Exploring Anaerobic Bacteria for Industrial Biotechnology - Diversity Studies, Screening and Biorefinery Applications

Depletion of easily accessible fossil energy resources, threat of climate change and political priority to achieve energy self-sufficiency and sustainable solutions prioritize a conscious and smart use of renewable resources to generate a bio-based economy. Bio-based compounds can replace chemicals and fuels that are now mainly produced from crude oil. Efficient processes for the conversion of plant biomass into compounds of interest to the biorefinery industry occur naturally in anaerobic environments such as in the forestomach of herbivores. Exploration of anaerobic microorganisms for industrial biotechnological applications creates the possibility to convey efficient and flexible processes, with lower implementation and running costs, making it also applicable to developing and emerging economies. Despite the growing interest in anaerobic microorganisms for applications in industrial biotechnology, there is less information available concerning their diversity and function compared to what is known for their aerobic counterparts. To counter this, microbial diversity studies on an unexplored environment for microbial applications, was investigated by molecular and traditional cultivation techniques. The bacterial diversity of the forestomach of the llama, showed differences in the prokaryotes populations according to the complexity of the material type digested. Bacterial isolates were selected by their ability to produce compounds such as organic acids and alcohols and hydrolytic enzymes. Also, a new strategy for cultivation of anaerobic microorganisms with the potential for an improved isolation rate and screening has been developed in this thesis work. The technique is based on single cell entrapment in alginate microbeads. A method was optimized for simple preparation under anaerobic conditions and successful cultivation of single cells was observed. Further applications of anaerobic bacteria towards the biorefinery were also studied. The production of 1,3-propanediol, a compound used as building block for polymer materials, was investigated from selected llama isolates. The use of wheat straw as co-substrate and/or support material improved the concentration of 1,3-propanediol by 29% for C. butyricum BSL59 and 65% for C. butyricum BSL61 in comparison to using sole glycerol in the medium. The use of wheat straw was also superior in comparison to addition of pure sugars. Moreover, the solid residue from sequence batch fermentation using wheat straw as co-substrate showed to have high methane potential yield. Demonstrating that agriculture residue can be used in an integrated process for the production of valuable chemical compounds and energy carriers. Finally, a new method for cell immobilization forming a macroporous material was evaluated for butanol production which reaches high yields and allows repeated use of the cell-based material

Multiple phosphorus acquisition strategies adopted by fine roots in low-fertility soils in Central Amazonia

This is the final version. Available from Springer Verlag via the DOI in this record.Background and aims Ancient Amazon soils are characterised by low concentrations of soil phosphorus (P). Therefore, it is hypothesised that plants may invest a substantial proportion of their resources belowground to adjust their P-uptake strategies, including root morphological, physiological (phosphatase enzyme activities) and biotic (arbuscular mycorrhizal (AM) associations) adaptations. Since these strategies are energy demanding, we hypothesise that trade-offs between morphological traits and root phosphatase exudation and symbiotic associations would occur. Specifically, we expected that plants which invest in finer roots, and therefore have greater ability to explore large soil volumes, would have a high investment in physiological adaptations such as enhanced phosphatase production. In contrast, we expected that plants with predominantly thicker roots would invest more in symbiotic associations, in which carbon is traded for P acquired from AM fungal communities. Methods We collected absorptive roots (<2 mm diameter) from a lowland Central Amazon forest near Manaus, Brazil. We measured fine root diameter, specific root length (SRL), specific root area (SRA), root tissue density (RTD), root phosphatase activity (APase) and arbuscular mycorrhizal (AM) fungi colonisation. Results Root morphological traits were related to APase activity, with higher APase activity in roots with higher SRL and SRA but lower RTD. However, the degree of AM colonisation was not related to any measured root morphological trait. Conclusions Fine absorptive roots likely benefit from having low RTD, high SRL, SRA and APase exudation to acquire P efficiently. However, because AM colonisation was not related to root morphology, we suggest that investment in multiple P-uptake strategies is required for maintaining productivity in Central Amazon forests.Natural Environment Research Council (NERC)Brazilian National Council for Scientific and Technological Development (CNPq)Australian Research Counci

Characterization of paspalum accessions as ornamental Lawn .

The lawn industry involves hundreds of millions of dollars around the world. Today, Brazilian lawns are formed mainly by Paspalum notatum. The native genus Paspalum contains a wide genetic variability between and within species. These germplasms are an important source to select genotypes with high potential for use as lawns. This study aimed to select the best Paspalum accession for use as cover for ornamental purposes. Main selection criteria are rapid expansion and low need for mowing under the conditions in northeastern Brazil. Five pre-selected Paspalum accessions (0102254, 043566, 023728, 010006 and 019178) were planted and field evaluations were made at two weeks intervals for two consecutive years after establishment. Soil coverage rate, ornamental quality (general appearance) of the lawn, extension of lateral growth, color homogeneity and weed invasion were rated by means of a visual appraisal. Based on the results obtained at the end of the 2nd year of cultivation, the accession 023728 had the highest scores and demonstrated an ornamental quality equal to or greater than the control

Direct evidence for phosphorus limitation on Amazon forest productivity

The productivity of rainforests growing on highly weathered tropical soils is expected to be limited by phosphorus availability1. Yet, controlled fertilization experiments have been unable to demonstrate a dominant role for phosphorus in controlling tropical forest net primary productivity. Recent syntheses have demonstrated that responses to nitrogen addition are as large as to phosphorus2, and adaptations to low phosphorus availability appear to enable net primary productivity to be maintained across major soil phosphorus gradients3. Thus, the extent to which phosphorus availability limits tropical forest productivity is highly uncertain. The majority of the Amazonia, however, is characterized by soils that are more depleted in phosphorus than those in which most tropical fertilization experiments have taken place2. Thus, we established a phosphorus, nitrogen and base cation addition experiment in an old growth Amazon rainforest, with a low soil phosphorus content that is representative of approximately 60% of the Amazon basin. Here we show that net primary productivity increased exclusively with phosphorus addition. After 2 years, strong responses were observed in fine root (+29%) and canopy productivity (+19%), but not stem growth. The direct evidence of phosphorus limitation of net primary productivity suggests that phosphorus availability may restrict Amazon forest responses to CO2 fertilization4, with major implications for future carbon sequestration and forest resilience to climate change.The authors acknowledge funding from the UK Natural Environment Research Council (NERC), grant number NE/L007223/1. This is publication 850 in the technical series of the BDFFP. C.A.Q. acknowledges the grants from Brazilian National Council for Scientific and Technological Development (CNPq) CNPq/LBA 68/2013, CNPq/MCTI/FNDCT no. 18/2021 and his productivity grant. C.A.Q., H.F.V.C., F.D.S., I.A., L.F.L., E.O.M. and S.G. acknowledge the AmazonFACE programme for financial support in cooperation with Coordination for the Improvement of Higher Education Personnel (CAPES) and the National Institute of Amazonian Research as part of the grants CAPES-INPA/88887.154643/2017-00 and 88881.154644/2017-01. T.F.D. acknowledges funds from FundacAo de Amparo a Pesquisa do Estado de SAo Paulo (FAPESP), grant 2015/50488-5, and the Partnership for Enhanced Engagement in Research (PEER) programme grant AID-OAA-A-11-00012. L.E.O.C.A. thanks CNPq (314416/2020-0)

Refolding by High Pressure of a Toxin Containing Seven Disulfide Bonds: Bothropstoxin-1 from Bothrops jararacussu

Aggregation is a serious obstacle for recovery of biologically active heterologous proteins from inclusion bodies (IBs) produced by recombinant bacteria. E. coli transformed with a vector containing the cDNA for Bothropstoxin-1 (BthTx-1) expressed the recombinant product as IBs. In order to obtain the native toxin, insoluble and aggregated protein was refolded using high hydrostatic pressure (HHP). IBs were dissolved and refolded (2 kbar, 16 h), and the effects of protein concentration, as well as changes in ratio and concentration of oxido-shuffling reagents, guanidine hydrochloride (GdnHCl), and pH in the refolding buffer, were assayed. A 32% yield (7.6 mg per liter of bacterial culture) in refolding of the native BthTx-1 was obtained using optimal conditions of the refolding buffer (Tris–HCl buffer, pH 7.5, containing 3 mM of a 2:3 ratio of GSH/GSSG, and 1 M GdnHCl). Scanning electron microscopy (SEM) showed that that disaggregation of part of IBs particles occurred upon compression and that the morphology of the remaining IBs, spherical particles, was not substantially altered. Dose-dependent cytotoxic activity of high-pressure refolded BthTx-1 was shown in C2C12 muscle cells

Second asymptomatic carotid surgery trial (ACST-2): a randomised comparison of carotid artery stenting versus carotid endarterectomy

Background: Among asymptomatic patients with severe carotid artery stenosis but no recent stroke or transient cerebral ischaemia, either carotid artery stenting (CAS) or carotid endarterectomy (CEA) can restore patency and reduce long-term stroke risks. However, from recent national registry data, each option causes about 1% procedural risk of disabling stroke or death. Comparison of their long-term protective effects requires large-scale randomised evidence. Methods: ACST-2 is an international multicentre randomised trial of CAS versus CEA among asymptomatic patients with severe stenosis thought to require intervention, interpreted with all other relevant trials. Patients were eligible if they had severe unilateral or bilateral carotid artery stenosis and both doctor and patient agreed that a carotid procedure should be undertaken, but they were substantially uncertain which one to choose. Patients were randomly allocated to CAS or CEA and followed up at 1 month and then annually, for a mean 5 years. Procedural events were those within 30 days of the intervention. Intention-to-treat analyses are provided. Analyses including procedural hazards use tabular methods. Analyses and meta-analyses of non-procedural strokes use Kaplan-Meier and log-rank methods. The trial is registered with the ISRCTN registry, ISRCTN21144362. Findings: Between Jan 15, 2008, and Dec 31, 2020, 3625 patients in 130 centres were randomly allocated, 1811 to CAS and 1814 to CEA, with good compliance, good medical therapy and a mean 5 years of follow-up. Overall, 1% had disabling stroke or death procedurally (15 allocated to CAS and 18 to CEA) and 2% had non-disabling procedural stroke (48 allocated to CAS and 29 to CEA). Kaplan-Meier estimates of 5-year non-procedural stroke were 2·5% in each group for fatal or disabling stroke, and 5·3% with CAS versus 4·5% with CEA for any stroke (rate ratio [RR] 1·16, 95% CI 0·86–1·57; p=0·33). Combining RRs for any non-procedural stroke in all CAS versus CEA trials, the RR was similar in symptomatic and asymptomatic patients (overall RR 1·11, 95% CI 0·91–1·32; p=0·21). Interpretation: Serious complications are similarly uncommon after competent CAS and CEA, and the long-term effects of these two carotid artery procedures on fatal or disabling stroke are comparable. Funding: UK Medical Research Council and Health Technology Assessment Programme