Selection and optimization of a suitable pretreatment method for miscanthus and poplar raw material

Miscanthus and poplar are very promising second‐generation feedstocks due to the high growth rates and low nutrient demand. The aim of the study was to develop a systematic approach for choosing suitable pretreatment methods evaluated with the modified severity factor (log ). Optimal pretreatment results in a high delignification grade, low cellulose solubilization and increased accessibility for enzymatic hydrolysis while revealing minimal log values. In order to do so, several reaction approaches were compared. Acid‐catalyzed organosolv processing carried out for miscanthus and poplar revealed the highest delignification grade leading to a relatively high glucose yield after enzymatic saccharification. In both cases, a design of experiments approach was used to study the influence of relevant parameters. Modeling the data resulted in the identification of optimum pretreatment conditions for miscanthus with concentrations of 0.16% H₂SO₄ and 50% EtOH at 185°C for a retention time of 60 min. Experimental validation of these conditions revealed an even higher delignification degree (88%) and glucose yield (85%) than predicted. 0.19% H₂SO₄ and 50% EtOH were determined as optimum concentrations, 182°C and 48 min identified as optimum pretreatment conditions for poplar; the delignification degree was 84% and the resulting glucose yield 70%.     

<Emphasis Type="Italic">Candida guilliermondii</Emphasis> as a potential biocatalyst for the production of long-chain α,ω-dicarboxylic acids

Objectives

To explore Candida guilliermondii for the production of long-chain dicarboxylic acids (DCA), we performed metabolic pathway engineering aiming to prevent DCA consumption during β-oxidation, but also to increase its production via the ω-oxidation pathway.

Results

We identified the major β- and ω-oxidation pathway genes in C. guilliermondii and performed first steps in the strain improvement. A double pox disruption mutant was created that slowed growth with oleic acid but showed accelerated DCA degradation. Increase in DCA production was achieved by homologous overexpression of a plasmid borne cytochrome P450 monooxygenase gene.

Conclusion

C. guilliermondii is a promising biocatalyst for DCA production but further insight into its fatty acid metabolism is necessary.

     

Localized Functional Chemical Stimulation of TE 671 Cells Cultured on Nanoporous Membrane by Calcein and Acetylcholine

Acetylcholine sensitive TE 671 cells were cultured on nanoporous membranes and chemically stimulated by localized application of i), calcein-AM and ii), acetylcholine, respectively, onto the bottom face of the membrane employing an ink jet print head. Stimulus correlated response of cells was recorded by fluorescence microscopy with temporal and spatial resolution. Calcein fluorescence develops as a result of intracellular enzymatic conversion of calcein-AM, whereas Ca²⁺ imaging using fluo-4 dye was employed to visualize cellular response to acetylcholine stimulation. Using 25 pl droplets and substance concentration ranging from 10 μM to 1 mM on Nucleopore membranes with pore diameters between 50 nm and 1 μm, a resolution on the order of 50 μm was achieved.     

In vitro synthesis and characterization of guanosine 3',5'-bis(diphosphate)

The intracellular alarmone guanosine 3′,5′-bis(diphosphate) (ppGpp) has been thoroughly investigated over the past 40 years and has become one of the best-known effectors in bacterial physiology. ppGpp is also of great importance for biotechnological applications. Systems biology research, involving experimental and mathematical approaches, has contributed a great deal to uncovering the alarmone’s complex regulatory effects. HPLC analysis and UV detection are used to quantify intracellular ppGpp. The samples analyzed also contain other phosphorylated guanine nucleotides and, therefore, are spiked with a standard ppGpp solution. A rapidly growing number of laboratories are turning to synthesizing the nucleotide in vitro involving time-consuming protocols and yielding only low amounts of ppGpp. The current article provides a protocol for the preparation of large quantities of a ribosome extract that contains high ppGpp synthesis activity. The demonstrated upscaling from shaking flask to bioreactor cultivation involves the continuous and refrigerated harvest of the biomass. ¹³C NMR analysis enabled the structural characterization of the synthesis product and was complemented by mass spectrometry and methods that are commonly used to identify ppGpp.     

Localized functional chemical stimulation of TE 671 cells cultured on nanoporous membrane by calcein and acetylcholine

Acetylcholine sensitive TE 671 cells were cultured on nanoporous membranes and chemically stimulated by localized application of i), calcein-AM and ii), acetylcholine, respectively, onto the bottom face of the membrane employing an ink jet print head. Stimulus correlated response of cells was recorded by fluorescence microscopy with temporal and spatial resolution. Calcein fluorescence develops as a result of intracellular enzymatic conversion of calcein-AM, whereas Ca(2+) imaging using fluo-4 dye was employed to visualize cellular response to acetylcholine stimulation. Using 25 pl droplets and substance concentration ranging from 10 microM to 1 mM on Nucleopore membranes with pore diameters between 50 nm and 1 microm, a resolution on the order of 50 microm was achieved.     

Biotechnological production of bio-based long-chain dicarboxylic acids with oleogenious yeasts

Long-chain α,ω-dicarboxylic acids (DCAs) are versatile chemical intermediates of industrial importance used as building blocks for the production of polymers, lubricants, or adhesives. The majority of industrial long-chain DCAs is produced from petro-chemical resources. An alternative is their biotechnological production from renewable materials like plant oil fatty acids by microbial fermentation using oleogenious yeasts. Oleogenious yeasts are natural long-chain DCA producers, which have to be genetically engineered for high-yield DCA production. Although, some commercialized fermentation processes using engineered yeasts are reported, bio-based long-chain DCAs are still far from being a mass product. Further progress in bioprocess engineering and rational strain design is necessary to advance their further commercialization. The present article reviews the basic strategies, as well as novel approaches in the strain design of oleogenious yeasts, such as the combination of traditional metabolic engineering with system biology strategies for high-yield long-chain DCA production. Therefore a detailed overview of the involved metabolic processes for the biochemical long-chain DCA synthesis is given.     

Conversion of aliphatic 2-acetoxynitriles by nitrile-hydrolysing bacteria

The enzymatic hydrolysis of the nitrile group of different 2-acetoxynitriles was investigated in order to obtain catalysts that chemoselectively hydrolyse nitriles in the presence of ester groups. The biotransformation of four 2-acetoxynitriles [2-acetoxybutenenitrile (ABN), 2-acetoxyheptanenitrile (AHN), 2-acetoxy-2-(2-furyl)acetonitrile (AFN), and 2-acetoxy-2,3,3-trimethylbutanenitrile (ATMB)] by different bacterial strains that synthesise nitrilases or nitrile hydratases was studied. ABN, AHN and AFN were converted by various microorganisms belonging to different bacterial genera (e.g. Pseudomonas or Rhodococcus) expressing either nitrilase or nitrile hydratase activities. In contrast, no metabolism of the sterically hindered substrate ATMB was observed. All wild-type strains investigated formed considerable amounts of cyanide and aldehydes from the 2-acetoxynitriles. This indicated the presence of esterases converting the 2-acetoxynitriles to 2-hydroxynitriles, which then spontaneously decomposed to the corresponding aldehydes and cyanide. In order to suppress unwanted side-reactions, biotransformations were performed with recombinant Escherichia coli strains that heterologously expressed nitrilase activities originating from Pseudomonas, Rhodococcus, or Synechocystis strains. The attempted conversion of the 2-acetoxynitriles to almost stoichiometric amounts of the corresponding 2-acetoxycarboxylic acids was finally achieved by using either a recombinant E. coli strain that highly overexpressed the nitrilase gene from the pseudomonad or the purified enzyme derived from this strain.     

Integrated lignocellulosic value chains in a growing bioeconomy: Status quo and perspectives

Lignocellulose is the most abundant biomass on Earth, with an estimated 181.5 billion tonnes produced annually. Of the 8.2 billion tonnes that are currently used, about 7 billion tonnes are produced from dedicated agricultural, grass and forest land and another 1.2 billion tonnes stem from agricultural residues. Economic and environmentally efficient pathways for production and utilization of lignocellulose for chemical products and energy are needed to expand the bioeconomy. This opinion paper arose from the research network “Lignocellulose as new resource platform for novel materials and products” funded by the German federal state of Baden‐Württemberg and summarizes original research presented in this special issue. It first discusses how the supply of lignocellulosic biomass can be organized sustainably and suggests that perennial biomass crops (PBC) are likely to play an important role in future regional biomass supply to European lignocellulosic biorefineries. Dedicated PBC production has the advantage of delivering biomass with reliable quantity and quality. The tailoring of PBC quality through crop breeding and management can support the integration of lignocellulosic value chains. Two biorefinery concepts using lignocellulosic biomass are then compared and discussed: the syngas biorefinery and the lignocellulosic biorefinery. Syngas biorefineries are less sensitive to biomass qualities and are technically relatively advanced, but require high investments and large‐scale facilities to be economically feasible. Lignocellulosic biorefineries require multiple processing steps to separate the recalcitrant lignin from cellulose and hemicellulose and convert the intermediates into valuable products. The refining processes for high‐quality lignin and hemicellulose fractions still need to be further developed. A concept of a modular lignocellulosic biorefinery is presented that could be flexibly adapted for a range of feedstock and products by combining appropriate technologies either at the same location or in a decentralized form.