Application of MALDI-MS for characterization of fucoidan hydrolysates and screening of endo-fucoidanase activity.

Brown macroalgae synthesize large amounts of fucoidans, sulfated fucose-containing polysaccharides, in the ocean. Fucoidans are of importance for their recently discovered contribution to marine carbon dioxide sequestration and due to their potential applications in biotechnology and biomedicine. However, fucoidans have high intra- and intermolecular diversity that challenges assignment of structure to biological function and the development of applications. Fucoidan-active enzymes may be used to simplify this diversity by producing defined oligosaccharides more applicable for structural refinement, characterization, and structure to function assignment for example via bioassays. In this study, we combined MALDI mass spectrometry with biocatalysis to show that the endo-fucoidanases P5AFcnA and Wv323 can produce defined oligosaccharide structures directly from unrefined macroalgal biomass. P5AFcnA released oligosaccharides from seven commercial fucoidan extracts in addition to unrefined biomass of three macroalgae species indicating a broadly applicable approach reproducible across 10 species. Both MALDI-TOF/TOF and AP-MALDI-Orbitrap systems were used, demonstrating that the approach is not instrument-specific and exploiting their combined high-throughput and high-resolution capabilities. Overall, the combination of MALDI-MS and endo-fucoidanase assays offers high-throughput evaluation of fucoidan samples and also enables extraction of defined oligosaccharides of known structure from unrefined seaweed biomass.

Raman spectroscopy online monitoring of biomass production, intracellular metabolites and carbon substrates during submerged fermentation of oleaginous and carotenogenic microorganisms.

BACKGROUND: Monitoring and control of both growth media and microbial biomass is extremely important for the development of economical bioprocesses. Unfortunately, process monitoring is still dependent on a limited number of standard parameters (pH, temperature, gasses etc.), while the critical process parameters, such as biomass, product and substrate concentrations, are rarely assessable in-line. Bioprocess optimization and monitoring will greatly benefit from advanced spectroscopy-based sensors that enable real-time monitoring and control. Here, Fourier transform (FT) Raman spectroscopy measurement via flow cell in a recirculatory loop, in combination with predictive data modeling, was assessed as a fast, low-cost, and highly sensitive process analytical technology (PAT) system for online monitoring of critical process parameters. To show the general applicability of the method, submerged fermentation was monitored using two different oleaginous and carotenogenic microorganisms grown on two different carbon substrates: glucose fermentation by yeast Rhodotorula toruloides and glycerol fermentation by marine thraustochytrid Schizochytrium sp. Additionally, the online FT-Raman spectroscopy approach was compared with two at-line spectroscopic methods, namely FT-Raman and FT-infrared spectroscopies in high throughput screening (HTS) setups. RESULTS: The system can provide real-time concentration data on carbon substrate (glucose and glycerol) utilization, and production of biomass, carotenoid pigments, and lipids (triglycerides and free fatty acids). Robust multivariate regression models were developed and showed high level of correlation between the online FT-Raman spectral data and reference measurements, with coefficients of determination (R2) in the 0.94-0.99 and 0.89-0.99 range for all concentration parameters of Rhodotorula and Schizochytrium fermentation, respectively. The online FT-Raman spectroscopy approach was superior to the at-line methods since the obtained information was more comprehensive, timely and provided more precise concentration profiles. CONCLUSIONS: The FT-Raman spectroscopy system with a flow measurement cell in a recirculatory loop, in combination with prediction models, can simultaneously provide real-time concentration data on carbon substrate utilization, and production of biomass, carotenoid pigments, and lipids. This data enables monitoring of dynamic behaviour of oleaginous and carotenogenic microorganisms, and thus can provide critical process parameters for process optimization and control. Overall, this study demonstrated the feasibility of using FT-Raman spectroscopy for online monitoring of fermentation processes.

Culture adaptation for enhanced biogas production from birch wood applying stable carbon isotope analysis to monitor changes in the microbial community.

Birch wood is a potential feedstock for biogas production in Northern Europe; however, the lignocellulosic matrix is recalcitrant preventing efficient conversion to methane. To improve digestibility, birch wood was thermally pre-treated using steam explosion at 220 °C for 10 min. The steam-exploded birch wood (SEBW) was co-digested with cow manure for a period of 120 days in continuously fed CSTRs where the microbial community adapted to the SEBW feedstock. Changes in the microbial community were tracked by stable carbon isotopes- and 16S r RNA analyses. The results showed that the adapted microbial culture could increase methane production up to 365 mL/g VS day, which is higher than previously reported methane production from pre-treated SEBW. This study also revealed that the microbial adaptation significantly increased the tolerance of the microbial community against the inhibitors furfural and HMF which were formed during pre-treatment of birch. The results of the microbial analysis indicated that the relative amount of cellulosic hydrolytic microorganisms (e.g. Actinobacteriota and Fibrobacterota) increased and replaced syntrophic acetate bacteria (e.g. Cloacimonadota, Dethiobacteraceae, and Syntrophomonadaceae) as a function of time. Moreover, the stable carbon isotope analysis indicated that the acetoclastic pathway became the main route for methane production after long-term adaptation. The shift in methane production pathway and change in microbial community shows that for anaerobic digestion of SEBW, the hydrolysis step is important. Although acetoclastic methanogens became dominant after 120 days, a potential route for methane production could also be a direct electron transfer among Sedimentibacter and methanogen archaea.

Production of docosahexaenoic acid from spruce sugars using Aurantiochytrium limacinum.

In this study lignocellulosic sugars from Norway spruce were used for production of docosahexaenoic acid (DHA) by the marine thraustochytrid Aurantiochytrium limacinum SR21. Enzymatically prepared spruce hydrolysate was combined with a complex nitrogen source and different amounts of salts. Shake flask batch cultivations revealed that addition of extra salts was not needed for optimal growth. Upscaling to fed-batch bioreactors yielded up to 55 g/L cell dry mass and a total fatty acid content of 44% (w/w) out of which 1/3 was DHA. Fourier transform infrared spectroscopy was successfully applied as a rapid method for monitoring lipid accumulation in A. limacinum SR21. Thus, this proof-of-principle study clearly demonstrates that crude spruce hydrolysates can be directly used as a novel and sustainable resource for production of DHA.

Developing a biogas centralised circular bioeconomy using agricultural residues - Challenges and opportunities.

Anaerobic digestion (AD) can be used as a stand-alone process or integrated as part of a larger biorefining process to produce biofuels, biochemicals and fertiliser, and has the potential to play a central role in the emerging circular bioeconomy (CBE). Agricultural residues, such as animal slurry, straw, and grass silage, represent an important resource and have a huge potential to boost biogas and methane yields. Under the CBE concept, there is a need to assess the long-term impact and investigate the potential accumulation of specific unwanted substances. Thus, a comprehensive literature review to summarise the benefits and environmental impacts of using agricultural residues for AD is needed. This review analyses the benefits and potential adverse effects related to developing biogas-centred CBE. The identified potential risks/challenges for developing biogas CBE include GHG emission, nutrient management, pollutants, etc. In general, the environmental risks are highly dependent on the input feedstocks and resulting digestate. Integrated treatment processes should be developed as these could both minimise risks and improve the economic perspective.

Extraction of high purity fucoidans from brown seaweeds using cellulases and alginate lyases.

Fucoidans are fucose rich sulfated polysaccharides that are found in the cell wall of brown seaweeds and have been shown to have several beneficial bioactivities. In the present study, we report a new enzymatic extraction technique for the production of pure and intact fucoidans from the two brown seaweeds Saccharina latissima and Alaria esculenta. This new extraction protocol uses the commercial cellulase blend Cellic® CTec2 in combination with endo- and exo-acting thermophilic alginate lyases. The fucoidans obtained by this extraction technique are compared to traditionally extracted fucoidans in terms of chemical compositions and molecular weights and are shown to contain significantly higher amounts of fucose and sulfate, the main components of fucoidans, while cellulose, laminarin, and alginate contamination is low. Thus, by using this combination of enzymes, the extracted fucoidans do not undergo depolymerization during extraction and additional purification steps are not needed. The high purity fucoidans isolated by this new enzymatic extraction technique can be used to provide insight into the different fucoidan structures and biological activities.

Stimulating biogas production from steam-exploded birch wood using Fenton reaction and fungal pretreatment.

Delignification of steam-exploded birch wood (SEBW) was stimulated using a pretreatment method including Fenton reaction (FR) and fungi. SEBW was employed as a substrate to optimize the Fe(III) and Fe(II) dosage in FR. Maximum iron-binding to SEBW was obtained at pH 3.5. FR pretreatment increased biological methane yields from 257 mL/g vS in control to 383 and 352 mL/ g vS in samples with 0.5 mM Fe(II) and 1.0 mM Fe(III), respectively. Further enzymatic pretreatment using a commercial cellulase cocktail clearly improved methane production rate but only increased the final methane yields by 2-9 %. Finally, pretreatments with the fungi Pleurotus ostreatus (PO) and Lentinula edodes (LE), alone or in combination with FR, were carried out. SEBW pretreated with only LE and samples pretreated with PO and1 mM Fe(III) + H2O2 increased the methane production yield to 420 and 419 mL/g vS respectively. These pretreatments delignified SEBW up to 25 %.

Starch-Rich Microalgae as an Active Ingredient in Beer Brewing.

Microalgal biomass is widely studied for its possible application in food and human nutrition due to its multiple potential health benefits, and to address raising sustainability concerns. An interesting field whereby to further explore the application of microalgae is that of beer brewing, due to the capacity of some species to accumulate large amounts of starch under specific growth conditions. The marine species Tetraselmis chui is a well-known starch producer, and was selected in this study for the production of biomass to be explored as an active ingredient in beer brewing. Cultivation was performed under nitrogen deprivation in 250 L tubular photobioreactors, producing a biomass containing 50% starch. The properties of high-starch microalgal biomass in a traditional mashing process were then assessed to identify critical steps and challenges, test the efficiency of fermentable sugar release, and develop a protocol for small-scale brewing trials. Finally, T. chui was successfully integrated at a small scale into the brewing process as an active ingredient, producing microalgae-enriched beer containing up to 20% algal biomass. The addition of microalgae had a noticeable effect on the beer properties, resulting in a product with distinct sensory properties. Regulation of pH proved to be a key parameter in the process.

Metabolic pathways for biosynthesis and degradation of starch in Tetraselmis chui during nitrogen deprivation and recovery.

Tetraselmis chui is known to accumulate starch when subjected to stress. This phenomenon is widely studied for the purpose of industrial production and process development. Yet, knowledge about the metabolic pathways involved is still immature. Hence, in this study, transcription of 27 starch-related genes was monitored under nitrogen deprivation and resupply in 25 L tubular photobioreactors. T. chui proved to be an efficient starch producer under nitrogen deprivation, accumulating starch up to 56% of relative biomass content. The prolonged absence of nitrogen led to an overall down-regulation of the tested genes, in most instances maintained even after nitrogen replenishment when starch was actively degraded. These gene expression patterns suggest post-transcriptional regulatory mechanisms play a key role in T. chui under nutrient stress. Finally, the high productivity combined with an efficient recovery after nitrogen restitution makes this species a suitable candidate for industrial production of high-starch biomass.

Fecal Excretion and Whole-Body Retention of Macro and Micro Minerals in Atlantic Salmon Fed Torula Yeast Grown on Sugar Kelp Hydrolysate.

Yeast is a microbial feed ingredient that can be produced from non-food biomasses. Brown seaweed contains high levels of complex carbohydrates that are not digested to any extent by monogastric animals but can be used as carbon sources for yeast production. The objective of this study was to investigate how minerals originating from brown macroalgae (Saccharina latissima) are incorporated in Cyberlindnera jadinii yeast and to assess the bioavailability of these different minerals as well as their accumulation into different organs of Atlantic salmon. The yeast C. jadinii was produced on a seaweed hydrolysate mixed with a sugar-rich wood hydrolysate in a 9:1 volume ratio and fed to Atlantic salmon (Salmo salar) in two different experiments: a digestibility experiment with 30% dietary inclusion of yeast and a retention experiment with increasing inclusion of yeast (5, 10, and 20%). Seaweed minerals such as zinc (Zn), copper (Cu), iodine (I), manganese (Mn), and cobalt (Co) were incorporated to a high degree in the yeast. The apparent fecal excretion of minerals was similar in both experiments, in general, with low excretion of, I, bromine (Br), and arsenic (As) (ranging from 18.0% to 63.5%) and high excretion of iron (Fe), Cu, Mn, aluminum (Al), cadmium (Cd) and lead (Pb) (ranging from 56.9% to <100%), despite the different fish size and fecal sampling method. High levels of Cu, I, Br, and Co in the yeast resulted in a linear decrease (p < 0.05) in retention of these minerals in salmon fed increasing levels of yeast. Despite increasing amounts of these minerals in the feed, whole-body levels of Cu and Mn remained stable, whereas whole-body levels of Co, somewhat unexpectedly, decreased with increased dietary yeast inclusion. The Cd from the yeast had low bioavailability but was concentrated more in the kidney (0.038 mg kg-1) and liver (0.025 mg kg-1) than in muscle (0.0009 mg kg-1). The given Cd level in fish strengthens the indication that it is safe to feed salmon with up to 20% inclusion of seaweed yeast without exceeding the maximum limit for Cd of 0.05 mg kg-1 w.w. in fish meat. The level and retention (p < 0.05) of As were lower in the yeast compared to fishmeal. The high level of iodine in S. latissima (3900 mg kg-1) was partly transferred to the yeast, and salmon fed increasing levels of yeast displayed a linear increase in whole-body I content (p < 0.05). There is, however, a need for a growth experiment with larger fish to draw any firm conclusions regarding food safety. Overall, this study shows that yeast grown on hydrolyzed seaweed can be a suitable mineral source for Atlantic salmon, especially when diets are low in fishmeal.

Impact of operational conditions on methane yield and microbial community composition during biological methanation in in situ and hybrid reactor systems.

BACKGROUND: Biogas can be upgraded to methane biologically by adding H2 to biogas reactors. The process is called biological methanation (BM) and can be done in situ in a regular biogas reactor or the biogas can be transferred to a separate ex situ upgrading reactor. The hybrid BM concept, a combination of in situ and ex situ BM, has received little attention, and only a few studies have been reported. The hybrid BM has the advantage of resolving the issue of pH increment during in situ BM, while the size of the ex situ BM reactor could be reduced. RESULTS: In this study, the efficiency of in situ and hybrid biological methanation (BM) for upgrading raw biogas was investigated. The hybrid BM system achieved a CH4 yield of 257 mL gVS-1 when degrading a feedstock blend of manure and cheese waste. This represented an increase in methane yield of 76% when compared to the control reactor with no H2 addition. A 2:1 H2:CO2 ratio resulted in stable reactor performance, while a 4:1 ratio resulted in a high accumulation of volatile fatty acids. H2 consumption rate was improved when a low manure-cheese waste ratio (90%:10%) was applied. Furthermore, feeding less frequently (every 48 h) resulted in a higher CH4 production from CO2 and H2. Methanothermobacter was found to dominate the archaeal community in the in situ BM reactor, and its relative abundance increased over the experimental time. Methanosarcina abundance was negatively affected by H2 addition and was nearly non-existent at the end of the experiment. CONCLUSIONS: Our results show that hybrid BM outperforms in situ BM in terms of total CH4 production and content of CH4 in the biogas. In comparison to in situ BM, the use of hybrid BM increased CH4 yield by up to 42%. Furthermore, addition of H2 at 2:1 H2:CO2 ratio in in situ BM resulted in stable reactor operation.

Calcium Affects Polyphosphate and Lipid Accumulation in Mucoromycota Fungi.

Calcium controls important processes in fungal metabolism, such as hyphae growth, cell wall synthesis, and stress tolerance. Recently, it was reported that calcium affects polyphosphate and lipid accumulation in fungi. The purpose of this study was to assess the effect of calcium on the accumulation of lipids and polyphosphate for six oleaginous Mucoromycota fungi grown under different phosphorus/pH conditions. A Duetz microtiter plate system (Duetz MTPS) was used for the cultivation. The compositional profile of the microbial biomass was recorded using Fourier-transform infrared spectroscopy, the high throughput screening extension (FTIR-HTS). Lipid content and fatty acid profiles were determined using gas chromatography (GC). Cellular phosphorus was determined using assay-based UV-Vis spectroscopy, and accumulated phosphates were characterized using solid-state 31P nuclear magnetic resonance spectroscopy. Glucose consumption was estimated by FTIR-attenuated total reflection (FTIR-ATR). Overall, the data indicated that calcium availability enhances polyphosphate accumulation in Mucoromycota fungi, while calcium deficiency increases lipid production, especially under acidic conditions (pH 2-3) caused by the phosphorus limitation. In addition, it was observed that under acidic conditions, calcium deficiency leads to increase in carotenoid production. It can be concluded that calcium availability can be used as an optimization parameter in fungal fermentation processes to enhance the production of lipids or polyphosphates.

Impact of down-stream processing on functional properties of yeasts and the implications on gut health of Atlantic salmon (Salmo salar).

Yeasts are becoming popular as novel ingredients in fish feeds because of their potential to support better growth and concomitantly ensure good fish health. Here, three species of yeasts (Cyberlindnera jadinii, Blastobotrys adeninivorans and Wickerhamomyces anomalus), grown on wood sugars and hydrolysates of chicken were subjected to two down-stream processes, either direct heat-inactivation or autolysis, and the feed potential of the resulting yeast preparations was assessed through a feeding trial with Atlantic salmon fry. Histological examination of distal intestine based on widening of lamina propria, showed that autolyzed W. anomalus was effective in alleviating mild intestinal enteritis, while only limited effects were observed for other yeasts. Our results showed that the functionality of yeast in counteracting intestinal enteritis in Atlantic salmon was dependent on both the type of yeast and the down-stream processing method, and demonstrated that C. jadinii and W. anomalus have promising effects on gut health of Atlantic salmon.

In situ measurements of oxidation-reduction potential and hydrogen peroxide concentration as tools for revealing LPMO inactivation during enzymatic saccharification of cellulose.

BACKGROUND: Biochemical conversion of lignocellulosic biomass to simple sugars at commercial scale is hampered by the high cost of saccharifying enzymes. Lytic polysaccharide monooxygenases (LPMOs) may hold the key to overcome economic barriers. Recent studies have shown that controlled activation of LPMOs by a continuous H2O2 supply can boost saccharification yields, while overdosing H2O2 may lead to enzyme inactivation and reduce overall sugar yields. While following LPMO action by ex situ analysis of LPMO products confirms enzyme inactivation, currently no preventive measures are available to intervene before complete inactivation. RESULTS: Here, we carried out enzymatic saccharification of the model cellulose Avicel with an LPMO-containing enzyme preparation (Cellic CTec3) and H2O2 feed at 1 L bioreactor scale and followed the oxidation-reduction potential and H2O2 concentration in situ with corresponding electrode probes. The rate of oxidation of the reductant as well as the estimation of the amount of H2O2 consumed by LPMOs indicate that, in addition to oxidative depolymerization of cellulose, LPMOs consume H2O2 in a futile non-catalytic cycle, and that inactivation of LPMOs happens gradually and starts long before the accumulation of LPMO-generated oxidative products comes to a halt. CONCLUSION: Our results indicate that, in this model system, the collapse of the LPMO-catalyzed reaction may be predicted by the rate of oxidation of the reductant, the accumulation of H2O2 in the reactor or, indirectly, by a clear increase in the oxidation-reduction potential. Being able to monitor the state of the LPMO activity in situ may help maximizing the benefit of LPMO action during saccharification. Overcoming enzyme inactivation could allow improving overall saccharification yields beyond the state of the art while lowering LPMO and, potentially, cellulase loads, both of which would have beneficial consequences on process economics.

Metal and Phosphate Ions Show Remarkable Influence on the Biomass Production and Lipid Accumulation in Oleaginous Mucor circinelloides.

The biomass of Mucor circinelloides, a dimorphic oleaginous filamentous fungus, has a significant nutritional value and can be used for single cell oil production. Metal ions are micronutrients supporting fungal growth and metabolic activity of cellular processes. We investigated the effect of 140 different substrates, with varying amounts of metal and phosphate ions concentration, on the growth, cell chemistry, lipid accumulation, and lipid profile of M. circinelloides. A high-throughput set-up consisting of a Duetz microcultivation system coupled to Fourier transform infrared spectroscopy was utilized. Lipids were extracted by a modified Lewis method and analyzed using gas chromatography. It was observed that Mg and Zn ions were essential for the growth and metabolic activity of M. circinelloides. An increase in Fe ion concentration inhibited fungal growth, while higher concentrations of Cu, Co, and Zn ions enhanced the growth and lipid accumulation. Lack of Ca and Cu ions, as well as higher amounts of Zn and Mn ions, enhanced lipid accumulation in M. circinelloides. Generally, the fatty acid profile of M. circinelloides lipids was quite consistent, irrespective of media composition. Increasing the amount of Ca ions enhanced polyphosphates accumulation, while lack of it showed fall in polyphosphate.

Fourier-transform infrared spectroscopy for monitoring proteolytic reactions using dry-films treated with trifluoroacetic acid.

In this study we explore the potential of using Fourier-transform infrared (FTIR) spectra of trifluoroacetate-protein and peptide complexes for monitoring proteolytic reactions. The idea of treating dry-films of protein hydrolysates with trifluoroacetic acid (TFA) prior to FTIR analysis is based on the unique properties of TFA. By adding a large excess of TFA to protein hydrolysate samples, the possible protonation sites of the proteins and peptides will be saturated. In addition, TFA has a low boiling point when protonated as well as complex-forming abilities. When forming TFA-treated dry-films of protein hydrolysates, the excess TFA will evaporate and the deprotonated acid (CF3COO-) will interact as a counter ion with the positive charges on the sample materials. In the study, spectral changes in TFA-treated dry-films of protein hydrolysates from a pure protein and poultry by-products, were compared to the FTIR fingerprints of untreated dry-films. The results show that time-dependent information related to proteolytic reactions and, consequently, on the characteristics of the protein hydrolysates can be obtained. With additional developments, FTIR on dry-films treated with TFA may be regarded as a potential future tool for the analysis of all types of proteolytic reactions in the laboratory as well as in industry.

The use of lytic polysaccharide monooxygenases in anaerobic digestion of lignocellulosic materials.

BACKGROUND: The recent discovery that LPMOs can work under anaerobic conditions when supplied with low amounts H2O2 opens the possibility of using LPMOs as enzyme aids in biogas reactors to increase methane yields from lignocellulosic materials. We have explored this possibility by studying anaerobic digestion of various lignocellulosic materials: Avicel, milled spruce and birch wood, and a lignin-rich hydrolysis residue from steam-exploded birch. The digestions were added LPMOs and various cellulolytic enzyme cocktails and were carried out with or without addition of H2O2. RESULTS: In several cases, enzyme addition had a beneficial effect on methane production, which was partly due to components present in the enzyme preparations. It was possible to detect LPMO activity during the initial phases of the anaerobic digestions of Avicel, and in some cases LPMO activity could be correlated with improved methane production from lignocellulosic materials. However, a positive effect on methane production was only seen when LPMOs were added together with cellulases, and never upon addition of LPMOs only. Generally, the experimental outcomes showed substrate-dependent variations in process efficiency and the importance of LPMOs and added H2O2. These differences could relate to variations in the type and content of lignin, which again will affect the activity of the LPMO, the fate of the added H2O2 and the generation of potentially damaging reactive-oxygen species. The observed effects showed that the interplay between cellulases and LPMOs is important for the overall efficiency of the process. CONCLUSION: This study shows that it may be possible to harness the power of LPMOs in anaerobic digestion processes and improve biogas production, but also highlight the complexity of the reaction systems at hand. One complicating factor was that the enzymes themselves and other organic components in the enzyme preparations acted as substrates for biogas production, meaning that good control reactions were essential to detect effects caused by enzyme activity. As also observed during regular aerobic enzymatic digestion of lignocellulosic biomass, the type and contents of lignin in the substrates likely plays a major role in determining the impact of LPMOs and of cellulolytic enzymes in general. More work is needed to unravel the interplay between LPMOs, O2, H2O2, and the multitude of redox-active components found in anaerobic bioreactors degrading lignocellulosic substrates.

FTIR-based hierarchical modeling for prediction of average molecular weights of protein hydrolysates.

In the presented study, Fourier-transform infrared (FTIR) spectroscopy is used to predict the average molecular weight of protein hydrolysates produced from protein-rich by-products from food industry using commercial enzymes. Enzymatic protein hydrolysis is a well-established method for production of protein-rich formulations, recognized for its potential to valorize food-processing by-products. The monitoring of such processes is still a significant challenge as the existing classical analytical methods are not easily applicable to industrial setups. In this study, we are reporting a generic FTIR-based approach for monitoring the average molecular weights of proteins during enzymatic hydrolysis of by-products from the food industry. A total of 885 hydrolysate samples from enzymatic protein hydrolysis reactions of poultry and fish by-products using different enzymes were studied. FTIR spectra acquired from dry-films of the hydrolysates were used to build partial least squares regression (PLSR) models. The most accurate predictions were obtained using a hierarchical PLSR approach involving supervised classification of the FTIR spectra according to raw material quality and enzyme used in the hydrolysis process, and subsequent local regression models tuned to specific enzyme-raw material combinations. The results clearly underline the potential of using FTIR for monitoring protein sizes during enzymatic protein hydrolysis in industrial settings, while also paving the way for measurements of protein sizes in other applications.

Effects of H2:CO2 ratio and H2 supply fluctuation on methane content and microbial community composition during in-situ biological biogas upgrading.

BACKGROUND: Commercial biogas upgrading facilities are expensive and consume energy. Biological biogas upgrading may serve as a low-cost approach because it can be easily integrated with existing facilities at biogas plants. The microbial communities found in anaerobic digesters typically contain hydrogenotrophic methanogens, which can use hydrogen (H2) as a reducing agent for conversion of carbon dioxide (CO2) into methane (CH4). Thus, biological biogas upgrading through the exogenous addition of H2 into biogas digesters for the conversion of CO2 into CH4 can increase CH4 yield and lower CO2 emission. RESULTS: The addition of 4 mol of H2 per mol of CO2 was optimal for batch biogas reactors and increased the CH4 content of the biogas from 67 to 94%. The CO2 content of the biogas was reduced from 33 to 3% and the average residual H2 content was 3%. At molar H2:CO2 ratios > 4:1, all CO2 was converted into CH4, but the pH increased above 8 due to depletion of CO2, which negatively influenced the process stability. Additionally, high residual H2 content in these reactors was unfavourable, causing volatile fatty acid accumulation and reduced CH4 yields. The reactor microbial communities shifted in composition over time, which corresponded to changes in the reactor variables. Numerous taxa responded to the H2 inputs, and in particular the hydrogenotrophic methanogen Methanobacterium increased in abundance with addition of H2. In addition, the apparent rapid response of hydrogenotrophic methanogens to intermittent H2 feeding indicates the suitability of biological methanation for variable H2 inputs, aligning well with fluctuations in renewable electricity production that may be used to produce H2. CONCLUSIONS: Our research demonstrates that the H2:CO2 ratio has a significant effect on reactor performance during in situ biological methanation. Consequently, the H2:CO2 molar ratio should be kept at 4:1 to avoid process instability. A shift toward hydrogenotrophic methanogenesis was indicated by an increase in the abundance of the obligate hydrogenotrophic methanogen Methanobacterium.