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- ItemDNA and RNA extraction and quantitative real-time PCR-based assays for biogas biocenoses in an interlaboratory comparison(Basel : MDPI, 2016) Lebuhn, Michael; Derenkó, Jaqueline; Rademacher, Antje; Helbig, Susanne; Munk, Bernhard; Pechtl, Alexander; Stolze, Yvonne; Prowe, Steffen; Schwarz, Wolfgang H.; Schlüter, Andreas; Liebl, Wolfgang; Klocke, MichaelFive institutional partners participated in an interlaboratory comparison of nucleic acid extraction, RNA preservation and quantitative Real-Time PCR (qPCR) based assays for biogas biocenoses derived from different grass silage digesting laboratory and pilot scale fermenters. A kit format DNA extraction system based on physical and chemical lysis with excellent extraction efficiency yielded highly reproducible results among the partners and clearly outperformed a traditional CTAB/chloroform/isoamylalcohol based method. Analytical purpose, sample texture, consistency and upstream pretreatment steps determine the modifications that should be applied to achieve maximum efficiency in the trade-off between extract purity and nucleic acid recovery rate. RNA extraction was much more variable, and the destination of the extract determines the method to be used. RNA stabilization with quaternary ammonium salts was an as satisfactory approach as flash freezing in liquid N2. Due to co-eluted impurities, spectrophotometry proved to be of limited value for nucleic acid qualification and quantification in extracts obtained with the kit, and picoGreen® based quantification was more trustworthy. Absorbance at 230 nm can be extremely high in the presence of certain chaotropic guanidine salts, but guanidinium isothiocyanate does not affect (q)PCR. Absolute quantification by qPCR requires application of a reliable internal standard for which correct PCR efficiency and Y-intercept values are important and must be reported.
- ItemUncertainty in the measurement of indoor temperature and humidity in naturally ventilated dairy buildings as influenced by measurement technique and data variability(Amsterdam : Elsevier, 2017) Hempel, Sabrina; König, Marcel; Menz, Christoph; Janke, David; Amon, Barbara; Banhazi, Thomas M.; Estellés, Fernando; Amon, ThomasThe microclimatic conditions in dairy buildings affect animal welfare and gaseous emissions. Measurements are highly variable due to the inhomogeneous distribution of heat and humidity sources (related to farm management) and the turbulent inflow (associated with meteorologic boundary conditions). The selection of the measurement strategy (number and position of the sensors) and the analysis methodology adds to the uncertainty of the applied measurement technique. To assess the suitability of different sensor positions, in situations where monitoring in the direct vicinity of the animals is not possible, we collected long-term data in two naturally ventilated dairy barns in Germany between March 2015 and April 2016 (horizontal and vertical profiles with 10 to 5 min temporal resolution). Uncertainties related to the measurement setup were assessed by comparing the device outputs under lab conditions after the on-farm experiments. We found out that the uncertainty in measurements of relative humidity is of particular importance when assessing heat stress risk and resulting economic losses in terms of temperature-humidity index. Measurements at a height of approximately 3 m–3.5 m turned out to be a good approximation for the microclimatic conditions in the animal occupied zone (including the air volume close to the emission active zone). However, further investigation along this cross-section is required to reduce uncertainties related to the inhomogeneous distribution of humidity. In addition, a regular sound cleaning (and if possible recalibration after few months) of the measurement devices is crucial to reduce the instrumentation uncertainty in long-term monitoring of relative humidity in dairy barns. © 2017 The Authors
- ItemCharacterization of Bathyarchaeota genomes assembled from metagenomes of biofilms residing in mesophilic and thermophilic biogas reactors(London : BioMed Central Ltd., 2018) Maus, I.; Rumming, M.; Bergmann, I.; Heeg, K.; Pohl, M.; Nettmann, E.; Jaenicke, S.; Blom, J.; Pühler, A.; Schlüter, A.; Sczyrba, A.; Klocke, M.Background: Previous studies on the Miscellaneous Crenarchaeota Group, recently assigned to the novel archaeal phylum Bathyarchaeota, reported on the dominance of these Archaea within the anaerobic carbohydrate cycle performed by the deep marine biosphere. For the first time, members of this phylum were identified also in mesophilic and thermophilic biogas-forming biofilms and characterized in detail. Results: Metagenome shotgun libraries of biofilm microbiomes were sequenced using the Illumina MiSeq system. Taxonomic classification revealed that between 0.1 and 2% of all classified sequences were assigned to Bathyarchaeota. Individual metagenome assemblies followed by genome binning resulted in the reconstruction of five metagenome-assembled genomes (MAGs) of Bathyarchaeota. MAGs were estimated to be 65-92% complete, ranging in their genome sizes from 1.1 to 2.0 Mb. Phylogenetic classification based on core gene sets confirmed their placement within the phylum Bathyarchaeota clustering as a separate group diverging from most of the recently known Bathyarchaeota clusters. The genetic repertoire of these MAGs indicated an energy metabolism based on carbohydrate and amino acid fermentation featuring the potential for extracellular hydrolysis of cellulose, cellobiose as well as proteins. In addition, corresponding transporter systems were identified. Furthermore, genes encoding enzymes for the utilization of carbon monoxide and/or carbon dioxide via the Wood-Ljungdahl pathway were detected. Conclusions: For the members of Bathyarchaeota detected in the biofilm microbiomes, a hydrolytic lifestyle is proposed. This is the first study indicating that Bathyarchaeota members contribute presumably to hydrolysis and subsequent fermentation of organic substrates within biotechnological biogas production processes.
- ItemAssessing the organic fraction of municipal solid wastes for the production of lactic acid(Amsterdam [u.a.] : Elsevier, 2019) López-Gómez, J. Pablo; Latorre-Sánchez, Marcos; Unger, Peter; Schneider, Roland; Coll Lozano, Caterina; Venus, JoachimWith an estimated yearly production of about 140 Mt in the EU, conventionally, the organic fraction of municipal solid wastes (OFMSW) has been disposed in landfills with negative environmental effects. Nonetheless, the chemical composition of this residue make it a substrate with great bioconversion potential. In this study, OFMSW from Spanish municipal treatment plants, were evaluated for the production of LA. Samples were identified according to the sorting mechanisms employed for their collection in: (A) separately collected, (B) non-separately collected and (C) separately collected+paper/cardboard. Enzymatic hydrolysis was used to produce hydrolysates A, B and C accordingly. Hydrolysate A showed the highest total sugars and glucose content with values of 70 and 55 g·L−1, respectively. Following the characterisation, a screening showed that growth of B. coagulans was possible in all three hydrolysates. Furthermore, lab scale fermentations showed that LA final concentrations could reach around 60 g·L−1, with yields from total sugars of above 0.60 g·g−1. A technical scale fermentation of the hydrolysate A resulted in a final LA concentration of 60.7 g·L−1, a yield of 0.71 g·g−1 with a productivity of 2.68 g·L−1·h−1. Overall, it was estimated that 0.23 g of LA could be produced from one g of dry OFMSW.
- ItemRapid determination of lime requirement by mid-infrared spectroscopy: A promising approach for precision agriculture(Weinheim : Wiley-VCH, 2019) Leenen, Matthias; Welp, Gerhard; Gebbers, Robin; Pätzold, StefanMid-infrared spectroscopy (MIRS) has proven to be a cost-effective, high throughput measurement technique for soil analysis. After multivariate calibration mid-infrared spectra can be used to predict various soil properties, some of which are related to lime requirement (LR). The objective of this study was to test the performance of MIRS for recommending variable rate liming on typical Central European soils in view of precision agriculture applications. In Germany, LR of arable topsoils is commonly derived from the parameters organic matter content (SOM), clay content, and soil pH (CaCl2) as recommended by the Association of German Agricultural Analytical and Research Institutes (VDLUFA). We analysed a total of 458 samples from six locations across Germany, which all revealed large within-field soil heterogeneity. Calcareous topsoils were observed at some positions of three locations (79 samples). To exclude such samples from LR determination, peak height at 2513 cm−1 of the MIR spectrum was used for identification. Spectra-based identification was accurate for carbonate contents > 0.5%. Subsequent LR derivation (LRSPP) from MIRS-PLSR predictions of SOM, clay, and pH (CaCl2) for non-calcareous soil samples using the VDLUFA look-up tables was successful for all locations (R2 = 0.54–0.82; RMSE = 857–1414 kg CaO ha−1). Alternatively, we tested direct LR prediction (LRDP) by MIRS-PLSR and also achieved satisfactory performance (R2 = 0.52–0.77; RMSE = 811–1420 kg CaO ha−1; RPD = 1.44–2.08). Further improvement was achieved by refining the VDLUFA tables towards a stepless algorithm. It can be concluded that MIRS provides a promising approach for precise LR estimation on heterogeneous arable fields. Large sample numbers can be processed with low effort which is an essential prerequisite for variable rate liming in precision agriculture. © 2019 The Authors. Journal of Plant Nutrition and Soil Science published by WILEY-VCH Verlag GmbH & Co. KGaA
- ItemRelevance of interactions between starch-based coatings and plum fruit surfaces: A physical-chemical analysis(Basel : MDPI AG, 2019) Basiak, Ewelina; Geyer, Martin; Debeaufort, Frédéric; Lenart, Andrzej; Linke, ManfredIn order to extend the shelf life of the fruit, improve appearance, and to keep all nutrition properties of the plum from diminishing, edible coatings comprised of wheat starch and wheat starch–whey protein isolate (in ratio 80/20) were created. Stand-alone films were produced to assess properties which helped to understand the phenomena occurring on the surface level of coated plums. The properties of coatings based on starch are similar to starch coatings containing oil because the natural epicuticular wax layer of plums merges with coating materials. Adding oil doubled the contact angle value and the dispersive component of the surface tension. The workings of adhesion and cohesion, spreading coefficient, water absorption, water content, and solubility in water of the films decreased. Similar processes were observed on the fruits’ surface. In appearance, the coating process is similar to polishing the plum surface for removing crystalline wax. The color parameters of coated fruits did not significantly change. Newly formed bonds or interactions established between starch, whey proteins, water, glycerol, and oil are displayed by Fourier transform infrared (FTIR) analysis. This work revealed how the interactions between the epicuticular wax on the fruit’s surface and the hydrocolloid-based coatings affect the efficiency of the coatings. © 2019 by the authors. Licensee MDPI, Basel, Switzerland.
- ItemModel-based characterisation of growth performance and l-lactic acid production with high optical purity by thermophilic Bacillus coagulans in a lignin-supplemented mixed substrate medium(New York, NY [u.a.] : Elsevier, 2017) Glaser, Robert; Venus, JoachimThree Bacillus coagulans strains were characterised in terms of their ability to grow in lignin-containing fermentation media and to consume the lignocellulose-related sugars glucose, xylose, and arabinose. An optical-density high-throughput screening was used for precharacterisation by means of different mathematical models for comparison (Logistic, Gompertz, Baranyi, Richards & Stannard, and Schnute). The growth response was characterised by the maximum growth rate and lag time. For a comparison of the screening and fermentation results, an unstructured mathematical model was proposed to characterise the lactate production, bacterial growth and substrate consumption. The growth model was then applied to fermentation procedures using wheat straw hydrolysates. The results indicated that the unstructured growth model can be used to evaluate lactate producing fermentation. Under the experimental fermentation conditions, one strain showed the ability to tolerate a high lignin concentration (2.5 g/L) but lacked the capacity for sufficient pentose uptake. The lactate yield of the strains that were able to consume all sugar fractions of glucose, xylose and arabinose was ∼83.4%. A photometric measurement at 280 nm revealed a dynamic change in alkali-lignin concentrations during lactate producing fermentation. A test of decolourisation of vanillin, ferulic acid, and alkali-lignin samples also showed the decolourisation performance of the B. coagulans strains under study. © 2017 The Author(s)
- ItemMetaproteome analysis reveals that syntrophy, competition, and phage-host interaction shape microbial communities in biogas plants(London : Biomed Central, 2019) Heyer, R.; Schallert, K.; Siewert, C.; Kohrs, F.; Greve, J.; Maus, I.; Klang, J.; Klocke, M.; Heiermann, M.; Hoffmann, M.; Püttker, S.; Calusinska, M.; Zoun, R.; Saake, G.; Benndorf, D.; Reichl, U.Background: In biogas plants, complex microbial communities produce methane and carbon dioxide by anaerobic digestion of biomass. For the characterization of the microbial functional networks, samples of 11 reactors were analyzed using a high-resolution metaproteomics pipeline. Results: Examined methanogenesis archaeal communities were either mixotrophic or strictly hydrogenotrophic in syntrophy with bacterial acetate oxidizers. Mapping of identified metaproteins with process steps described by the Anaerobic Digestion Model 1 confirmed its main assumptions and also proposed some extensions such as syntrophic acetate oxidation or fermentation of alcohols. Results indicate that the microbial communities were shaped by syntrophy as well as competition and phage-host interactions causing cell lysis. For the families Bacillaceae, Enterobacteriaceae, and Clostridiaceae, the number of phages exceeded up to 20-fold the number of host cells. Conclusion: Phage-induced cell lysis might slow down the conversion of substrates to biogas, though, it could support the growth of auxotrophic microbes by cycling of nutrients. © 2019 The Author(s).
- ItemMembrane technologies for lactic acid separation from fermentation broths derived from renewable resources(Basel : MDPI AG, 2018) Alexandri, M.; Schneider, R.; Venus, J.Lactic acid (LA) was produced on a pilot scale using a defined medium with glucose, acid whey, sugar bread and crust bread. The fermentation broths were then subjected to micro-and nanofiltration. Microfiltration efficiently separated the microbial cells. The highest average permeate flow flux was achieved for the defined medium (263.3 L/m2/h) and the lowest for the crust bread-based medium (103.8 L/m2/h). No LA losses were observed during microfiltration of the acid whey, whilst the highest retention of LA was 21.5% for crust bread. Nanofiltration led to high rejections of residual sugars, proteins and ions (sulphate, magnesium, calcium), with a low retention of LA. Unconverted sugar rejections were 100% and 63% for crust bread and sugar bread media respectively, with corresponding LA losses of 22.4% and 2.5%. The membrane retained more than 50% of the ions and proteins present in all media and more than 60% of phosphorus. The average flux was highly affected by the nature of the medium as well as by the final concentration of LA and sugars. The results of this study indicate that micro-and nanofiltration could be industrially employed as primary separation steps for the biotechnologically produced LA.
- ItemMetagenome, metatranscriptome, and metaproteome approaches unraveled compositions and functional relationships of microbial communities residing in biogas plants(Berlin ; Heidelberg ; New York : Springer, 2018-4-30) Hassa, Julia; Maus, Irena; Off, Sandra; Pühler, Alfred; Scherer, Paul; Klocke, Michael; Schlüter, AndreasThe production of biogas by anaerobic digestion (AD) of agricultural residues, organic wastes, animal excrements, municipal sludge, and energy crops has a firm place in sustainable energy production and bio-economy strategies. Focusing on the microbial community involved in biomass conversion offers the opportunity to control and engineer the biogas process with the objective to optimize its efficiency. Taxonomic profiling of biogas producing communities by means of high-throughput 16S rRNA gene amplicon sequencing provided high-resolution insights into bacterial and archaeal structures of AD assemblages and their linkages to fed substrates and process parameters. Commonly, the bacterial phyla Firmicutes and Bacteroidetes appeared to dominate biogas communities in varying abundances depending on the apparent process conditions. Regarding the community of methanogenic Archaea, their diversity was mainly affected by the nature and composition of the substrates, availability of nutrients and ammonium/ammonia contents, but not by the temperature. It also appeared that a high proportion of 16S rRNA sequences can only be classified on higher taxonomic ranks indicating that many community members and their participation in AD within functional networks are still unknown. Although cultivation-based approaches to isolate microorganisms from biogas fermentation samples yielded hundreds of novel species and strains, this approach intrinsically is limited to the cultivable fraction of the community. To obtain genome sequence information of non-cultivable biogas community members, metagenome sequencing including assembly and binning strategies was highly valuable. Corresponding research has led to the compilation of hundreds of metagenome-assembled genomes (MAGs) frequently representing novel taxa whose metabolism and lifestyle could be reconstructed based on nucleotide sequence information. In contrast to metagenome analyses revealing the genetic potential of microbial communities, metatranscriptome sequencing provided insights into the metabolically active community. Taking advantage of genome sequence information, transcriptional activities were evaluated considering the microorganism’s genetic background. Metaproteome studies uncovered enzyme profiles expressed by biogas community members. Enzymes involved in cellulose and hemicellulose decomposition and utilization of other complex biopolymers were identified. Future studies on biogas functional microbial networks will increasingly involve integrated multi-omics analyses evaluating metagenome, transcriptome, proteome, and metabolome datasets. © 2018, The Author(s).
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