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.

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.

What does calorimetry and thermodynamics of living cells tell us?

This article presents and compares several thermodynamic methods for the quantitative interpretation of data from calorimetric measurements. Heat generation and absorption are universal features of microbial growth and product formation as well as of cell cultures from animals, plants and insects. The heat production rate reflects metabolic changes in real time and is measurable on-line. The detection limit of commercially available calorimetric instruments can be low enough to measure the heat of 100,000 aerobically growing bacteria or of 100 myocardial cells. Heat can be monitored in reaction vessels ranging from a few nanoliters up to many cubic meters. Most important the heat flux measurement does not interfere with the biological process under investigation. The practical advantages of calorimetry include the waiver of labeling and reactants. It is further possible to assemble the thermal transducer in a protected way that reduces aging and thereby signal drifts. Calorimetry works with optically opaque solutions. All of these advantages make calorimetry an interesting method for many applications in medicine, environmental sciences, ecology, biochemistry and biotechnology, just to mention a few. However, in many cases the heat signal is merely used to monitor biological processes but only rarely to quantitatively interpret the data. Therefore, a significant proportion of the information potential of calorimetry remains unutilized. To fill this information gap and to motivate the reader using the full information potential of calorimetry, various methods for quantitative data interpretations are presented, evaluated and compared with each other. Possible errors of interpretation and limitations of quantitative data analysis are also discussed.

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.

Fungal Lignocellulose Utilisation Strategies from a Bioenergetic Perspective: Quantification of Related Functional Traits Using Biocalorimetry.

In the present study, we investigated whether a non-invasive metabolic heat flux analysis could serve the determination of the functional traits in free-living saprotrophic decomposer fungi and aid the prediction of fungal influences on ecosystem processes. For this, seven fungi, including ascomycete, basidiomycete, and zygomycete species, were investigated in a standardised laboratory environment, employing wheat straw as a globally relevant lignocellulosic substrate. Our study demonstrates that biocalorimetry can be employed successfully to determine growth-related fungal activity parameters, such as apparent maximum growth rates (AMGR), cultivation times until the observable onset of fungal growth at AMGR (tAMGR), quotients formed from the AMGR and tAMGR (herein referred to as competitive growth potential, CGP), and heat yield coefficients (YQ/X), the latter indicating the degree of resource investment into fungal biomass versus other functional attributes. These parameters seem suitable to link fungal potentials for biomass production to corresponding ecological strategies employed during resource utilisation, and therefore may be considered as fungal life history traits. A close connection exists between the CGP and YQ/X values, which suggests an interpretation that relates to fungal life history strategies.

Photocalorespirometry (Photo-CR): A Novel Method for Access to Photosynthetic Energy Conversion Efficiency.

One key parameter for assessing the CO2 fixation in aquatic ecosystems but also for the productivity of photobioreactors is the energy conversion efficiency (PE) by the photosynthetic apparatus. PE strictly depends on a range of different fluctuating environmental conditions and is therefore highly variable. PE is the result of complex metabolic control. At the moment PE can only be determined indirectly. Furthermore, the currently available techniques either capture only short time processes, thus reflecting only parts of the photosynthetic engine, or quantify the total process but only with limited time resolution. To close this gap, we suggest for the first time the direct measurement of the fixed energy combined with respirometry, called photocalorespirometry (Photo-CR). The proof of the principle of Photo-CR was established with the microalga Chlamydomonas reinhardtii. The simultaneous measurement of oxygen production and energy fixation provides an calorespirometric ratio of -(437.9 ± 0.7) kJ mol-1 under low light conditions. The elevated calorespirometric ratio under high light conditions provides an indication of photo-protective mechanisms. The Photo-CR delivers the PE in real time, depending on the light intensity. Energetic differences less than 0.14% at radiation densities of up to 800 µE m-2 s-1 can be quantified. Other photosynthetic growth parameters (e.g. the specific growth rate of 0.071 h-1, the cell specific energy conservation of 30.9 ± 1.3 pW cell-1 at 150 µE m-2 s-1 and the number of photons (86.8) required to fix one molecule of CO2) can easily be derived from the Photo-CR data.

A simple method for the measurement of metabolic heat production rates during solid-state fermentations using ß-carotene production with Blakeslea trispora as a model system.

Solid-state fermentation (SSF) technology has been rapidly developed for the past 10 years as a production platform for secondary metabolites, biofuels, food, and pharmaceuticals. Yet, the main drawback of SSF is the local temperature rise of up to 20 K, which potentially reduces the strain activity and inactivates heat sensible products. Due to the low heat capacity and thermal conductivity of mixtures of air with plant material, in comparison to aqueous suspensions in submerged fermentations, heat from metabolic processes is less efficiently dissipated. The exact knowledge of the metabolic heat generation during SSF processes is crucial to guide strategies against overheating. In this work, a simple method using a cost-efficient multichannel instrument is proposed, which allows the determination of heat generation during SSF processes. This method was successfully tested and validated with Blakeslea trispora producing ß-carotene during growth on barley. Additionally, the consequences of the generated metabolic heat during SSF on temperature rise and water evaporation were discussed. Finally, changes in growth and product concentration could also be detected by the heat signal, implying the potential as a timesaving screening method.