Impact of altered cell wall composition on saccharification efficiency in stem tissue of Arabidopsis RABA GTPase-deficient knockout mutants

Use of biomass for second-generation biofuel production is severely hindered by the inherent recalcitrance of the plant cell wall to digestion. Trafficking is crucial for compartmentalisation within the cell. This process is partly regulated by small Rab GTPase proteins. In particular, control of trafficking to the cell wall is regulated through the RABA clade. Manipulation of this regulatory system offers tantalising opportunities for manipulation of cell wall composition and hence recalcitrance. Trafficking-defective rabA mutants have already been shown to impact cell wall composition. To study the impacts of these mutants on cell wall digestion, we developed a saccharification process for Arabidopsis based on the hot water method. We then showed that following pre-treatment, stems from the T-DNA knockouts of the three RABA4 genes expressed in Arabidopsis stem show an increased sugar release on saccharification. These rabA4 mutants have been shown to impact the “hemicellulose-rich” fraction during cell wall fractionation. Furthermore, we go on to show that these mutant lines also show increased sugar release when subjected to saccharification without pre-treatment. Finally, we used X-ray diffraction to show that rabA4 mutants had no impact on cellulose crystallinity, thus supporting the hypothesis that the increases in saccharification were not due to alterations of the cellulose microfibrils but were a direct effect of reduced hemicellulose levels. We also present data to show that the growth characteristics of these plants were unaffected. The data obtained from these lines are most easily explained by the reported alteration in hemicellulose increasing pre-treatment efficiency

Succession of physiological stages hallmarks the transcriptomic response of the fungus Aspergillus niger to lignocellulose.

BackgroundUnderstanding how fungi degrade lignocellulose is a cornerstone of improving renewables-based biotechnology, in particular for the production of hydrolytic enzymes. Considerable progress has been made in investigating fungal degradation during time-points where CAZyme expression peaks. However, a robust understanding of the fungal survival strategies over its life time on lignocellulose is thereby missed. Here we aimed to uncover the physiological responses of the biotechnological workhorse and enzyme producer Aspergillus niger over its life time to six substrates important for biofuel production.ResultsWe analysed the response of A. niger to the feedstock Miscanthus and compared it with our previous study on wheat straw, alone or in combination with hydrothermal or ionic liquid feedstock pretreatments. Conserved (substrate-independent) metabolic responses as well as those affected by pretreatment and feedstock were identified via multivariate analysis of genome-wide transcriptomics combined with targeted transcript and protein analyses and mapping to a metabolic model. Initial exposure to all substrates increased fatty acid beta-oxidation and lipid metabolism transcripts. In a strain carrying a deletion of the ortholog of the Aspergillus nidulans fatty acid beta-oxidation transcriptional regulator farA, there was a reduction in expression of selected lignocellulose degradative CAZyme-encoding genes suggesting that beta-oxidation contributes to adaptation to lignocellulose. Mannan degradation expression was wheat straw feedstock-dependent and pectin degradation was higher on the untreated substrates. In the later life stages, known and novel secondary metabolite gene clusters were activated, which are of high interest due to their potential to synthesize bioactive compounds.ConclusionIn this study, which includes the first transcriptional response of Aspergilli to Miscanthus, we highlighted that life time as well as substrate composition and structure (via variations in pretreatment and feedstock) influence the fungal responses to lignocellulose. We also demonstrated that the fungal response contains physiological stages that are conserved across substrates and are typically found outside of the conditions with high CAZyme expression, as exemplified by the stages that are dominated by lipid and secondary metabolism

Understanding the mechanisms of cooperative physico-chemical treatment and mechanical disintegration of biomass as a route for enhancing enzyme saccharification

A novel chemico-kinetic disintegration model has been applied to study the cooperative relationship between physico-chemical treatment and supplementary wet-state milling of biomass, as an efficient process route to achieve high enzyme accessibility. Wheat straw, Miscanthus and short-rotation willow were studied as three contrasting biomass species, which were subjected to controlled hydrothermal pretreatment using a microwave reactor, followed by controlled wet-state ball-milling. Comparative particle disintegration behaviour and related enzyme digestibilities have been interpreted on the basis of model parameters and with evaluation of textural and chemical differences in tissue structures, aided by the application of specific material characterisation techniques. Supplementary milling led to a 1.3×, 1.6× and 3× enhancement in glucose saccharification yield after 24 h for straw, Miscanthus and willow, respectively, following a standardised 10-min hydrothermal treatment, with corresponding milling energy savings of 98, 97 and 91% predicted from the model, compared to the unmilled case. The results confirm the viability of pretreatment combined with supplementary wet-milling as an efficient process route. The results will be valuable in understanding the key parameters for process design and optimisation and also the key phenotypical parameters for feedstock breeding and selection for highest saccharification yield

Experimental Validation of Specialised Questioning Techniques in Conservation

Conservation increasingly relies on social science tools to understand human behavior. Specialized questioning techniques (SQTs) are a suite of methods designed to reduce bias in social surveys and are widely used to collect data on sensitive topics, including compliance with conservation rules. Most SQTs have been developed in Western, industrialized, educated, rich, and democratic countries, meaning their suitability in other contexts may be limited. Whether these techniques perform better than conventional direct questioning is important for those considering their use. We designed an experiment to validate the performance of four SQTs (unmatched count technique, randomized response technique, crosswise model, and bean method) against direct questions when asking about a commonly researched sensitive behavior in conservation, wildlife hunting. We developed fictional characters, and for each method asked respondents to report the answers that each fictional character should give when asked if they hunt wildlife. We collected data from 609 individuals living close to protected areas in two different cultural and socioeconomic contexts (Indonesia and Tanzania) to quantify the extent to which respondents understood and followed SQT instructions and to explore the sociodemographic factors that influenced a correct response. Data were modeled using binomial general linear mixed models. Participants were more likely to refuse to answer questions asked using SQTs compared with direct questions. Model results suggested that SQTs were harder for participants to understand. Demographic factors (e.g., age and education level) significantly influenced response accuracy. When sensitive responses to sensitive questions were required, all SQTs (excluding the bean method) outperformed direct questions, demonstrating that SQTs can successfully reduce sensitivity bias. However, when reviewing each method, most respondents (59–89%) reported they would feel uncomfortable using them to provide information on their own hunting behavior, highlighting the considerable challenge of encouraging truthful reporting on sensitive topics. Our results demonstrate the importance of assessing the suitability of social science methods prior to their implementation in conservation contexts

Evaluation of Multiprocessor Interconnection Networks

This report describes the work carried out under the EMIN project to set up a testbed for simulating multiprocessor networks. All levels from low level hardware to the software interfaces affect performance, and so the initial simulation testbed provided an MPI interface on top of a cycle level simulator. The networks modelled included a crossbar and the Cray T3D network. Meaningful simulations at this level of detail proved infeasible, however, and an alternative approach was to use microbenchmarking, of both shared memory and message passing network primitives, as a means of characterising network performance in a way which is meaningful to programmers. This led to a refined simulation testbed which cleanly separates workload models from network models, using an interface based on the microbenchmarking work. In a further development, a web version of the testbed was developed and the value of this approach to modelling is evaluated, in particular the accessibility of the simulation m..

Board size and financial transparency : an investigation of Australian not-for-profit organisations

Headlines regarding allegations of fund misappropriation by not-for-profit organisations (NFPs) have led to concerns about the governance and financial transparency of NFPs. Australian NFPs have been no exception to these concerns. Contemporary NFP studies examining board size and financial statement disclosures have focused on USA (Saxton et al. 2012) and Malaysian contexts (Zainon et al. 2014). Extant studies have investigated the influence of board mechanisms on the accuracy of financial statements (Yetman and Yetman 2012) and on financial web disclosures by NFPs in the USA (Lee and Joseph 2013). Other studies have explored the impact of governance boards on annual report disclosures made by Malaysian (Arshad et al. 2013) and Portuguese NFPs (Carvalho et al. 2017). This paper adds to current NFP literature by investigating the influence of board size on the financial transparency of publicly reporting Australian NFPs. The paper addresses its main research question using a resource dependence theoretical (RDT) framework, as introduced by Pfeffer and Salancik (1978). The main observation of this paper is a statistically significant relationship between board size and financial statement disclosures. Larger boards potentially have greater collective expertise, facilitating the use of financial statement disclosures to meet the information needs of resource providers and to support the financial sustainability of NFPs. The paper concludes publicly reporting NFPs need to pay more attention to their board size as the latter impacts their financial transparency and financial sustainability