Crude Feedstock Research Articles

Opportunities and challenges in Upscaling the production of N,N-dimethylaminoethanol from bio-based glycolaldehyde

In recent years, significant focus has been placed on the sustainable chemical production of ethanolamines using bio-based feedstocks. Thorough investigations have been conducted to explore and refine laboratory-scale methodologies. However, important parameters for successfully scaling up these processes to meet industrial productivity standards are often overlooked. In this article, the feasibility of scaling up the production of N,N-dimethylaminoethanol (DMAE) from bio-derived industrial glycolaldehyde (GA) was investigated. By determining a minimum kLa value of 0.27 s−1, a 97 % selectivity towards DMAE at complete GA conversion was ensured, irrespective of the reactor configuration. This improvement led to a substantial productivity increase, reaching up to 1526 kg DMAE/m3.h. Additionally, it was observed that impurities present in the industrial crude GA feedstock lowered the DMAE selectivity to 76 %. Notably, the presence of acidic impurities within the crude feedstock was identified as the main cause of the imbalance between the hydrogenation, dehydration, and tautomerization reaction, ultimately resulting in the formation of undesired diamines. This article elucidates potential engineering approaches to enhance the selectivity and productivity for the industrial production of bio-based ethanolamines.

Development of an itaconic acid production process with Ustilaginaceae on alternative feedstocks

BackgroundCurrently, Aspergillus terreus is used for the industrial production of itaconic acid. Although, alternative feedstock use in fermentations is crucial for cost-efficient and sustainable itaconic acid production, their utilisation with A. terreus most often requires expensive pretreatment. Ustilaginacea are robust alternatives for itaconic acid production, evading the challenges, including the pretreatment of crude feedstocks regarding reduction of manganese concentration, that A. terreus poses.ResultsIn this study, five different Ustilago strains were screened for their growth and production of itaconic acid on defined media. The most promising strains were then used to find a suitable alternative feedstock, based on the local food industry. U. cynodontis ITA Max pH, a highly engineered production strain, was selected to determine the biologically available nitrogen concentration in thick juice and molasses. Based on these findings, thick juice was chosen as feedstock to ensure the necessary nitrogen limitation for itaconic acid production. U. cynodontis ITA Max pH was further characterised regarding osmotolerance and product inhibition and a successful scale-up to a 2 L stirred tank reactor was accomplished. A titer of 106.4 gitaconic acid/L with a theoretical yield of 0.50 gitaconic acid/gsucrose and a space-time yield of 0.72 gitaconic acid/L/h was reached.ConclusionsThis study demonstrates the utilisation of alternative feedstocks to produce ITA with Ustilaginaceae, without drawbacks in either titer or yield, compared to glucose fermentations.

A fluidized-bed-riser adsorption system for continuous bioproduct recovery from crude feedstock.

In this work, a novel technique for continuous purification of biologics from a crude feedstock is demonstrated with equipment referred to as Fluidized Bed Adsorption System (FBRAS). The development and validation of such unit operations were performed utilizing lysozyme as a model protein and Relisorb™ SP405/EB as a carrier. The performance of FBRAS to carry out combined clarification and purification was evaluated by capturing of antifungal peptides directly from the lysed broth. The novel technique reduced the number of process unit operations from six to three without having an impact on purity. Overall productivity increased by 250% in comparison to the existing downstream processing routine.

Efficient crystallization process of dodecanedioic acid by a pneumatically agitated crystallizer

Dodecanedioic acid (DC12) is mainly prepared via microbial synthesis in industrial production, however, the refined products have many drawbacks, such as low purity, poor crystal size distribution (CSD), and heavily agglomerated character, to name a few. Therefore, the refining process is a crucial step in DC12 preparation by microbial production. In this study, a novel pneumatically agitated crystallizer for DC12 refinery by gassing crystallization was developed. A batch cooling crystallization process was developed, providing the final crystal in 82.0% yield and purity increment to 99.20% at the end of crystallization process. Optimized conditions were determined as the initial supersaturation ratio of 300:3000 (g/mL) and gassing air flow rate of 4 L/min with solution preparation temperature of 85 °C. The crystal products with significantly better morphology characteristics than the starting crude feedstocks, including crystal uniformity and color brightness, were harvested. These obtained results indicated that the pneumatically agitated crystallizer-based crystallization process without any mechanical agitation was a robust and economically feasible technique. To our knowledge, this is also the first DC12 crystallization process with sole gassing agitation for unseeded batch cooling crystallization.

Characterisation and separation of infectious bursal disease virus-like particles using aqueous two-phase systems

Infectious bursal disease (IBD) causes considerable economic losses in the commercial poultry industry worldwide. The principal way to control IBD virus (IBDV), the causative agent of IBD, is still through vaccination programs. Virus-like particles (VLPs) are recognised as a safe and potent recombinant vaccine platform. This research work explores the characterisation and separation of infectious bursal disease virus-like particles (IBD-VLPs) from crude feedstock. Various characteristics were studied with high-performance size-exclusion chromatography (HP-SEC), sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE) and transmission electron microscopy (TEM) analyses. Subsequently, the separation of IBD-VLPs using polyethylene glycol (PEG)/sodium citrate-based aqueous two-phase systems (ATPSs) was conducted and optimised. Moreover, a scale-up study of the best ATPS constituted of 15% PEG 6000, 11% sodium citrate and 10% crude feedstock was performed to compare the separation performance of IBD-VLPs with and without centrifugation-assisted. The results indicated that the optimised ATPS with centrifugation-assisted for both 5 g and 50 g systems showed good recovery of IBD-VLPs of >97% in the interphase between the PEG-rich top and salt-rich bottom phases. These optimised systems also showed high removal efficiencies of impurities of >95%. The results demonstrated that aqueous two-phase extraction could be a promising technology for efficient VLPs separation.

Production of gamma-aminobutyric acid (GABA) by Bacillus subtilis BBEL02 fermentation using nitrogen-rich industrial wastes as crude feedstocks

Industrial-scale fermentation of γ-aminobutyric acid (GABA) is often limited by high cost of the starting raw materials. Increasing commercial applications of GABA in various industries have urged the research on finding the alternative substrates for the GABA production. The effects of concentration of different types of carbon and nitrogen sources; concentration of monosodium glutamate (MSG) in the defined medium for GABA production using the newly isolated Bacillus subtilis BBEL02 were studied. The feasibility of corn steep liquor (CSL) and soybean hydrolysate (SBH) as the replacement of nitrogen sources in the fermentation media for GABA production was also investigated. Highest GABA content (10.9 gL−1) and productivity (3.7 gL−1d−1) were achieved with defined medium containing 1 % (w/v) glucose, 2 % (v/v) SBH and 5 % (w/v) MSG. Dissolved oxygen (DO) of 80 % in 2-L fermenter presented the highest productivity (4.2 gL−1d−1) and GABA concentration (12.5 gL−1), however DO of 50 % was preferred because of the lower rotation speed to prevent cells deterioration.

Geochemistry of oils and condensates from the lower Eagle Ford formation, south Texas. Part 1: Crude assay measurements and SimDist modeling

Fifty-five oils and condensates produced from unconventional reservoirs in lower Eagle Ford Shale were characterized. Most of the samples are from the play area east of the Maverick Basin and west of the San Marcos Arch, while five samples are from wells east of the Arch. Crude assay measurements (API gravity, wt% sulfur, wt% nitrogen, viscosity at 25 and 40 °C, pour point, simulated distillation [SimDist] and C15+ chemical group-type fractionation) were conducted on dead oil samples using standard methods. All bulk properties were found to correlate well with each other. Relationships between crude assay measurement and production depth suggest that thermal maturation accounts for most of the variance in composition; however, different depth trends are observed in oils from the Karnes Trough area compared to locations west of that region.Refinery valuation and process models rely heavily on the fractional composition of crude feedstock defined by boiling point distribution. We constructed a model using seven crude assay parameters (API gravity, % S, % N, viscosity at 40 °C, and % C15+ saturates, % C15+ aromatics and % C15+ resins) as inputs for predicting SimDist boiling point curves. Using optimized parameters derived from this model, we found that SimDist curves can be modeled with nearly the same degree of accuracy using only API gravity and %S because of their strong correlation with the other bulk properties. This offers the prospect for refiners to identify wells producing specific grades of crude across the entire lower Eagle Ford play. The ability to construct a SimDist predictive model based only on the most basic assay measurements suggest that the compositional variations are due mostly to thermal maturity and the heterogeneity in source facies is insignificant in the lower Eagle Ford west of the San Marcos Arch. The SimDist predictive model is specific to the Eagle Ford shale oils and condensates, but the methodology could be used to construct formation specific models in other unconventional plays.

Evaluation of ionic liquids/salt aqueous biphasic flotation system on recovery of Kytococcus sedentarius TWHKC01 keratinase from crude feedstock

BackgroundIonic liquids (IL) is a unique environmental benign solvent with attractive characteristics which can be used in the formation of aqueous biphasic system. Aqueous biphasic flotation (ABF) system can improve the recovery yields of target biomolecules by incorporating the gas bubbles into the aqueous biphasic system (ABS). Keratinase has been widely applied in numerous industries due to the capability to degrade the keratinous wastes and convert them into valuable products via bioconversion. MethodsThe feasibility of IL-based ABF system to recover the Kytococcus sedentarius TWHKC01 keratinase from crude feedstock was investigated in this study. FindingsIL-based ABF system formed with 25.0% (w/w) 1-ethyl-3-methylimidazolium ethyl sulphate, (EMIM)(ESO4), 12.5% (w/w) sodium carbonate and 20% (w/w) crude feedstock demonstrated the optimum yield (Y) of 91.28% ± 0.64 and partition coefficient (Ke) of 8.39 ± 0.64. Keratinase was transferred to the IL-rich top phase of the ABF system which was operating at an airflow rate of 20 cc/min for 5 min. The keratinase selectivity (S) of 2.48 ± 0.31 and purification fold (PF) of 2.12 ± 0.04 were recorded. The IL-based ABF system is therefore highly recommended as a one-step operation to recover and purify the microbial keratinase due to the cost-saving and environmentally friendly properties.

Evaluation of Aqueous Biphasic Electrophoresis System Based on Halide-Free Ionic Liquids for Direct Recovery of Keratinase.

Aqueous biphasic electrophoresis system (ABES) incorporates electric fields into the biphasic system to separate the target biomolecules from crude feedstock. Ionic liquid (IL) is regarded as an excellent candidate as the phase-forming components for ABES because of the great electrical conductivity, which can promote the electromigration of biomolecules in ABES, and thereby enhances the separation efficiency of the target biomolecules from crude feedstock. The application of electric fields to the conventional biphasic system expedites the phase settling time of the biphasic system, which eases the subsequent scaling-up steps and reduces the overall processing time of the recovery process. Alkyl sulphate-based IL is a green and economical halide-free surfactant when compared to the other halide-containing IL. The feasibility of halide-free IL-based ABES to recover Kytococcus sedentarius TWHK01 keratinase was studied. Optimum partition coefficient (Ke = 7.53 ± 0.35) and yield (YT = 80.36% ± 0.71) were recorded with IL-ABES comprised of 15.0% (w/w) [EMIM][ESO4], 20.0% (w/w) sodium carbonate and 15% (w/w) crude feedstock. Selectivity (S) of 5.75 ± 0.27 was obtained with the IL-ABES operated at operation time of 5 min with 10 V voltage supplied. Halide-free IL is proven to be a potential phase-forming component of IL-ABES for large-scale recovery of keratinase.

Incorporation of electric fields to ionic liquids-based aqueous biphasic system for enhanced recovery of extracellular Kytococcus sedentarius TWHKC01 keratinase

BackgroundThe purification of keratinase is essential to enhance the catalytic efficiency of keratinase enzyme for industrial applications. Biphasic system incorporated with electric fields is an effective purification tool for the recovery of biomolecules. Ionic liquids (IL) with good conductivity are ideal phase-forming components for biphasic system which promotes the separation of charged biomolecules via electromigration. MethodsThis study aimed to evaluate the effect of electric fields on enhanced recovery of Kytococcus sedentarius TWHKC01 keratinase using IL-based biphasic system. FindingsKeratinase was recovered with biphasic system incorporated with anode in salt-rich bottom phase and cathode in IL-rich top phase operated at 5 V for 5 min. A total of 95.82% ± 0.17 of keratinase was attained with the biphasic system at pH 9 comprising of 20% (w/w) 1‑butyl‑3-methylimidazolium tetrafluoroborate ((Bmim)BF4) and 25% (w/w) ammonium sulphate loaded with 15% (w/w) K. sedentarius TWHKC01 crude feedstock. The selectivity (S) and purification fold (PFT) of keratinase were increased from 7.25 ± 1.50 to 10.87 ± 0.61 and 5.94 ± 0.22 to 8.22 ± 0.44, respectively with the incorporation of electrodes, demonstrating the efficiency of electric fields in enhancing the purity of recovered keratinase.

Leakage of gasoil from side cut piping in crude distillation unit of a petroleum refinery

This paper reports failure of elbow from side cut piping after three years of refinery gasoil service and piping metallurgy was SS316L austenitic stainless steel. Systematic investigations including visual inspection coupled with laboratory studies were carried out to determine the cause of failure. The samples from failure location were examined for chemical analysis, hardness, fractography, elemental analysis and metallographic evaluation. Further, crude feedstock was evaluated as well. Various characterization studies concluded that presence of material defect encapsulated in elbow prior to start of initial distillation column (side cut piping) operation was the root cause of failure. Localized stresses induced from internal pressure act on remaining wall thickness have lead to brittle fracture and resulted in gasoil leakage. The existence of material defect in failed elbow prior to initial operation had revealed poor pre-commissioning inspection practice. The origination of detected material defect was discussed and similar leakages shall be avoided by defect free piping components provided with proper support system at critical piping locations. The results in this article intended for petroleum refinery piping/energy pipeline operators for use of qualified piping components at critical locations especially during plant/piping commissioning stage.

Surface energetics to assess influence of biomass-type and biomass\u2013adsorbent interactions in expanded beds

In integrated bioprocessing applications, expanded bed adsorption (EBA) chromatography presents an opportunity to harvest biomolecules directly from the crude feedstock. However, unfavorable biomass interactions with adsorbent usually leads to fouling, which reduces its protein binding capacity as it alters column hydrodynamics and binding site availability. In this work, a detailed study on biomass adhesion behavior of four different industrially relevant microorganisms on 26 different, most commonly occurring adsorbent surfaces with varying degrees of surface energy and surface charge has been conducted. The results showed the derivation of a relative “stickiness” factor for every microorganism, which further classifies each organism based on their general degree of adhesion to surfaces with respect to one another. The obtained results can help to better understand the effect of biomass homogenization on biomass–adsorbent interactions in EBA. The data of surface energy and charge for the surfaces investigated in this work can be used to calculate the stickiness factor of other microorganisms of interest and may assist in the development of novel adsorbent materials for EBA chromatography.

Novel Process Simulation of Biodiesel Production from Crude Castor Oil

A continuous process flowsheet for biodiesel production from crude castor oil and methanol was developed on Aspen HYSYS simulation environment. It consists of an acid-catalysed sub-process for pre-treatment of the crude feedstock and a base-catalysed one for trans-esterification of treated oil. Reactions involved were defined using kinetic parameters obtained from lab-scale experiments for increased realism, and reactor setup was decided based on basic technological and economical assessments. Specifications of other unit operations and flows were obtained from different approximation methods, literature adaptation and trade-off consideration. Input and output quality controls were achieved via simple adjustments with tight tolerances. The use of crude feedstock reduced the cost of raw material while expanding the scale of the process, which managed to produce 8000 tons of biodiesel having purity of over 99 % a year. Specifically, the process could remove up to 90.83 % amount of free fatty acids in the crude oil and convert up to 93.82 % of oil to biodiesel.

Primary capture of Bacillus subtilis xylanase from crude feedstock using alcohol/salt liquid biphasic flotation

Abstract Liquid biphasic flotation (LBF) offers advanced downstream processing of bioproducts by floating of gas bubbles within the biphasic system to enhance the separation efficiency of target bioproducts. In this study, the feasibility of alcohol/salt LBF for primary capture of extracellular Bacillus subtilis xylanase from crude feedstock was investigated. The effects of types of phase-forming components; phase composition; concentration of crude feedstock load; and flotation time on the recovery performance of xylanase in LBF were evaluated. The recovery of xylanase was achieved in LBF comprised of 28 % (w/w) 2-propanol, 22 % (w/w) ammonium sulfate and 10 % (w/w) B. subtilis crude feedstock load. A maximum partition coefficient (Ke) of 13.29 ± 0.14, selectivity (S) of 24.71 ± 0.96 and recovery yield (YT) of 90.03 % ± 0.10 were recorded in the optimum 2-propanol/sulfate LBF with flotation time of 20 min. SDS-PAGE analysis revealed that B. subtilis xylanase was successfully recovered in the 2-propanol-rich top phase with high purity. The findings demonstrated that LBF is a practicable technology for the large-scale production of microbial xylanase in a one-step operation.

Efficiency of polymer/salt aqueous two-phase electrophoresis system for recovery of extracellular Kytococcus sedentarius TWHKC01 keratinase

Keratinase is widely applied in the bioconversion of feather waste to produce value-added bioproducts. The wide applications of industrial keratinase have urged the advancement in producing keratinase with high productivity and yields. The efficiency of aqueous two-phase electrophoresis system (ATPES) to recover extracellular Kytococcus sedentarius TWHKC01 keratinase was investigated. The operation time of 10 min and voltage of 10 V were applied for the recovery of K. sedentarius TWHKC01 keratinase with the PEG 1000/sulfate ATPES. Keratinase was recovered in the PEG-rich phase of the ATPES consists of 23% (w/w) PEG 1000, 18.5% (w/w) ammonium sulfate of pH 9, 20% (w/w) crude feedstock and with 3% (w/w) of NaCl added. Highest partition coefficient (Ke) of 4.63 ± 0.17, recovery yield (YT) of 78.75% ± 0.61, and purification fold (PFT) of 3.89 ± 0.84 were obtained in the optimal ATPES coupled with cathode electrode in top phase and anode electrode in the bottom phase. The introduction of electric field to the ATPS has enhanced the selectivity (S) of the system towards the extracellular keratinase by two-fold from 2.79 ± 0.46 to 5.68 ± 0.43, demonstrating the efficiency of ATPES in separating the keratinase from crude feedstock when compared to conventional two-phase system.

Effective purification of lysozyme from chicken egg white by tris(hydroxymethyl)aminomethane affinity nanofiber membrane

Polyacrylonitrile nanofiber membrane functionalized with tris(hydroxymethyl)aminomethane (P-Tris) was used in affinity membrane chromatography for lysozyme adsorption. The effects of pH and protein concentration on lysozyme adsorption were investigated. Based on Langmuir model, the adsorption capacity of P-Tris nanofiber membrane was estimated to be 345.83 mg/g. For the operation of dynamic membrane chromatography with three-layer P-Tris nanofiber membranes, the optimal operating conditions were at pH 9, 1.0 mL/min of feed flow rate, and 2 mg/mL of feed concentration. Chicken egg white (CEW) was applied as the crude feedstock of lysozyme in the optimized dynamic membrane chromatography. The percent recovery and purification factor of lysozyme obtained from the chromatography were 93.28% and 103.98 folds, respectively. Our findings demonstrated the effectiveness of P-Tris affinity nanofiber membrane for the recovery of lysozyme from complex CEW solution.

Primary purification of intracellular Halomonas salina ectoine using ionic liquids-based aqueous biphasic system

Ectoine is a zwitterionic amino acid derivative that can be naturally sourced from halophilic microorganisms. The increasing demands of ectoine in various industries have urged the researches on the cost-effective approaches on production of ectoine. Ionic liquids-based aqueous biphasic system (ILABS) was applied to recover Halomonas salina ectoine from cells hydrolysate. The 1-butyl-3-methylimidazolium tetrafluoroborate (Bmim)BF4 was used in the ILABS and the recovery efficiency of ILABS to recover ectoine from H.salina cells lysate was evaluated by determining the effects of phase composition; pHs; crude loading and additional neutral salt (NaCl). The hydrophilic ectoine was targeted to partition to the hydrophilic salt-rich phase. A total yield (YB) of 96.32% ± 1.08 of ectoine was obtained with ILABS of phase composition of 20% (w/w) (Bmim)BF4 and 30% (w/w) sulfate salts; system pH of 5.5 when the 20% (w/w) of crude feedstock was applied to the ILABS. There was no significant enhancement on the ectoine recovery efficiency using the ILABS when NaCl was added, therefore the ILABS composition without the additional neutral salt was recommended for the primary purification of ectoine. Partition coefficient (KE) of 30.80 ± 0.42, purity (PE) of 95.82% and enrichment factor (Ef) of 1.92 were recorded with the optimum (Bmim)BF4/sulfate ILABS. These findings have provided an insight on the feasibility of recovery of intracellular biomolecules using the green solvent-based aqueous system in one single-step operation.

Optimal design and operation of refinery hydrogen systems under multi-scale uncertainties

Robust design of hydrogen systems for large-scale refineries under multi-scale uncertainties has great potential for energy saving and cost reduction. The operation of hydrogen network is greatly affected by the rapidly changing market, deteriorating crude feedstock and varying operating conditions. In this paper, we propose a two-stage stochastic programming with flexibility constraints modeling framework that accounts for multi-scale uncertainties in an integrated strategy, aiming to achieve a good trade-off between the economic performance and the robustness of operation. A tailored-made global optimization algorithm based on multiparametric disaggregation technique is developed to tackle the computational challenge of the resulting MINLP problem with numerous bilinear terms. To illustrate the applicability of the proposed modeling framework and solution algorithm, studies on the optimal design of an industrial hydrogen system are presented. Multi-scenario operation strategy increases the operational flexibility and greatly reduces the total annualized cost in terms of the value of stochastic solution.

Integration of semi-batch cultivation and extraction for maximal lipid production in Chlamydomonas sp. Tai-03

Rapid improvements in bioseparation technology, new regulatory directives, product quality constraints, and the production efficiency have necessitated the development of more advanced and powerful downstream bioprocesses for biotechnology and biopharmaceuticals industrial. This has transformed in dramatically improvements in traditional bioseparation processes as well as the development of entirely new approaches. In this paper, we highlight some of these recent advances of integration of semi-batch cultivation and extraction for maximal lipid production in Chlamydomonas sp. Tai-03. This includes extractive cultivation, extractive bioconversion aqueous two-phase system, aqueous two-phase flotation, and newly developed liquid biphasic flotation. Alcohol/salt liquid biphasic flotation (LBF) with aid of ultrasonication which have the ability of killing two birds with one stone, it not only capable in cell rupturing, it also able to recover bioproducts simultaneously and continuously. The effect of varying crude feedstock concentration, flotation time, type of salt, concentration of salt, type of alcohol, concentration of alcohol, initial volumes of salt and alcohol were investigated. Microalgal biofuels or generation three biofuels have been widely recognized as potential replacements of fossil fuels. One of the most attractive option is the partial or full replacement of diesel fuel with microalgal biodiesel. Here, Chlamydomonas sp. Tai-03 was cultured using semi-batch cultivation to enhance its lipid production. Upon lowering the culture replacement fraction to 25%, the greatest biomass and lipid productivities were obtained at 1.23 ± 0.02 g/L/d and 239.6 ± 24.8 mg/L/d. After transesterification, palmitic acid (C16:0), oleic acid (C18:1), and linoleic acid (C18:2) were the main fatty acid methyl esters (FAMEs) present. These short-chain FAMEs and high productivities of Chlamydomonas sp. Tai-03 are suitable for biodiesel output.

Precipitation of complex antibody solutions: influence of contaminant composition and cell culture medium on the precipitation behavior

Preparative protein precipitation is known as a cost-efficient and easy-to-use alternative to chromatographic purification steps. This said, at the moment, there is no process for monoclonal antibodies (mAb) on the market, although especially polyethylene glycol-induced precipitation has shown great potential. One reason might be the highly complex behavior of each component of a crude feedstock during the precipitation process. For different investigated mAbs, significant variations in the host cell protein (HCP) reduction are observed. In contrast to the precipitation behavior of single components, the interactions and interplay in a complex feedstock are not fully understood yet. This work discusses the influence of contaminants on the precipitation behavior of two different mAbs, an IgG1, and an IgG2. By spiking the mAbs with mock solution, a complex feedstock could successfully be mimicked. Spiking contaminants influenced the yield and purity of the mAbs after the precipitation step, compared to the precipitation behavior of the single components. The mixture showed a decrease in the contaminant and mAb solubility. By re-buffering the mock solution prior to spiking, special salts, small molecules like amino acids, vitamins, or sugars could be depleted while larger ones like HCP or DNA were still present. Therefore, it was possible to distinguish the influence of small molecules and larger ones. Hence, mAb–macromolecular interaction could be identified as a possible reason for the observed higher precipitation propensity, while small molecules of the cell culture medium were identified as solubilisation factors during the precipitation process.