for at least 8 h.The pilot scale biomass was produced in an SBR (120 L working volume) operated with automatic control . The SBR was inoculated with activated sludge from Treviso full-scale WWTP . The PHA-accumulating biomass exhibited typical accumulating potential between 0.40 to 0.46 gPHA/gVSS. Full details of the pilot SBR operations and performances are reported elsewhere .v/v) mixture of the organic fraction of municipal solid waste (OFMSW) and excess waste activated sludge (WAS). Both organic input streams were produced and managed within the Treviso municipality. The fermentate used to produce the biomass was characterized with 75% soluble COD as VFA, and a VFA composition dominated by butyric and acetic acids followed by propionic and other organic acids. The SBR was operated continuously (6-h cycle and 1 d HRT and SRT) with no biomass settling phase. Aeration, with linear membrane blowers (Bibus EL-S-250), provided oxygen and mixing. The 6-h aerobic cycle was as follows: (a) surplus biomass (mixed liquor) withdrawal (0.5 min), (b) delay (10 min), (c) feed (0.5 min), and (d) reaction (349 min). Temperature  and pH (8.0–8.5) trends were monitored, but not controlled. OLR with VFA was 3.3 ± 0.7 gCOD/L/d with variations due to VFA level and composition fluctuations from the feedstock fermentation process [The selection SBR was fed with filtrate coming from fermentation of a 33:67  over a few hours with continuous aeration and mixing. The famine condition for the biomass was confirmed with assessment for negligible biomass PHA content (FTIR measurements).A stock of mixed liquor with the famine biomass was maintained aerated and well-mixed at constant temperature (24 ± 1 °C). For each pulse-stimulation experiment a grab sample of mixed liquor of defined dilution and volume (50 to 100 mL) was mixed to target a biomass suspended solids concentration measured as VSS (volatile suspended solids). Dilutions were made with the same media  or matrix  of the mixed liquor, containing no or negligible biomass and no added organic substrate. pH was at the level established in the respective SBRs and not adjusted. For the pilot scale mixed liquor, biomass suspended solids were separated by gravity separation and decantation to generate dilution media with the same matrix. The source of dilution media was maintained similarly to the famine biomass source with constant aeration and at the same temperature. Respiration experiments were conducted with and without constant aeration, and initial substrate concentrations over a range from 5 to 500 mgCOD/L. Biomass concentrations were in the order of 1 and 2 gVSS/L for the laboratory and pilot biomass experiments, respectively.For each experiment, the defined mixed liquor volume containing famine biomass was disposed directly to a vessel with magnetic coupled stirrer (600 rpm). This vessel was maintained with a pre-set level of constant aeration (0 to 1 mL/min air). Air flow from standard aquarium pumps was regulated through a Dwyer Series VF flowmeter. DO and pH trends were logged starting upon mixed liquor addition to the vessel and for a period of pre-aeration lasting at least 2-min. Then, a defined volume (pulse) of substrate (0.20 to 2 mL) from a concentrated stock solution was injected as an impulse to the defined mixed liquor volume. The time point of substrate injection was tagged to the log file. DO and pH trends were followed until dissolved oxygen concentration increased again asymptotically upon substrate consumption. Such a pulse-stimulation experiment lasted anywhere from a few minutes to the better part of 1 h. This duration was expected to be dependent on the biomass source, the mass of substrate added, and the concentration of the biomass. The added aliquots of substrate stock solutions were defined mixtures of acetic acid and/or sodium acetate generated from 2 M stock solutions of acetic acid and sodium acetate trihydrate (Sigma-Aldrich > 99%), respectively.g for 10 min). The supernatant was decanted to another Eppendorf tube by a single inversion, and residual hanging liquid was carefully wicked out of the tube with tissue. The pellet was either resuspended back to 1.5 mL with deionized water, or it was dried overnight at 105 °C. The suspended solids concentration was evaluated based on chemical oxygen demand (COD), for the resuspended pellet solution. The mixed liquor supernatant was stored at 4 °C pending ammonia analysis. COD measurements were with Spectroquant®  25–1500 and 300–3500 mg/L kits and procedure in combination with a Spectroquant® NOV60A spectrophotometer . Calibration was based on an acetic acid stock solution dilution series. The COD of the famine biomass was converted to volatile suspended solids (VSS) based on the assumed biomass composition of C5H7O2N with 1.42 mgCOD/mgVSS. Ammonium ions were measured on supernatant samples spectrophotometrically at 420 nm by the direct Nessler method [Suspended solids from selected famine mixed liquor samples (1.5 mL Eppendorf) having negligible soluble COD were separated by microcentrifugation  and high  steady state DO levels. Shift up and down trends were evaluated in triplicate, and the first order response time constants were estimated for the pH and DO probes from least squares non-linear regression analysis of the first order exponential asymptotic trends . The probe first order time constants were found to be 0.35 ± 0.02 Dissolved oxygen and pH-millivolt signals were measured continuously during experiments and logged at 0.1 Hz . The pH-millivolt signal was 4-point calibrated . Probe dynamic response characteristic was based on measured instantaneous shift-up and shift-down changes: pH was logged for the trends in shifts between 0.01 molar phosphate buffer solutions. Phosphate buffer solutions were prepared with KHky-Golay  second oky-Golay .oC is the DO concentration (mgO2/L) as a function of time (t in minutes), osQ is the DO uptake rate (mgO2/L/min) due to substrate consumption, opQ is the DO uptake rate (mgO2/L/min) due to activity on stored polymer, oeQ is the biomass endogenous respiration rate (mgO2/L/min), and oaQ is the DO supply rate (mgO2/L/min) due to constant air flow with coarse bubble aeration.The model for the data analysis was based on a mass balance for dissolved oxygen and chemical oxygen demand concentrations. It considered a defined impulse addition of substrate to a defined amount of active biomass (VSS), in a constant volume system with constant aeration. The initial biomass was a famine biomass meaning that the biomass existed with a low background level of endogenous aerobic respiration after being subjected to a long period without exogenous organic substrate supply. The mass balance assumed that the famine biomass would exhibit a feast response. A feast response means a sudden increase in aerobic respiration levels due to the added organic substrate. This increased level of aerobic respiration was assumed to be due to substrate uptake for PHA storage only (no active biomass growth). In particular, exogenous supplied acetic acid is converted into intracellular stored polyhydroxybutyrate granules (PHB) during the feast response. DO concentration changes in the control volume were linked to processes of aeration, endogenous aerobic respiration, aerobic respiration in substrate consumption, and aerobic respiration on stored PHA:sq, mgCOD/gVSS/min) was assumed to be coupled to oxygen uptake rate by a constant (average) yield (osY) for oxygen consumed per amount of substrate consumed (mgO2/mgCOD):osq is specific oxygen uptake rate (mgO2/gVSS/min), and aX is the initial (active) biomass concentration (gVSS/L). DO supply rate, governed by the conditions of volume, aeration, and mixing, was:ak is the estimated aeration oxygen mass transfer rate (1/min) with constant air flow and volume, and 2/L). Aerobic respiration activity on stored polymer was assumed to ensue upon depletion of exogenous substrate. As substrate supply becomes exhausted, respiration shifts rapidly to famine and the subsequent consumption of stored polymer [sk is the Monod apparent affinity constant on substrate concentration (mgCOD/L), ok is the Monod apparent affinity constant on oxygen concentration (mgO2/L), sC is the exogenous substrate concentration (mgCOD/L) as a function of time, 2/gVSS/min), and min is minimum of a and b. Thus, maximum aerobic respiration levels required that DO concentration was not limiting.The specific substrate uptake rate ( polymer . A switcopQ.Aerobic respiration rate (DO consumption rate) on polymer was expected to be a function of the amount of polymer stored by the biomass . RespirapX is the PHA concentration as function of time (mgCOD/L), and siC is the initial substrate concentration (mgCOD/L).For the COD mass balance, amount of polymer produced was determined from the average yield on substrate given polymer storage and assuming no active growth following similar previous model developments :(5)Xp=1−hk is the apparent Haldane substrate inhibition factor (mgCOD/L), and siC. The biomass extant maximum substrate uptake rate is a function of the stimulating (upshift) substrate concentration (siC) and time. The transient phase could be described explicitly as a function of time [if represents the initial respiration level with respect to the maximum reached substrate uptake rate (0 < if < 1), and ik is the respiration induction substrate concentration constant (mgCOD/L). The initial substrate uptake :uk is an apparent half-saturation constant (mgCOD/L) for an upshift in substrate uptake rate given siC as the peak  exogenous substrate concentration.Specific uptake rates were estimated based on the measured stock biomass (gVSS/L) and substrate (gCOD/L) concentrations that were corrected for the known media dilutions that were used for each respiration experiment. Model exploration for assessing the mass balance with measured DO concentration trends was performed with Berkeley Madonna . Data analysis for estimation of model parameters, by nonlinear least squares regression analyses, was performed with custom scripts written in MATLAB , or with model equations defined and fitted by non-linear regression analyses in Prism .mV to a stimulation mixing zone sV receiving influent substrate. In sV, the influent substrate, and recirculated biomass mixed liquor, are mixed in proportions to reach elevated substrate concentrations to stimulate a maximal substrate uptake rate . In Vm,Equation ). The mosfC is disposed to sV at a flow rate of fQ and the flow mQ maintains a defined continuous exchange and recirculation of the mixed liquor from the main volume mV to the mixing zone of sV and back again. This recirculation generates the concentration ssC in sV. The mV hydraulic residence time is mHRT = m/QmV. Similarly, the maximum sHRT is s/QmV.In mV by an ideal separator. Process effluent from mV is at the influent flow rate of fQ. Effluent has substrate concentration smC. Concentration ssC of substrate in sV is a function of fQ, mQ, smC and sfC based on the flow and mass balances. Oxygen uptake rates were calculated and oxygen was assumed to be maintained at levels that were not limiting for microbial activity in mV. Influent flow rate was set to achieve a target organic loading rate with respect to the process theoretically maximum possible substrate uptake rate (Equation (Biomass suspended solids are retained in Equation ).The model applied the system of equations according to the above presented oxygen and chemical oxygen demand mass balances. Model parameters in Equations –9) used used9) usV were assumed to be negligible due to anticipated DO limitation.The respiration and substrate uptake rates for each individual biomass element was dependent on its history of exposure to dissolved substrate concentration. The capacity for substrate uptake rate for each biomass element was therefore determined by the highest most recently experienced substrate concentration (Equation ) in combuk dependent up-shift kinetics (Equation (sk dependent downshift kinetics (Equation (Uptake rates were driven with Equation ) for bioEquation ) for eles” and maintenance “m” control volume substrate concentrations:Numerical integration of governing differential equations was made for each time step to update stimulation “mV.The element specific substrate uptake rates, Equation  and withThe biomass elements were assumed to be ideally mixed in respective control volumes. Transport of biomass elements between ith biomass element, and fQ was adjusted to provide a mass input rate of substrate based on sfC to give a constant substrate organic loading rate with respect to the maximum biomass uptake capacity qsimto range . A valueto range  was applqsimto raTen sets of experiments were performed with the laboratory scale SBR biomass harvested after the feast phase with a subsequent applied period of famine. All the respiration experiments on the laboratory scale famine biomass were spread over 3 months of steady state SBR operations. Two sets of replicate experiments were performed for the pilot scale MMC biomass harvested after famine. Consistency of the biomass condition before experiments was evaluated. FTIR measurements were performed on dried biomass samples from the laboratory biomass directly after feast harvesting. The laboratory and pilot famine biomass were also assessed by FTIR before experiments. Consistency of added ammonia uptake after laboratory feast biomass harvesting was confirmed.4-N/L down from 62 mgNH4-N/L added just after feast harvesting. The pilot scale biomass was harvested after the famine cycle on site. Pilot scale famine biomass was similarly confirmed by FTIR to contain negligible PHA content before the start of the respiration experiments   . RespirasiC) larger than about 20 mgCOD/L, dissolved oxygen levels were insufficient for complete removal of added substrate (if and ik given in Equations (ok was negligible (<0.1 mgO2/L) for this MMC biomass dominated by finely dispersed suspended solids. A non-readily settling biomass was expected because enrichment was applied without requirement for settling as part of the selection pressure (HRT = SRT).For initial substrate concentrations (ubstrate A. For thquations  and 8)..Csi) larsiC levels (<20 mgCOD/L), initial dissolved oxygen concentrations were high enough to support complete added substrate COD removal  gO2/gCOD. This estimated yield coincides with the previously reported theoretical osY level of 0.26 gO2/gCOD [For lower starting  removal B. ParamegO2/gCOD  for PHA osY values can also be lower than the estimated theoretical level of 0.26 gO2/gCOD.Transport of acetate into the cell and its activation is estimated to cost 1 ATP per Cmol of acetate . Acetatesk value of 0.110 ± 0.004 mgCOD/L was estimated for the cases with low starting substrate concentrations. A low apparent substrate affinity constant (sk) was expected. In previous work [sk value of 0.2 Cmmol/L (6.2 mgCOD/L) was selected, and not estimated explicitly to avoid numerical problems in other parameter determination. The present work suggests an affinity constant for acetate in the substrate consumption )oeQ , was thopQ, the average respiration rate on stored polymer directly after substrate depletion, was then calculated )osQ, was then estimated ))Qos, wasosQ, the cumulative biochemical oxygen demand (sBOD) due to substrate removal, was estimated. Then the average yield osY and the trend of substrate concentration were calculated  were interpolated from the derived trend of substrate uptake rate as a function of interpreted substrate concentration. Remaining parameters were estimated by nonlinear least squares regression analysis ). Th). Th8)).ysis see .Each experimental data set was divided into 3 zones for the model parameter estimation see A: Zone 1The laboratory scale SBR biomass was characterised in triplicate with distinct famine biomass batches over a week of steady SBR operations A,C. The osY  was determined independently for each experiment with consistent outcomes. A lower air flow rate was applied for the pilot scale biomass experiments due to lower levels of respiration rate.The estimated average yield of oxygen on substrate Yos  reproducage only . The asssk) was observed for both biomass sources. The sk value, about 2 mgCOD/L, was higher than the value estimated for experiments reported above without active aeration at low initial siC values. The sk (without active aeration) was reproducible and estimated by non-linear least squares regression model analysis. The sk (with active aeration) was estimated pragmatically as the interpolated concentration at half the maximum substrate uptake rate from the 6-step derived trend of sC with time. The value of about 2 mgCOD/L was considered to be a conservative estimate of what may actually be a lower affinity constant in reality. A sk of 2 mgCOD/L is still much lower than previously assumed [sk has significant bearing on the performance of a flow through PHA accumulation bioprocess. A lower sk means, with all other parameters being equal, that similarly high polymer production rates can be maintained with lower substrate concentrations. Lower substrate concentration means less substrate lost in the effluent of a flow-through process giving a better substrate utilisation efficiency.A reproducible low substrate affinity constant ( assumed . ks has uk) was found to be significantly larger than sk  and dow than ks ) both sisiC. However, when dosing with acetic acid, Enrichment MMC biomass sourced from laboratory and pilot SBRs revealed a similar behaviour, but the pilot MMC exhibited about one third the specific maximum rate to assimilate substrate. The trends of maximum substrate uptake rate further suggested an inherent starting (resting) capacity for substrate uptake rate . Since,t to qsr B,C.aX concentration is shown in ik  was observed for acetic acid versus acetate additions in experiments for approximately the same starting COD concentration.The buffer capacity of the mixed liquor involves contribution from dissolved solids as well as the biomass suspended solids. Initial pH change due to acid addition could be characterized with respect to the specific acid addition. The buffer capacity for mmol/L wn in ki B. Thus, wn in ki C,D. A loaX) of 1 gVSS/L, 57 mgCOD/L of acetic acid would result in about 2.5 percent gPHA/gVSS. Even though the biomass PHA content was not measured explicitly, it was of interest to evaluate if the independently determined level of biomass respiration after substrate consumption corresponded to the model predicted level of PHA produced (pX). Directly after substrate consumption the residual elevated respiration rate opQ was estimated from Equations (opQ should be directly related to aerobic metabolism on stored PHA [pX/aX). This outcome suggests an internal model consistency and supports previous research findings for microbial activity during famine [The relatively small increment in PHA content that accumulated in the biomass after a small pulse of substrate was not measured directly as part of the study. The median applied substrate pulse for the experiments was 57 mgCOD/L acetic acid concentration. From quations . Qop shoored PHA ,38. The g famine .uk in Equation . The system settles, in this example, at “A” in An influence of stimulation-maintenance hysteresis is illustrated in Equation ). Csm wimHRT, flocs of biomass are more frequently and repeatedly stimulated to a higher respiration rate due to exposure to ssC in sV. Over a period of about 3 × mHRT, essentially all the biomass suspended solids should have been disposed to respiration stimulation in sV. Increasing mQ to give a 10-min mHRT, ssC in the order of 500 mgCOD/L in sV. This level is significant for stimulating maximal respiration rates for the laboratory scale biomass . HoweveEquation ), and suEquation ). In thiL “B” in . This lotrations A,C. For e “C” in . Thus, lThis strategy for engineering accumulation bioprocess with internal, or external, recirculation and contact to elevated substrate concentrations that exist in the influent mixing zone(s) becomes increasingly more relevant the lower the influent substrate concentration becomes. Stimulation of respective biomass elements is asynchronous in time because not all biomass elements experience “stimulation” at the same time. Monte Carlo modelling can be applied as a way to understand the nature of the distribution of activities for discrete floc elements. In contrast, the typical approach for PHA accumulation with laboratory and pilot scale systems in the reviewed research literature ,12 has bTypically, dissolved oxygen ,45,46, rAdditions of substrate in MMC accumulation cannot be too large due to a risk for inhibition . Larger Smaller pulse-wise substrate additions can be made in a fed-batch process with peak substrate concentrations that are just sufficient to stimulate high average substrate uptake rates . This seMethods supporting the development of industrial MMC PHA accumulation with continuous feed supply and as a flow through bioprocess have not been found to be reported in the research literature. Initial outcomes in published work for fed-batch methods applying constant feed based on predetermined rates without feedback control were not found to be satisfactory . Since tmHRT = 250 min in mHRT = 5 min in As an example, numerical simulations were performed to explore how the application of a stimulation zone could influence substrate utilisation efficiency while pushing the system towards theoretical limits of maximum PHA production rates with a feedstock VFA concentration of 1000 mgCOD/L . Higher The stochastic modelling of the observed MMC biomass response in stimulation of respiration and substrate uptake rates suggests potential to understand and develop optimal operational conditions for robust continuous feed methods at industrial scale. Characteristics for any biomass may be readily determined by the methods of the respiration experiments that were replicated for both the laboratory and pilot scale SBR enrichment biomass sources in the present investigation. This kind of characterisation of the biomass dynamic response is recommended towards innovation in the bioprocess that can address challenges for maximising productivity and for avoiding growth of flanking populations.In the present work, the dynamic response characterisation for two biomass sources revealed a similar inhibition effect of specific acid dosing B,C. ThissV using as low a mHRT as possible helps to initially stimulate all the process biomass as quickly as possible. Feeding exclusively to the stimulation zone becomes less critical to maintain if all the process biomass has been “stimulated”. Once all the process biomass is stimulated the process control objective is to ensure a feeding rate to sustain sufficient but still relatively low substrate concentration levels . Thus a challenge is to establish feedback control for optimum feeding rate without over or under feeding the process.Recirculation to sV and mV zones can be monitored in parallel with sensor signals  that are related directly or indirectly to substrate concentrations, biomass respiration levels, and/or PHA content. The sV zone provides a dynamic reference measurement because water quality parameters, including but not limited to, buffer capacity, and ionic composition will change in time. Changes will be due to the feedstock water quality and also the intense PHA accumulation biological activity in mV. So-called soft-sensing process control strategies [In the model simulations, the required influent flow rate was calculated. In practice, the feeding rate must be determined from the process monitoring. Recirculating to the stimulation zone with known flows, mixing ratios and characteristic time constants, can serve needs for monitoring and feed rate feedback control . Vs and rategies  are antiA property of hysteresis in the dynamic response of MMCs storing PHA could be demonstrated for two distinct enrichment cultures using dissolved oxygen and chemical oxygen demand mass balance experiments.This hysteresis could be modelled with readily identifiable parameters using Monod equations describing the distinct upshift and downshift dynamics in substrate uptake rates as a function of substrate concentrations and as a function of time. It was found that the substrate concentration, required to stimulate a substrate uptake rate, was higher than the substrate concentration required to maintain an attained substrate uptake rate.The system of equations in numerical simulations suggest for an opportunity to exploit this property of hysteresis in industrial scale bioprocesses for PHA production. MMC PHA production processes can be operated with continuous feeding strategies, even with low concentration feedstocks. The model simulations found that engineered stimulation zones can be applied in continuous flow PHA production bioprocesses as a strategy to reach maximum possible performance in volumetric productivity without sacrificing performance in substrate utilization efficiency.There are three main outcomes as conclusions from this study and with novelty in contributions from the investigation efforts to characterize and model dynamics of substrate uptake rate for MMCs during PHA accumulation:Applying ideas and principles revealed by the model simulations is a future challenge with applications in practice. Methods for process monitoring are required in combination with feedback control to maintain optimal organic loading rates during the PHA accumulation process.


The basic science research endeavor has been abundantly and astonishingly successful in the last three decades in elucidating the mechanisms of neuro-oncologic disease and in suggesting therapeutic strategies. Clinical successes have lagged behind, and translation of promising laboratory findings into clinical practice is rare. We hypothesize that one important reason for this discordance is the use of different paradigms for designing laboratory and clinical trials, and that utilizing clinically relevant procedures could improve laboratory study impact.We identified all pre-clinical neuro-oncology therapeutic trials published in four high-impact journals between 11/2018 and 4/2019 and assigned a level of evidence (LOE) to each study using the American Academy of Neurology evidence classification system. We then identified all phase III trials of therapeutics for COVID and performed the same analysis on all preclinical studies preceding the trials.Of the 26 neuro-oncology articles identified, 85% had a LOE of IV and 15% were class III. An analysis of successful human trials showed significantly more high quality laboratory studies supporting “successful” compared to “unsuccessful” trials (p=0.048). This same pattern was identified in phase III trials of COVID. Twenty antiviral studies failed to meet the primary endpoint; all were preceded by class III or IV LOE preclinical studies. Eight evaluable phase three studies of COVID vaccines were identified, all of which met their primary endpoints. These were supported with a mix of Class I/II (n=4) and III/IV (n=4) preclinical studies. Higher LOE by AAN criteria is associated with successful COVID therapeutic trials (p=0.0034).Despite rigorous, elegant, and enlightening laboratory experiments, successful translation to human therapeutics remains rare. Envisioning basic science research through the lens of clinical therapeutics represents a challenging but surmountable paradigm shift that may reverse this pattern and create a more successful research enterprise in neuro-oncology and beyond.


Culture expansion of primary cells evokes highly reproducible DNA methylation (DNAm) changes. We have identified CG dinucleotides (CpGs) that become continuously hyper- or hypomethylated during long-term culture of mesenchymal stem cells (MSCs) and other cell types. Bisulfite barcoded amplicon sequencing (BBA-seq) demonstrated that DNAm patterns of neighboring CpGs become more complex without evidence of continuous pattern development and without association to oligoclonal subpopulations. Circularized chromatin conformation capture (4C) revealed reproducible changes in nuclear organization between early and late passages, while there was no enriched interaction with other genomic regions that also harbor culture-associated DNAm changes. Chromatin immunoprecipitation of CTCF did not show significant differences during long-term culture of MSCs, however culture-associated hypermethylation was enriched at CTCF binding sites and hypomethylated CpGs were devoid of CTCF. Taken together, our results support the notion that DNAm changes during culture-expansion are not directly regulated by a targeted mechanism but rather resemble epigenetic drift. Julia Franzen et al. investigate if changes in DNA methylation at specific genetic loci during cell culture expansion are due to a specific mechanism or gradual deregulation of an epigenetic state. Their results suggest that changes in CpG methylation are due to indirect epigenetic drift, rather than a consequence of targeting by DNA methyltransferases. This is of particular relevance for the more than 1000 registered clinical trials with mesenchymal stem/stromal cells , which usually use high cell doses (often about 106 cells/kg body weight) and therefore necessitate excessive culture expansion4. Furthermore, long-term culture of cells is often required for basic research and this may hamper the reproducibility of results. Tracking the impact of long-term culture is therefore an important aspect for quality control of cell preparations.Cell preparations are often expanded in vitro for many passages to achieve enough material for basic research or cellular therapy. However, culture expansion has a severe and continuous impact on the growth, morphology, gene expression, metabolomics, and function of primary cells, until they ultimately enter a state of replicative senescence6. We have previously demonstrated that DNAm levels at only six cytosine/guanine dinucleotides (CpG sites) can be used to estimate passage numbers and cumulative population doublings (cPD)8. It therefore provides a reliable biomarker to estimate the state of culture-associated modifications. In our previous studies, we referred to these epigenetic modifications as senescence-associated DNAm changes, albeit it is unclear if the culture-associated DNAm changes are linked to the state of cellular senescence—we therefore changed the terminology into culture-associated DNAm changes. Furthermore, it is so far unclear how DNAm patterns evolve during culture expansion and why they occur at specific genomic regions.Long-term culture is reflected by highly reproducible DNA methylation (DNAm) changes at specific sites in the genome9. In fact, there is some overlap with epigenetic clocks for aging, but the two processes can be clearly discerned and it is yet unclear how the DNAm changes are governed. It is generally anticipated that DNAm changes during development are regulated by epigenetic writers—such as the de novo methyltransferases DNMT3A and DNMT3B, or TET methylcytosine dioxygenases10. If an epigenetic writer is targeted to a specific site in the genome the neighboring CpGs will most likely also be modified. Alternatively, the DNAm changes that accumulate during culture expansion might not be directly regulated but rather reflect dysregulation, as suggested for epigenetic drift during aging of the organism13. A better understanding of how DNAm changes evolve in culture expansion might shed light into the underlying process.At first sight, the culture-associated DNAm changes seem to be related to the DNAm changes that are acquired during the aging of the organism14. Using hairpin-bisulfite PCR15 several studies have demonstrated that, despite the general preference for concordant DNAm on both strands, certain sites are specifically methylated on only one strand18. Such hemimethylation can be stably inherited over several passages and has been associated with CCCTC-binding factor (CTCF)/cohesin binding sites19. CTCF has been linked with changes in chromatin conformation and cellular senescence21. We have demonstrated that senescence entry upon extensive culture expansion is associated with a reorganization of CTCF into large senescence-induced CTCF clusters (SICCs)20. However, it remains unclear if and how the binding of CTCF changes during long-term culture.Furthermore, it is generally thought that DNAm patterns are identical on the forward and the reverse DNA strand (Watson and Crick strands)In this study, we further investigated if culture-associated DNAm changes are caused by a targeted regulatory mechanism or rather by epigenetic drift.7. In continuation of this work, we utilized the meanwhile available 450k Illumina BeadChip datasets, which interrogate ~16 times more CpGs than the 27k version of the chip, and we also considered datasets of additional types of primary cells. We compiled 63 DNAm profiles of human primary MSCs (n = 45), fibroblasts (n = 5), and human umbilical vein endothelial cells (n = 13) with precise information on passage numbers  to select 15 hypermethylated and 15 hypomethylated sites  based on 27k Illumina BeadChip datasets, and thereby established a 6 CpG predictor to estimate passage numbers specifically for MSCsArachidonate 12-Lipoxygenase , Docking Protein 6 , Leukotriene C4 Synthase , and TNNI3 Interacting Kinase . Samples of the training set were all cultured until growth arrest and senescence was checked by staining with senescence-associated β-galactosidase  of 3.9 passages. Subsequently, our epigenetic predictor for long-term culture was validated on an additional independent set of samples  levels, which cannot be distinguished from 5-methylcytosine (5mC) by bisulfite treatment. To address this question, we used the TrueMethyl Array kit on three MSC donors in early (passage 4) and late (passage 10) passages to specifically look at the percentage of 5hmC at our 646 hyper- and 2442 hypomethylated culture-associated CpGs. Overall, we did not detect high levels of hydroxymethylation at early or late passages (mean estimated 5hmC levels about 0.3%) and neither hyper- nor hypomethylated culture-associated CpGs showed higher mean levels of 5hmC than other CpGs .Importantly, culture-associated DNAm changes are distinct from age-associated DNAm changes, which correlate with chronological age rather than with passage numbersics Fig.  and the 23  there is an inverse switch in epigenetic patterns of pluripotency and culture expansion around day 7. While the long-term culture-associated DNAm changes are then continuously acquired upon differentiation of iPSCs, this is not observed for age-associated signatures23 Fig. . Convers 23 Fig. . Taken t30, which might contribute to culture-associated DNAm changes. Therefore, we aimed for a better understanding how DNAm patterns at neighboring CpGs evolve during culture expansion and how this is affected by the clonal composition of MSCs. We anticipated that tracking of DNAm patterns over several passages would provide insights into the changing composition of subclones within MSC preparations. To address this question, we used samples from a previously published study:30 Umbilical cord-derived MSCs from two donors were transduced with lentiviral vectors containing random barcodes and three different fluorescent proteins. Flow cytometry and deep sequencing demonstrated that the diversity of cellular subsets declines and that senescent passages became oligoclonal 7. The combined BBA-seq measurements were then used to predict passage numbers. Taking all passages into account, the epigenetic estimations correlated well with the number of passages —the other amplicons hardly comprised neighboring CpGs and could therefore not be included into this analysis. DNAm patterns overall remained stable or became even more diverse during culture expansion, which is also reflected by a moderate increase of the Shannon index in most amplicons  in the loop region to adjust for potential PCR bias . As a control, we considered an additional genomic region that was generally methylated (associated with the genes C12orf12). The accuracy of epigenetic predictions of passage numbers was similar when reads with the same UMI were only considered once  using circular chromatin conformation capture (4C). Two independent MSC preparations at early (P2 and P3) and late (P7 and P9) passages revealed overall reproducible interaction profiles : A similar number of interactions revealed such significant gains and losses during culture expansion  was previously shown to have an exceptionally large differentially methylated region during culture expansion, which may explain this enrichment in the differentially methylated nearbait region22. However, this finding might also be coincidental since the significant gains of interaction at KRTAP13.3 included only 6 CpGs of the Illumina Bead Chip. Overall, our results did not indicate that the four culture-associated genomic regions revealed clear enrichment of interaction with other culture-associated CpGs at nearbait or cis-contacts.Next, we investigated if chromatin interactions preferentially occurred at other genomic regions that become methylated or demethylated during culture expansion. As mentioned above, we used the 63 DNAm datasets of cells at different passages . In contrast, such overlap with LADs was rather depleted for hypermethylated regions  we used a publicly available dataset of fibroblasts20. When we analyzed the distribution of CTCF in MSCs by fluorescence microscopy we also observed increased co-localization of CTCF in the larger nuclei of senescent MSCs . Overall, the ChIP seq peaks were in line with previous data of embryonic stem cell-derived MSCs35  in HUVEC and IMR90 cellsSCs Fig. . We thenALOX12 and LTC4S) exhibited significant enrichment of binding motifs for CTCF and CCCTC-binding factor like  for all three donors  Resetting of culture-associated DNAm during reprogramming into iPSCs seems to occur synchronously with the epigenetic changes in pluripotency genes, which is directly linked to the epigenetic transition itself. Furthermore, it occurs simultaneously with resetting of age-associated DNAm, which has also been attributed to epigenetic drift13. (2) The culture-associated DNAm patterns do not reflect MSC clonality and become even more diverse in oligoclonal cell preparations at late passages. If these epigenetic modifications were evoked by targeted regulation in individual subclones specific patterns should become dominant, too. (3) BBA-seq analysis demonstrated that culture-associated DNAm patterns do not develop in an additive manner at neighboring CpGs and this was further supported by hairpin sequencing of the complementary DNA strands. If an epigenetic writer is targeted to a specific site in the genome the neighboring CpGs will most likely also be modified, as observed for CRISPR-guided approaches of epigenetic writers that coherently modify neighboring CpGs37. (4) There was no evidence that culture-associated DNAm is coherently modified at interacting chromatin domains. It is known that DNMTs accumulate in replication foci or punctate heterochromatic foci39 and hence, it might be speculated that a targeted mechanism of an epigenetic writer would also involve the interaction of culture-associated chromatin domains. If culture-associated DNAm changes are not governed by targeting of epigenetic writers, there needs to be another—yet unknown— mechanism that disposes specific genomic regions to epigenetic drift.The continuous and highly reproducible nature of culture-associated DNAm changes may suggest that this process is tightly controlled41. Nuclear depletion of HMGB2 and its induction of CTCF clustering are early events on the path to replicative senescence, which disturb the chromosomal 3D organization20. Our 4C analysis supports the notion that the 3D chromatin structure undergoes highly reproducible changes during culture expansion, while these changes do not seem to be associated with changes in DNAm. It has been demonstrated that CTCF occupancy, which is to some extent cell-type specific, is also linked to differential DNAm42. Furthermore, some CTCF sites may function as a bifurcation point defining the differential methylation landscape43. In this regard, it was unexpected that we did not observe clear differences in the CTCF ChIP-seq data of early versus late passages. On the other hand, hypermethylation seems to occur preferentially at CTCF binding sites, whereas hypomethylation occurs apart from CTCF binding sites and preferentially at LADs. This might partly be attributed to the fact that the hypermethylated CpGs have rather low DNAm levels at early passage, which may support binding of CTCF to the corresponding motive at early passage43.Cell culture is evidently associated with major changes in chromatin structure. The nucleus becomes much larger at later passages, while chromatin volume decreases due to extensive reorganization of hetero- and euchromatin conformation44. Thus, the binding of these transcription factors might be affected by DNAm or vice versa44. It is also conceivable that DNAm changes during long-term culture are indirectly mediated by the histone code, insulators, chromatin loops, and the overarching nuclear structure. Furthermore, the culture-associated differentially methylated regions might be related to nucleosome size. However, for such analysis it needs to be considered that the Illumina Bead Chip data has the limitation that it does not address all CpGs of the genome. Furthermore, we have only exemplarily analyzed few amplicons with BBA-seq, hemimethylation analysis, and 4C. It is therefore unclear if the findings of our study are really representative of the entire genome. Importantly, the culture-associated DNAm changes might also be influenced by specific culture conditions or cell types and this deserves further analysis in the future.Taken together, the results of this study support the notion that the given chromatin conformation favors site-specific epigenetic drift over subsequent passages—but they also provide room for additional hypotheses, which have not been addressed with our current study. We have previously demonstrated that some transcription factor binding motifs  were enriched in senescence-associated differentially methylated regions and in the promoters of differentially expressed genes8. Thus, a direct link to a specific gene promotor is often not possible. It has been shown that culture-associated DNAm is only partly associated with gene expression changes of the corresponding genes46. Yet, it would be an oversimplification to only consider transcriptional regulation as being functionally relevant. Chromatin conformation, loop structures, histone modifications, and LADs may favor modulation of DNAm at specific sites in the genome—and on the other hand, culture-associated DNAm may stabilize such chromatin features. In future research, different levels of chromatin organization should be considered to fully understand the underlying mechanism that drives epigenetic drift during long-term culture.Last but not least, the functional relevance of DNAm changes is still unclear. Overall, culture-associated CpGs are rather depleted from CpG islands and their shore/self-regions, while they are enriched in open sea, 3′UTR and intergenic regions47, respectively. An outlier test using the R package car was performed resulting in the exclusion of one sample (GSM1004625). Initially, we selected culture-associated CpGs based on Pearson correlation of DNAm levels  and passage numbers at a threshold of R > 0.7 or R < −0.7 22 as well as DNAm profiles during re-differentiation of iPSCs to iMSCs (GSE54767)23. We focused on three different sets of culture-associated CpGs: (1) 30 CpGs ; (2) 3088 CpGs ; and (3) 9 CpGs of our epigenetic signatures 7. Furthermore, we focused on 1432 pluripotency-associated CpGs27 and 99 age-associated CpGs25 of the Illumina 450k BeadChip. Heatmaps were produced with R package gplots, Pearson correlation, and corresponding p-values were calculated with R package stats.The kinetics of culture-associated DNAm changes during reprogramming of fibroblasts into iPSCs were investigated using the dataset of Ohnuki et al. n = 11; BM-MSC RWTH Aachen)48, from bone marrow aspirates of allogeneic hematopoietic stem cell donors 49, from subcutaneous adipose tissue lipoaspirates 50 and from umbilical cord pieces 30. Fibroblasts were isolated from dermis 7. Human umbilical vein endothelial cells were isolated from umbilical cords of healthy donors after cesarean sections 51 or obtained from Lonza 8. All samples were taken after informed and written consent and the study was specifically approved by the ethics committees of the corresponding Universities. All cell preparations were thoroughly characterized  and culture conditions were used as described in detail in our previous work50. In addition, HUVECS were cultured on 0.1% gelatin in M199 medium  supplemented with 20% fetal calf serum (FCS) (Gibco Thermo Fisher), 1% penicillin/streptomycin (PAA), 0.1% heparin  and 50 µg/ml endothelial cell growth supplement (ECGS) . For long-term culture all cells were passaged at ~90% confluency and reseeded at 10,000 cells/cm2. Beta-galactosidase  staining was performed using the Senescence Detection Kit ab65351 . cPD were calculated as described before50.All human cell samples were taken after informed and written consent was obtained from donors and the study was specifically approved by the ethics committees of RWTH Aachen University Medical School , University of Heidelberg, and Hannover Medical School. MSCs were isolated from the bone marrow of donors undergoing orthopedic surgery  according to the manufacturer’s instructions. In short, three MSC donors of early passage (passage 4) and late passage (passage 10) were each divided into two samples: one sample was oxidized and bisulfite converted (OxBS), the other sample was just bisulfite converted (BS). Oxidation prior to bisulfite treatment leads to the conversion of 5hmC to 5-formylcytosine (5fC), which is decarbonylated and deaminated to uracil upon bisulfite treatment, similar to non-methylated cytosines. Both OxBS and BS samples were finally investigated by Illumina 450k BeadChip microarrays . Beta values of the microarrays were provided by Illumina’s GenomeStudio software and hydroxymethylation was calculated as the difference of beta values of BS samples subtracted by the beta values of OxBS samples.β-values) at the four CpGs in α = ALOX12 (cg03762994), β = DOK6 (cg25968937), γ = LTC4S (cg26683398) and δ = TNNI3K (cg05264232) was as follows:52 to perform 10-fold cross-validation on the training dataset.Genomic DNA was isolated with the NucleoSpin Tissue kit  and bisulfite converted using the EZ DNA Methylation kit . Pyrosequencing was performed on a PyroMark ID System . Primers for pyrosequencing were designed with the PSQ assay design software  and by deep sequencing of PCR amplified barcodes using ion torrent sequencing. Area plots were produced with the R package ggplot2. The Shannon-Index was calculated with the following formula:Primary MSCs in umbilical cord tissue were transduced with three different lentiviral vectors containing the fluorescent proteins mCherry (red), Venus (green) or Cerulean (blue) and a barcode of 16 random and 15 vector-backbone-specific nucleotides, as previously published8. Amplicons were pooled and sequenced on an Illumina MiSeq lane with the v2 nano reagents (Illumina) in 250 PE mode. Bisulfite converted sequencing data were analyzed using TrimGalore, Bismark53 and bowtie254. Mean sequencing coverage of amplicons was ~3900 reads per amplicon  based on their binary sequel of methylated and unmethylated CpG sites. Probabilities of passage numbers were based on linear regression models at each CpG site, retrieved from the training dataset. Finally, we calculated the mean passage number for each sample based on all sequencing reads. Further details on the rational and derivation of the mathematical model are provided in our previous work31.Bisulfite-converted DNA was used for a nested PCR using the PyroMark PCR kit  was digested with restriction enzymes that cut close to our CpG of interest: AccI , DdeI  and CviQI . Hairpin linkers  and cis interaction analysis and k = 20 for trans chromosomal interactions. We focused particularly on the high-interacting reads that were called in both replicates. Differential interactions were called with 4Cker, which uses DESeq2 and a FDR corrected p-value < 0.5 to call significant differential interactions. Circos-plots were generated with the R package RCircos58. For comparison we used DNAm profiles of MSCs at early (n = 5) and late passages 22. CTCF and CTCFL enrichment in interacting regions was tested with the RGT motif enrichment tool . Motif enrichment was tested within high-interacting regions called by 4Cker ranging approximately from 1.1 to 336 kilobases and a mean region length of 23 kilobases within nearbait and from 1.3 to 950 kilobases with a mean region length of 86 kilobases within cis regions. Enrichment of interactions with LADs of human fibroblasts32 were analyzed in comparison to randomly chosen regions of similar sizes by Fisher’s exact test using R stats.Ten million cells from two MSC preparations at early (P2 and 3) and late (P7 and 9) passages were cross-linked with 4% paraformaldehyde  for 10 min., harvested into ice cold PBS with 0.125 M glycine, and frozen under a protease inhibitor cocktail . 4C-seq was performed as described before20. For image acquisition, a widefield LeicaDMI 6000B with a HCX PL APO 63x/1.40 (Oil) objective was used.Staining of CTCF  and counterstaining with DAPI was performed as described in detail beforen = 3) and late passage  were cross-linked with 1.2% formaldehyde  for 15 min at room temperature. After cell lysis, chromatin fragmentation was performed for 20 min  with a Biorupter® Pico Sonicator Device (Diagenode). 5–33 µg of sonicated chromatin was incubated with 4 µg ChIP-validated CTCF antibody (Active Motif) overnight on an end-to-end rotator at 4 °C. For immunoprecipitation, samples were incubated with protein G agarose beads (Active Motife) at 4 °C for 3 h. Finally, the cross-link was reversed, the DNA was proteinase K digested and purified.Chromatin Immunoprecipitation of CTCF was performed using the ChIP-IT PBMC kit with the fixation protocol of the ChIP-IT High Sensitivity kit . In brief, MSCs of early passage  before reads were aligned to the hg19 genome assembly using bowtie254. Reads approximately exhibited a 97% concordant alignment rate in all samples and sequencing depth ranged from 38 to 170 million reads. We used MACS259 for peak calling on each sample with the default parameters against the input control. All the peaks were filtered by signal >50 and selected with the overlapping of potential CTCF binding motif, which was obtained by RGT motif analysis . Enrichment of interactions between 4C high-interacting regions and CTCF ChIP-seq peaks of early and late passages were analyzed in comparison to randomly chosen regions of similar sizes by Fisher’s exact test using R stats. The normalized coverage of each sample was calculated by Deeptools60 bamCoverage with normalization of Reads Per Kilobase per Million mapped reads (RPKM), these normalized coverages were used for down-stream analyses. The correlation heatmap across the samples was generated by Deeptools multiBigwigSummary. All the potential CTCF binding sites were evaluated by the normalized coverage profiles and shown in MA plot by comparing early passages with late passages. The lineplots were calculated by a sliding window (size = 200 bp with step = 100 bp) on the normalized read counts on the extended sites (length = 2000 bp for each direction) of either CTCF motifs or various sets of CpGs. Statistical analysis was performed against these random CpG sites using the Kruskal–Wallis test with post hoc Dunn’s test and Benjamini & Hochberg adjustment.Sequencing and library preparation were conducted by the IZKF Sequencing Core Facility of RWTH Aachen University Medical school. The samples were sequenced in paired-end mode on the Illumina NextSeq 500. The quality of the resulting reads was checked by FastQC (Available online at: Statistical tests were performed as described in the corresponding methods sections. All experiments were performed for at least two independent cell donors and at least four culture-associated CpGs.Further information on research design is available in the Peer Review FileSupplementary InformationDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2Supplementary Data 3Supplementary Data 4Reporting Summary


Acute ischemic thromboembolic stroke is one of the most feared complications of atrial fibrillation (AF), and the risk increases with higher CHA2DS2-VASc scores. Postoperative atrial fibrillation (POAF) is common after noncardiac surgery, particularly after thoracic surgery, and can result in significant morbidity and mortality. We report the case of an 85-year-old female with a history of untreated hypertension (HTN) and no prior history of AF, who presented 5 days after an elective repair of a paraesophageal hernia with recurrence of a large type III paraesophageal hiatal hernia, AF, and subsequent acute thromboembolic ischemic stroke. Patient’s AF resolved shortly after treatment with calcium channel blocker. The risk of stroke is high in patients who develop AF and a period of 48 h after onset of AF is usually considered safe as the risk of stroke is low in this time period. However, this may not be the case during the perioperative period and preventive measures such as preoperative calcium channel blocker could be considered. Our case highlights that acute ischemic thromboembolic stroke might develop earlier tha 48 h after onset of POAF in patients undergoing paraesophageal hernia repair. Initiation of a calcium channel blocker should be considered during preoperative evaluation for patients undergoing paraesophageal hernia repairs, especially in those with untreated HTN. Postoperative atrial fibrillation (POAF) is a common condition ensuing noncardiac surgery, with a reported incidence rate between 0.4 and 3.0%  who presented on post-op day five from paraesophageal hernia with recurrent large type III paraesophageal hiatal hernia, POAF, and subsequently developed acute thromboembolic ischemic stroke. The patient’s AF resolved shortly after administration of diltiazem, a non-dihydropyridine calcium channel blocker. We would like to highlight the potential use of POAF prophylaxis in high-risk patients undergoing paraesophageal hernia repair surgery, and propose a possible treatment regimen with non-dihydropyridine calcium channel blocker, particularly in those with untreated or uncontrolled HTN.An 85-year-old female with a past medical history significant for renal cell carcinoma, gastroesophageal reflux disease, and untreated hypertension presented to the intensive care unit on postoperative day five from a laparoscopic large type III paraesophageal hiatal hernia repair with gastropexy, with right-sided weakness, mild dysarthria, and right hemineglect  = 11). Upon arrival to the intensive care unit, she had a heart rate of 120–130 beats per minute, respiratory rate of 20–30 breaths per minute, oxygen saturation > 92% on room air, and blood pressure ranging from 160 to 180 mmHg systolic and 90–100 mmHg diastolic. The patient had been discharged 3 days after her surgery with cardiac telemetry showing normal sinus rhythm.A computed tomography of the head without contrast was performed and ruled out acute hemorrhage; however, tissue plasminogen activator was contraindicated in the setting of recent surgery. A magnetic resonance imaging of the brain exhibited scattered acute infarcts in bilateral frontal lobes, and subsequent head and neck magnetic resonance angiography revealed bilateral anterior cerebral artery occlusion.An electrocardiogram was performed and exhibited AF with rapid ventricular response of 120 bpm and a blood pressure of 165/90. The patient was immediately treated with diltiazem 10 mg intravenous followed by a diltiazem infusion with a resolution of the AF within 2 h which lead to discontinuation of the drip. Subsequent rhythm strips for the following 12 h showed normal sinus rhythm. The following day, the patient had another episode of AF with a rapid ventricular response, at this time, it was decided to use amiodarone to prevent any potential hemodynamic instability in the setting of a recent acute ischemic stroke. The patient was given amiodarone 150 mg intravenous and she was then placed on 3 days of amiodarone 200 mg twice daily through her nasogastric tube. A heparin drip with a partial thromboplastin time goal of 50–70 was also started for anticoagulation. A transthoracic echocardiogram with bubble study was performed and revealed moderate left atrial dilation. However, no clots were seen in the left atrial appendage.The patient was taken to the operating room for flexible esophagogastroduodenoscopy, gastric decompression, and placement of nasogastric tube when further imaging indicated rightward mediastinum shift due to recurrence of large type III paraesophageal hiatal hernia. The large type III hernia contained the entirety of the patient’s stomach, proximal duodenum, and transverse colon. The patient returned to the operating room 11 days after initial repair for repeat paraesophageal hiatal hernia repair as well as chest tube and percutaneous endoscopic gastrostomy tube placement. Heparin drip was held 12 h pre-operatively. Postoperative chest radiograph indicated resolution of mediastinal shift with re-expansion of the left lung. After her initial 3 days loading dose of amiodarone, the patient was continued on a 200-mg daily dose through her perioperative period to prevent additional episodes. She was discharged on amiodarone 200 mg daily and apixaban 2.5 mg twice daily 17 days after her admission to a long-term care facility.2DS2-VASc clinical stroke risk score, have been developed to aid with selecting those at high risk for embolic stroke with recommended anticoagulation for those with scores ≥ 2 in men and ≥ 3 in women , would allow this patient to benefit from prophylactic treatment with a rate-control agent such as diltiazem prior to surgery and avoid the risk of stroke sequelae.It is possible that both the recurrence of the hiatal hernia and her history of uncontrolled hypertension may have been the principal causes of AF in this patient and thus subsequent embolic stroke, although such conclusions cannot be proven. POAF is known to occur in association with numerous acute conditions such as hypo- and hypervolemia, electrolytes imbalances, anemia, infection, myocardial infarction, hypoxia, hypoglycemia, and intraoperative hypotension  even in low-risk patients undergoing hiatal hernia repair (Roy et al. 2DS2-VASc score could benefit from calcium channel blocker prophylaxis; however, further studies should be performed.Previous evidence suggests that transient POAF after non-cardiac surgery may carry a long-term stroke risk similar to any other AF diagnosis (Gialdini et al. Acute ischemic thromboembolic stroke might develop earlier that 48 h in postoperative paraesophageal hernia repair patients with POAF. Although HTN is not the sole independent risk factor, preventive measures such as calcium channel blocker should be considered during preoperative evaluation for patients undergoing paraesophageal hernia repairs, especially in patients with untreated HTN.