Continuous enzymatic hydrolysis of sugar beet pectin and l-arabinose recovery within an integrated biorefinery

Sugar beet pulp (SBP) fractionated by steam explosion, released sugar beet pectin (SB-pectin) which was selectively hydrolysed using a novel α-l-arabinofuranosidase (AF), yielding monomeric l-arabinose (Ara) and a galacturonic acid rich backbone (GABB). AF was immobilised on an epoxy-functionalised resin with 70% overall immobilisation yield. Pretreatment of SB-pectin, to remove coloured compounds, improved the stability of the immobilised AF, allowing its reutilisation for up to 10 reaction cycles in a stirred tank reactor. Continuous hydrolysis of SB-pectin was subsequently performed using a packed bed reactor (PBR) with immobilised AF. Reactor performance was evaluated using a Design of Experiment approach. Pretreated SB-pectin hydrolysis was run for 7 consecutive days maintaining 73% of PBR performance. Continuous separation of Ara from GABB was achieved by tangential flow ultrafiltration with 92% Ara recovery. These results demonstrate the feasibility of establishing a continuous bioprocess to obtain Ara from the inexpensive SBP biomass

Selective fractionation of sugar beet pulp for release of fermentation and chemical feedstocks; optimisation of thermo-chemical pre-treatment

The effect of time and pressure on the selective extraction of sugar beet pectin using steam pre-treatment on unprocessed Sugar Beet Pulp was evaluated using a design of experiments approach. This process gave the highest solubilisation of pectin oligomers at a relatively low pressure and longer time (5Bar, 24min), whilst leaving the majority of the cellulose fraction intact. This method of steam pre-treatment fits into the concept of a sugar beet biorefinery as it valorises an existing waste stream without requiring any further physical processing such as milling or dilution with water. The residual cellulose fraction was enriched in cellulose and could be effectively fermented into ethanol by yeast after enzymatic digestion, producing 0.48g ethanol per gram of glucose

Assessing the potential application of bacteria-based self-healing cementitious materials for enhancing durability of wastewater treatment infrastructure

Wastewater treatment plants (WWTPs) around the world are mainly built using concrete. The continuous exposure to wastewater affects the durability of concrete structures and requires costly maintenance or replacement. Concrete production and repair represents ∼8% of the global anthropogenic CO2 emissions due to the use of cement, thus contributing to climate change. Developing a more sustainable cementitious material is therefore required for this vital health infrastructure. In this study, the feasibility of using bacteria-based self-healing (BBSH) cementitious materials for WWTPs is assessed by exposing BBSH mortar prisms to a continuous municipal wastewater flow and comparing their self-healing capacity to equivalent mortar prisms exposed to tap water. Microscopy imaging, water-flow tests and micro-CT analyses were performed to evaluate the self-healing efficiency of the mortar prisms, while SEM-EDX and Raman spectroscopy were used to characterise the healing products. Our work represents the first systematic study of the healing potential of BBSH in mortar exposed to wastewater. The results indicate that the purposely added bacteria are able to induce calcium carbonate precipitation when exposed to wastewater conditions. Moreover, if additional sources of calcium and carbon are embedded within the cement matrix, the rich bacterial community inherently present in the wastewater is capable of inducing calcium carbonate precipitation, even if no bacteria are purposely added to the mortar. The results of this study offer promising avenues for the construction of more sustainable wastewater infrastructure, with the potential of significantly reducing costs and simplifying the production process of BBSH concretes for this specific application

Advancements in bacteria based self-healing concrete and the promise of modelling

In the last two decades self-healing of concrete through microbial based carbonate precipitation has emerged as a promising technology for making concrete structures more resilient and sustainable. Currently, progress in the field is achieved mainly through physical experiments, but their duration and cost are barriers to innovation and keep the number of large scale applications still very limited. Modelling and simulation of the phenomena underlying microbial based healing of concrete may provide a key to complement the experimental efforts, but their development is still in its infancy. In this review, we briefly present the field, introduce some key aspects emerged from the experiments, present the main ongoing developments in modelling and simulation of mineral and microbial systems, and discuss how their synergy may be accomplished to speed up progress in the near future

An Integrated Biorefinery Concept for Conversion of Sugar Beet Pulp into Value-added Chemicals and Pharmaceutical Intermediates

Over 8 million tonnes of sugar beet are grown annually in the UK. Sugar beet pulp (SBP) is the main by-product of sugar beet processing which is currently dried and sold as a low value animal feed. SBP is a rich source of carbohydrates, mainly in the form of cellulose and pectin, including D-glucose (Glu), L-arabinose (Ara) and D-galacturonic acid (GalAc). This work describes the technical feasibility of an integrated biorefinery concept for fractionation of SBP and conversion of these monosaccharides into value-added products. SBP fractionation is initially carried out by steam explosion under mild conditions to yield soluble pectin and insoluble cellulose fractions. The cellulose is readily hydrolysed by cellulases to release Glu that can then be fermented by a commercial Yeast strain to produce bioethanol with a high yield. The pectin fraction can be either fully hydrolysed, using physico-chemical methods, or selectively hydrolysed, using cloned arabinases and galacturonases, to yield Ara-rich and GalAc-rich streams. These monomers can be separated using either Centrifugal Partition Chromatography (CPC) or ultrafiltration into streams suitable for subsequent enzymatic upgrading. Building on our previous experience with transketolase (TK) and transaminase (TAm) enzymes, the conversion of Ara and GalAc into higher value products was explored. In particular the conversion of Ara into L-gluco-heptulose (GluHep), that has potential therapeutic applications in hypoglycaemia and cancer, using a mutant TK is described. Preliminary studies with TAm also suggest GluHep can be selectively aminated to the corresponding chiral aminopolyol. Current work is addressing upgrading of the remaining SBP monomer, GalAc, and modelling of the biorefinery concept to enable economic and Life Cycle Analysis (LCA)

Selective fractionation of sugar beet pulp for release of fermentation and chemical feedstocks; optimisation of thermo-chemical pre-treatment

The effect of time and pressure on the selective extraction of sugar beet pectin using steam pre-treatment on unprocessed Sugar Beet Pulp was evaluated using a design of experiments approach. This process gave the highest solubilisation of pectin oligomers at a relatively low pressure and longer time (5 Bar, 24 min), whilst leaving the majority of the cellulose fraction intact. This method of steam pre-treatment fits into the concept of a sugar beet biorefinery as it valorises an existing waste stream without requiring any further physical processing such as milling or dilution with water. The residual cellulose fraction was enriched in cellulose and could be effectively fermented into ethanol by yeast after enzymatic digestion, producing 0.48 g ethanol per gram of glucose.</p

Air-entraining admixtures as a protection method for bacterial spores in self-healing cementitious composites:Healing evaluation of early and later-age cracks

Costs associated with the encapsulation process of bacterial spores continue to be a limiting factor for the commercialisation of self-healing cementitious materials. The feasibility of using air-entraining admixtures (AEAs) as an economical and straightforward encapsulation method for bacterial spores was evaluated to heal cracks (∼0.50 mm) that were formed at an early (28 days) or later age (9 months). Three AEAs, commonly used in concrete industry, were compared to a successfully proven protection method (i.e., via aerated concrete granules (ACGs)). In this regard, only one of the three AEAs investigated improved the healing performance when compared to an equivalent mix using bacterial spores encapsulated in ACGs. Healing ratios obtained with this successful AEA were 59.6% and 46.2% higher than the results observed for the ACGs-containing mix when the cracking age was 28 days and 9 months, respectively. Moreover, water penetration resistance was increased by 18.1% or presented very similar values (∼84%) after 56 days of healing for early or later-formed cracks, respectively. Moreover, a simple cost analysis was conducted to confirm the significant economic benefits of using AEAs to protect directly added bacterial spores. In this regard, the cost of using AEAs is about 13 times lower than for ACGs. Therefore, this study provides for the first time, evidence of the feasibility of using AEAs to protect bacterial spores, opening the doors to the development of bespoke AEAs to design cost-efficient self-healing cementitious materials.</p

Development of an efficient technique for gene deletion and allelic exchange in Geobacillus spp.

Abstract Background Geobacillus thermoglucosidasius is a thermophilic, natural ethanol producer and a potential candidate for commercial bioethanol production. Previously, G. thermoglucosidasius has been genetically modified to create an industrially-relevant ethanol production strain. However, creating chromosomal integrations and deletions in Geobacillus spp. is laborious. Here we describe a new technique to create marker-less mutations in Geobacillus utilising a novel homologous recombination process. Results Our technique incorporates counter-selection using β-glucosidase and the synthetic substrate X-Glu, in combination with a two-step homologous recombination process where the first step is a selectable double-crossover event that deletes the target gene. We demonstrate how we have utilised this technique to delete two components of the proteinaceous shell of the Geobacillus propanediol-utilization microcompartment, and simultaneously introduce an oxygen-sensitive promoter in front of the remaining shell-component genes and confirm its functional incorporation. Conclusion The selectable deletion of the target gene in the first step of our process prevents re-creation of wild-type which can occur in most homologous recombination techniques, circumventing the need for PCR screening to identify mutants. Our new technique therefore offers a faster, more efficient method of creating mutants in Geobacillus