Biocalorimetry-aided monitoring of fungal pretreatment of lignocellulosic agricultural residues.

The present study aimed to investigate whether and how non-invasive biocalorimetric measurements could serve for process monitoring of fungal pretreatment during solid-state fermentation (SSF) of lignocellulosic agricultural residues such as wheat straw. Seven filamentous fungi representing different lignocellulose decay types were employed. Water-soluble sugars being immediately available after fungal pretreatment and those becoming water-extractable after enzymatic digestion of pretreated wheat straw with hydrolysing (hemi)cellulases were considered to constitute the total bioaccessible sugar fraction. The latter was used to indicate the success of pretreatments and linked to corresponding species-specific metabolic heat yield coefficients (YQ/X) derived from metabolic heat flux measurements during fungal wheat straw colonisation. An YQ/X range of about 120 to 140 kJ/g was seemingly optimal for pretreatment upon consideration of all investigated fungi and application of a non-linear Gaussian fitting model. Upon exclusion from analysis of the brown-rot basidiomycete Gloeophyllum trabeum, which differs from all other here investigated fungi in employing extracellular Fenton chemistry for lignocellulose decomposition, a linear relationship where amounts of total bioaccessible sugars were suggested to increase with increasing YQ/X values was obtained. It remains to be elucidated whether an YQ/X range being optimal for fungal pretreatment could firmly be established, or if the sugar accessibility for post-treatment generally increases with increasing YQ/X values as long as "conventional" enzymatic, i.e. (hemi)cellulase-based, lignocellulose decomposition mechanisms are operative. In any case, metabolic heat measurement-derived parameters such as YQ/X values may become very valuable tools supporting the assessment of the suitability of different fungal species for pretreatment of lignocellulosic substrates. KEY POINTS: • Biocalorimetry was used to monitor wheat straw pretreatment with seven filamentous fungi. • Metabolic heat yield coefficients (YQ/X) seem to indicate pretreatment success. • YQ/X values may support the selection of suitable fungal strains for pretreatment.

Applicability and information value of biocalorimetry for the monitoring of fungal solid-state fermentation of lignocellulosic agricultural by-products.

The applicability of biocalorimetry for monitoring fungal conversion of lignocellulosic agricultural by-products during solid-state fermentation (SSF) was substantiated through linking the non-invasive measurement of metabolic heat fluxes to conventional invasive determination of fungal activity (growth, substrate degradation, enzyme activity) parameters. For this, the fast-growing, cellulose-utilising ascomycete Stachybotrys chlorohalonata and the comparatively slow-growing litter-decay basidiomycete Stropharia rugosoannulata were investigated as model organisms during growth on solid wheat straw. Both biocalorimetric and non-calorimetric data may suggest R (ruderal)- and C (combative)-selected life history strategies in S. chlorohalonata and S. rugosoannulata, respectively. For both species, a strong linear correlation of the released metabolic heat with the corresponding fungal biomass was observed. Species-specific YQ/X values (metabolic heat released per fungal biomass unit) were obtained, which potentially enable use of biocalorimetric signals for the quantification of fungal biomass during single-species SSF processes. Moreover, YQ/X values may also indicate different fungal life history strategies and therefore be considered as useful parameters aiding fungal ecology research.

Biocalorimetric monitoring of glycoengineered P. pastoris cultivation for the production of recombinant huIFNα2b: A quantitative study based on mixed feeding strategies.

Real-time monitoring of glycoengineered Pichia pastoris by employing process analytical technology (PAT) tools is vital for gaining deeper insights into the therapeutic protein production process. The present study focuses on influence of mixed feed carbon substrates during the induction phases of glycoengineered P. pastoris cultivation, for recombinant human interferon α2b (huIFNα2b) production by employing calorimetric (biological heat rate, q B ) and respirometric (oxygen uptake rate and carbon dioxide evolution rate) measurements. Mixed feed stream of carbon substrates (methanol + glycerol, methanol + sorbitol) at a predetermined "C-molar ratios" were added during the induction phases. Methanol- and sorbitol-based mixed feeding approach resulted in an improved huIFNα2b titer of 288 mg/L by channeling of methanol predominantly towards an optimal functioning of AOX expression system. A stand-off between biomass yield YXSand biomass heat yieldYQX coefficient, degree of reduction of methanol and its cosubstrate (glycerol and sorbitol) determines the fraction of carbon energy channeled toward biomass and protein production, under strict aerobic conditions. Calorespirometric monitoring and assessment of thermal yields enables a reliable prediction of process variables, leading to futuristic efficient PAT-based feed rate control.

Effect of aeration and agitation on yeast inulinase production: a biocalorimetric investigation.

Air flow rate and agitation speed for inulinase production by Kluyveromyces marxianus were optimized based on metabolic heat release profiles. Shear stress and oxygen transfer (kLa) values were compared to assess the effects of aeration and agitation. At agitation rates of ≤ 100 rpm, the oxygen mass transfer rates were small and eventually led to less inulinase production, but at agitation rates > 150 rpm, loss of biomass resulted in less inulinase activity. Bio-reaction calorimeter (BioRc1e) experiment with aeration rates ≤ 0.5 lpm showed low kLa while at 1.5 lpm frothing of reactor contents caused loss of biomass and inulinase activity. The optimum conditions for aeration and agitation rate for K. marxianus in BioRc1e were 1 lpm and 150 rpm. Heat yield values obtained for the substrate, product and biomass reinstated the ongoing metabolic process. The heat release pattern could be a promising tool for optimization of bioprocess and in situ monitoring, with a possibility of interventions during the biotransformation process. At optimized aeration and agitation conditions, a two-fold increase in inulinase activity could be noticed.

Application of heat compensation calorimetry to an E. coli fed-batch process.

The application of biocalorimetry to fermentation processes offers advantageous insights, while being less complex compared to other, sophisticated PAT solutions. Although the general concept is established, calorimetric methods vary in detail. In this work, a special approach, called heat compensation calorimetry, was applied to an E. coli fed-batch process. Much work has been done for batch processes, proving the validity and accuracy of this calorimetric mode. However, the adaption of this strategy to fed-batch processes has some implications. In the first section of this work, batch fermentations were performed, comparing heat capacity calorimetry to the compensation mode. Both processes showed very good agreement by means of growth behavior. The heat related differences, e.g. temperature profiles, were obvious. In addition, the impact of the chosen mode on the calculation of in-process heat transfer coefficients was shown. Finally, a fed-batch fermentation was performed. The compensation mode was kept sufficiently, up to the point where the metabolic heat production accelerated strongly. Controller tuning was a neuralgic point, which would have needed further optimization under these conditions. Nevertheless, in the present work it was possible to realize a working compensation process while demonstrating critical aspects that must be considered when establishing such approach.

Metabolic pathway and role of individual species in the bacterial consortium for biodegradation of azo dye: A biocalorimetric investigation.

In this study, an attempt was made to investigate the functional role and metabolic behaviour of the monoculture (Staphylococcus lentus (SL), Bacillus flexus (BF) and Pseudomonas aeruginosa (PA)) in the bacterial biocenosis for biotransformation of an azo dye. The power-time profile obtained from consortia depicted three distinct peaks, which correlated well with the individual bacterial growth (PA > SL > BF), indicating the synergistic relation and division of labour in the biocenosis. The heat release pattern was used to identify the sequential behaviour of microbial consortia in real time. Yield calculation based on total heat liberated to the complete substrate utilization Y (Q/S) for PA, SL, and BF were 15.99, 16.68, 7.32 kJ/L respectively. Similarly, the oxy calorific values Y (Q/O) for the above species are respectively 386, 375, 440 kJ/mol and indicates the aerobic nature of microorganism employed. Further, the metabolome produced during the biotransformation were identified using Gas Chromatography-Mass Spectrometry (GC-MS), based on which a plausible pathway was predicted. The abundant metabolites were palmitic acid (m/z = 256) and diethyl phthalate (m/z = 222.2). The abundance of diethyl phthalate was much lesser in the consortia compared to the monoculture. Thus, the biocalorimetric heat yield calculation along with the stoichiometry and plausible pathway based biochemical elucidation provides a mechanistic basis for understanding the azo-dye biotransformation by the monocultures in consortia.

Modeling of exo-inulinase biosynthesis by Kluyveromyces marxianus in fed-batch mode: correlating production kinetics and metabolic heat fluxes.

A metabolic heat-based model was used for estimating the growth of Kluyveromyces marxianus, and the modified Luedeking-Piret kinetic model was used for describing the inulinase production kinetics. For the first time, a relationship was developed to relate inulinase production kinetics directly to metabolic heat generated, which corroborated well with the experimental data (with R 2 values of above 0.9). It also demonstrated the predominantly growth-associated nature of the inulinase production with Luedeking-Piret parameters α and ß, having values of 0.75 and 0.033, respectively, in the exponential feeding experiment. MATLAB was used for simulating the inulinase production kinetics which demonstrated the model's utility in performing real-time prediction of inulinase concentration with metabolic heat data as input. To validate the model predictions, a biocalorimetric (Bio RC1e) experiment for inulinase production by K. marxianus was performed. The inulinase concentration (IU/mL) values acquired from the model in were validated with the experimental values and the metabolic heat data. This modeling approach enabled the optimization, monitoring, and control of inulinase production process using the real-time biocalorimetric (Bio RC1e) data. Gas chromatography and mass spectrometry analysis were carried out to study the overflow metabolism taking place in K. marxianus inulinase production.

Calorespirometric feeding control enhances bioproduction from toxic feedstocks-Demonstration for biopolymer production out of methanol.

The sustainable production of fuels and industrial bulk chemicals by microorganisms in biotechnological processes is promising but still facing various challenges. In particular, toxic substrates require an efficient process control strategy. Methanol, as an example, has the potential to become a major future feedstock due to its availability from fossil and renewable resources. However, besides being toxic, methanol is highly volatile. To optimize its dosage during microbial cultivations, an innovative, predictive process control strategy based on calorespirometry, i.e., simultaneous measurements of heat and CO2 emission rates, was developed. This rarely used technique allows an online-estimation of growth parameters such as the specific growth rate and substrate consumption rate as well as a detection of shifts in microbial metabolism thus enabling an adapted feeding for different phases of growth. The calorespirometric control strategy is demonstrated exemplarily for growth of the methylotrophic bacterium Methylobacterium extorquens on methanol and compared to alternative control strategies. Applying the new approach, the methanol concentration could be maintained far below a critical limit, while increased growth rates of M. extorquens and higher final contents of the biopolymer polyhydroxybutyrate were obtained. Biotechnol. Bioeng. 2016;113: 2113-2121. © 2016 Wiley Periodicals, Inc.

Metabolism and biotransformation of azo dye by bacterial consortium studied in a bioreaction calorimeter.

Effluents from leather and textile industries are difficult for treatment owing to its recalcitrant nature. Since the volume of effluent generated are high, a robust and active microbial consortia is required for effective treatment. The focus in the present study is the calorimetric traceability of the metabolic behaviors of mixed microbial consortia, while it grows and degrades recalcitrant substance such as an azo dye acid blue 113. The consortium exhibited a syntrophic division of substrate and was effective in degrading dye up to 0.8g/l. Notably, it was able to degrade 93.7% of the azo dye in 12-16h whereas its monocultures required 48-72h to reach 82.1%. The products of biodegradation were analyzed and the chemical pathway substantiated using chemical thermodynamic and energy release patterns. MTT assay confirmed that emanates are eco-friendly. Heat profile pattern and bioenergetics provide fundamental data for a feasible application in commercial level.