Selection of Conditions for Cellulase and Xylanase Extraction from Switchgrass Colonized by Acidothermus cellulolyticus

Solid-state fermentation has been widely used for enzyme production. However, secreted enzymes often bind to the solid substrate preventing their detection and recovery. A series of screening studies was performed to examine the role of extraction buffer composition including NaCl, ethylene glycol, sodium acetate buffer, and Tween 80, on xylanase and cellulase recovery from switchgrass. Our results indicated that the selection of an extraction buffer is highly dependent on the nature and source of the enzyme being extracted. While a buffer containing 50 mM sodium acetate at pH 5 was found to have a positive effect on the recovery of commercial fungal-derived cellulase and xylanase amended to switchgrass, the same buffer had a significant negative effect on enzyme extraction from solid fermentation samples colonized by the bacterium Acidothermus cellulolyticus. Xylanase activity was more affected by components in the extraction buffers compared to cellulase. This study demonstrated that extraction followed by diafiltration is important for assessing enzyme recovery from solid fermentation samples. Reduction in activity due to compounds present in the switchgrass extracts is reversible when the compounds are removed via diafiltration

An efficient Foxtail mosaic virus vector system with reduced environmental risk

<p>Abstract</p> <p>Background</p> <p>Plant viral vectors offer high-yield expression of pharmaceutical and commercially important proteins with a minimum of cost and preparation time. The use of <it>Agrobacterium tumefaciens </it>has been introduced to deliver the viral vector as a transgene to each plant cell via a simple, nonsterile infiltration technique called "agroinoculation". With agroinoculation, a full length, systemically moving virus is no longer necessary for excellent protein yield, since the viral transgene is transcribed and replicates in every infiltrated cell. Viral genes may therefore be deleted to decrease the potential for accidental spread and persistence of the viral vector in the environment.</p> <p>Results</p> <p>In this study, both the coat protein (CP) and triple gene block (TGB) genetic segments were eliminated from <it>Foxtail mosaic virus </it>to create the "FECT" vector series, comprising a deletion of 29% of the genome. This viral vector is highly crippled and expresses little or no marker gene within the inoculated leaf. However, when co-agroinoculated with a silencing suppressor (p19 or HcPro), FECT expressed GFP at 40% total soluble protein in the tobacco host, <it>Nicotiana benthamiana</it>. The modified FoMV vector retained the full-length replicase ORF, the TGB1 subgenomic RNA leader sequence and either 0, 22 or 40 bases of TGB1 ORF (in vectors FECT0, FECT22 and FECT40, respectively). As well as <it>N. benthamiana</it>, infection of legumes was demonstrated. Despite many attempts, expression of GFP via syringe agroinoculation of various grass species was very low, reflecting the low <it>Agrobacterium</it>-mediated transformation rate of monocots.</p> <p>Conclusions</p> <p>The FECT/40 vector expresses foreign genes at a very high level, and yet has a greatly reduced biohazard potential. It can form no virions and can effectively replicate only in a plant with suppressed silencing.</p

Switchgrass (Panicum virgatum L.) polyubiquitin gene (PvUbi1 and PvUbi2) promoters for use in plant transformation

<p>Abstract</p> <p>Background</p> <p>The ubiquitin protein is present in all eukaryotic cells and promoters from ubiquitin genes are good candidates to regulate the constitutive expression of transgenes in plants. Therefore, two switchgrass (<it>Panicum virgatum </it>L.) ubiquitin genes (<it>PvUbi1 </it>and <it>PvUbi2</it>) were cloned and characterized. Reporter constructs were produced containing the isolated 5' upstream regulatory regions of the coding sequences (i.e. <it>PvUbi1 </it>and <it>PvUbi2 </it>promoters) fused to the <it>uidA </it>coding region (<it>GUS</it>) and tested for transient and stable expression in a variety of plant species and tissues.</p> <p>Results</p> <p><it>PvUbi1 </it>consists of 607 bp containing <it>cis</it>-acting regulatory elements, a 5' untranslated region (UTR) containing a 93 bp non-coding exon and a 1291 bp intron, and a 918 bp open reading frame (ORF) that encodes four tandem, head -to-tail ubiquitin monomer repeats followed by a 191 bp 3' UTR. <it>PvUbi2 </it>consists of 692 bp containing <it>cis</it>-acting regulatory elements, a 5' UTR containing a 97 bp non-coding exon and a 1072 bp intron, a 1146 bp ORF that encodes five tandem ubiquitin monomer repeats and a 183 bp 3' UTR. <it>PvUbi1 </it>and <it>PvUbi2 </it>were expressed in all examined switchgrass tissues as measured by qRT-PCR. Using biolistic bombardment, <it>PvUbi1 </it>and <it>PvUbi2 </it>promoters showed strong expression in switchgrass and rice callus, equaling or surpassing the expression levels of the CaMV <it>35S, 2x35S, ZmUbi1</it>, and <it>OsAct1 </it>promoters. GUS staining following stable transformation in rice demonstrated that the <it>PvUbi1 </it>and <it>PvUbi2 </it>promoters drove expression in all examined tissues. When stably transformed into tobacco (<it>Nicotiana tabacum</it>), the <it>PvUbi2+3 </it>and <it>PvUbi2+9 </it>promoter fusion variants showed expression in vascular and reproductive tissues.</p> <p>Conclusions</p> <p>The <it>PvUbi1 </it>and <it>PvUbi2 </it>promoters drive expression in switchgrass, rice and tobacco and are strong constitutive promoter candidates that will be useful in genetic transformation of monocots and dicots.</p

Soil Application of Almond Residue Biomass Following Black Soldier Fly Larvae Cultivation

Insect farming has the potential to transform abundant residual biomass into feed that is compatible with non-ruminant animal production systems. However, insect cultivation generates its own by-products. There is a need to find valuable and sustainable applications for this material to enable commercial-scale insect farming. Soil application of by-products, which may be either basic broadcasting incorporation or part of a sustainable soil borne pest management practice, such as biosolarization, could offer an agricultural outlet. The objective of this study was to assess the potential of applying black soldier fly larvae (BSFL)-digested substrate as soil amendment for soil biosolarization and evaluate its impact on soil health. Sandy loam (SL) and sandy clay loam (CL) soils amended with BSFL-digested almond processing residues, i.e., spent pollinator hulls (SPH), at 2% dry weight (dw) were incubated under aerobic and anaerobic conditions for 15 days under a daily fluctuating temperature-interval (30–50°C). The microbial respiration, pH, electrical conductivity, volatile fatty acids, macronutrients, and germination index using radish seeds (Raphanus sativus L.) were quantified to assess the soil health after amendment application. Incubation showed a statistically significant (p &lt; 0.05) increase in electrical conductivity related to amendment addition and a decrease potentially linked to microbiological activity, i.e., sequestering of ions. Under aerobic conditions, SPH addition increased the CO2-accumulation by a factor of 5–6 compared to the non-amended soils in SL and CL, respectively. This increase further suggests a higher microbiological activity and that SPH behaves like a partially stabilized organic material. Under anaerobic conditions, CO2-development remained unchanged. BSFL-digested residues significantly increased the carbon, nitrogen, C/N, phosphate, ammonium, and potassium in the two soil types, replenishing soils with essential macronutrients. However, greenhouse trials with lettuce seeds (Lactuca sativa) lasting 14 days resulted in a decrease of the biomass by 44.6 ± 35.4 and 35.2 ± 25.3% for SL and CL, respectively, compared to their respective non-amended soil samples. This reduction of the biomass resulted from residual phytotoxic compounds, indicating that BSFL-digested SPH have the potential to be used for biosolarization and as soil amendments, depending on the concentration and mitigation strategies. Application and environmental conditions must be carefully selected to minimize the persistence of soil phytotoxicity

Impact of thiamine metabolites and spent medium from Chlorella sorokiniana on metabolism in the green algae Auxenochlorella prototheciodes

Auxenochlorella protothecoides is a known thiamine auxotroph but our past work has shown that it can synthesize thiamine if provided with the precursor molecule 4-amino-5-hydroxymethyl-2-methylpyrimidine (HMP). Partial thiamine auxotrophy is common in microalgae with important ramifications for global phytoplankton productivity as well as engineering applications of algae. While thiamine deficiency can greatly depress algae growth and lipid content, the detailed metabolic impacts of thiamine deficiency are not well understood. We used metabolomics to study the response to thiamine-limited and replete conditions in mixotrophic A. protothecoides. We also investigated the impacts of exogenous HMP addition and the use of spent medium from another green algae, C. sorokiniana, as a source of thiamine metabolites. This is the first study, to our knowledge, that addresses metabolic impacts of thiamine deficiency and alleviation in green microalgae. Thiamine deficient cultures exhibited accumulation of pyruvate and α-ketoglutarate, indicating bottlenecks at the pyruvate dehydrogenase (PDH) and oxoglutarate dehydrogenase (OGDH) complexes. Both PDH and OGDH require thiamine pyrophosphate (TPP) as a cofactor. Transketolase also requires TPP but we only observed build-up of ribose-5-phosphate when glucose was supplied as a substrate. As expected, thiamine and HMP addition could alleviate these metabolic bottlenecks while greatly increasing algal growth, neutral lipid and starch content. Spent medium from C. sorokiniana only appeared to partially alleviate thiamine deficiency and resulted in build-up of isocitrate and glycolate, metabolites that appeared relatively unaffected by the presence or absence of thiamine. Interestingly, longer culture time of C. sorokiniana when preparing the spent medium led to much higher availability of thiamine metabolites. Thus, under the right conditions, it may be possible to co-culture mutually beneficial algae species and/or recycle spent cultivation medium to overcome auxotrophy in algae

Impact of thiamine metabolites and spent medium from Chlorella sorokiniana on metabolism in the green algae Auxenochlorella prototheciodes

Auxenochlorella protothecoides is a known thiamine auxotroph but our past work has shown that it can synthesize thiamine if provided with the precursor molecule 4-amino-5-hydroxymethyl-2-methylpyrimidine (HMP). Partial thiamine auxotrophy is common in microalgae with important ramifications for global phytoplankton productivity as well as engineering applications of algae. While thiamine deficiency can greatly depress algae growth and lipid content, the detailed metabolic impacts of thiamine deficiency are not well understood. We used metabolomics to study the response to thiamine-limited and replete conditions in mixotrophic A. protothecoides. We also investigated the impacts of exogenous HMP addition and the use of spent medium from another green algae, C. sorokiniana, as a source of thiamine metabolites. This is the first study, to our knowledge, that addresses metabolic impacts of thiamine deficiency and alleviation in green microalgae. Thiamine deficient cultures exhibited accumulation of pyruvate and α-ketoglutarate, indicating bottlenecks at the pyruvate dehydrogenase (PDH) and oxoglutarate dehydrogenase (OGDH) complexes. Both PDH and OGDH require thiamine pyrophosphate (TPP) as a cofactor. Transketolase also requires TPP but we only observed build-up of ribose-5-phosphate when glucose was supplied as a substrate. As expected, thiamine and HMP addition could alleviate these metabolic bottlenecks while greatly increasing algal growth, neutral lipid and starch content. Spent medium from C. sorokiniana only appeared to partially alleviate thiamine deficiency and resulted in build-up of isocitrate and glycolate, metabolites that appeared relatively unaffected by the presence or absence of thiamine. Interestingly, longer culture time of C. sorokiniana when preparing the spent medium led to much higher availability of thiamine metabolites. Thus, under the right conditions, it may be possible to co-culture mutually beneficial algae species and/or recycle spent cultivation medium to overcome auxotrophy in algae

Nitrogen amendment of green waste impacts microbial community, enzyme secretion and potential for lignocellulose decomposition

Microorganisms involved in biomass deconstruction are an important resource for organic waste recycling and enzymes for lignocellulose bioconversion. The goals of this study were to examine the impact of nitrogen amendment on microbial community restructuring, secretion of xylanases and endoglucanases, and potential for biomass deconstruction. Communities were cultivated aerobically at 55&nbsp;°C on green waste (GW) amended with varying levels of NH4Cl. Bacterial and fungal communities were determined using 16S rRNA and ITS region gene sequencing and PICRUSt (Phylogenetic Investigation of Communities by Reconstruction of Unobserved States) was applied to predict relative abundance of genes involved in lignocellulose hydrolysis. Nitrogen amendment significantly increased secretion of xylanases and endoglucanases, and microbial activity; enzyme activities and cumulative respiration were greatest when nitrogen level in GW was between 4.13–4.56&nbsp;wt% (g/g), but decreased with higher nitrogen levels. The microbial community shifted to one with increasing potential to decompose complex polymers as nitrogen increased with peak potential occurring between 3.79–4.45&nbsp;wt% (g/g) nitrogen amendment. The results will aid in informing the management of nitrogen level to foster microbial communities capable of secreting enzymes that hydrolyze recalcitrant polymers in lignocellulose and yield rapid decomposition of green waste