Engineering Rhodosporidium toruloides for sustainable production of value-added punicic acid from glucose and wood residues.

Rhodosporidium toruloides has emerged as a prominent candidate for producing single-cell oil from cost-effective feedstocks. In this study, the capability of R. toruloides to produce punicic acid (PuA), a representative plant unusual fatty acid, was investigated. The introduction of acyl lipid desaturase and conjugase (PgFADX) allowed R. toruloides to accumulate 3.7 % of total fatty acids as PuA. Delta-12 acyl lipid desaturase (PgFAD2) and diacylglycerol acyltransferase 2 were shown to benefit PuA production. The strain with PgFADX and PgFAD2 coexpression accumulated 12 % of its lipids as PuA from glucose, which translated into a PuA titer of 451.6 mg/L in shake flask condition. Utilizing wood hydrolysate as the feedstock, this strain produced 6.4 % PuA with a titer of 310 mg/L. Taken together, the results demonstrated that R. toruloides could serve as an ideal platform for the production of plant-derived high-value conjugated fatty acid using agricultural and forestry waste as feedstock.

Adapted feeding strategies in fed-batch fermentation improve sugar delivery and ethanol productivity.

Bioethanol is a renewable fuel widely used in road transportation and is generally regarded as a clean energy source. Although fermentation is one of the major processes in bioethanol production, studies on improving its efficiency through operational design are limited, especially compared to other steps (pretreatment and hydrolysis/saccharification). In this study, two adapted feeding strategies, in which feed medium addition (sugar delivery) was adjusted to increase the supply of fermentable sugar, were developed to improve ethanol productivity in 5-L fed-batch fermentation by Saccharomyces cerevisiae. Specifically, a linear adapted feeding strategy was established based on changes in cell biomass, and an exponential adapted feeding strategy was developed based on cell biomass accumulation. By implementing these two feeding strategies, the overall ethanol productivity reached 0.88±0.04 and 0.87±0.06 g/L/h, respectively. This corresponded to ~20% increases in ethanol productivity compared to fixed pulsed feeding operations. Additionally, there was no residual glucose at the end of fermentation, and final ethanol content reached 95±3 g/L under the linear adapted operation and 104±3 g/L under the exponential adapted feeding strategy. No statistical difference was observed in the overall ethanol yield (ethanol-to-sugar ratio) between fixed and adapted feeding strategies (~91%). These results demonstrate that sugar delivery controlled by adapted feeding strategies was more efficient than fixed feeding operations, leading to higher ethanol productivity. Overall, this study provides novel adapted feeding strategies to improve sugar delivery and ethanol productivity. Integration into the current practices of the ethanol industry could improve productivity and reduce production costs of fermentation processes.

Valorization strategies for hazardous proteinaceous waste from rendering production - Recent advances in specified risk materials (SRMs) conversion.

Strict bans on specific risk materials (SRMs) are in place to prevent the spread of bovine spongiform encephalopathy (BSE). SRMs are characterized as tissues in cattle where misfolded proteins, the potential source of BSE infection, are concentrated. As a result of these bans, SRMs must be strictly isolated and disposed of, resulting in great costs for rendering companies. The increasing yield and the landfill of SRMs also exacerbated the burden on the environment. To cope with the emergence of SRMs, novel disposal methods and feasible value-added conversion routes are needed. The focus of this review is on the valorization progress achieved in the conversion of peptides derived from SRMs via an alternative disposal method, thermal hydrolysis. Promising value-added conversion of SRM-derived peptides into tackifiers, wood adhesives, flocculants, and bioplastics, is introduced. The potential conjugation strategies that can be adapted to SRM-derived peptides for desired properties are also critically reviewed. The purpose of this review is to discover a technical platform through which other hazardous proteinaceous waste, SRMs, can be treated as a high-demand feedstock for the production of renewable materials.

Current progress in lipid-based biofuels: Feedstocks and production technologies.

The expanding use of fossil fuels has caused concern in terms of both energy security and environmental issues. Therefore, attempts have been made worldwide to promote the development of renewable energy sources, among which biofuel is especially attractive. Compared to other biofuels, lipid-derived biofuels have a higher energy density and better compatibility with existing infrastructure, and their performance can be readily improved by adjusting the chemical composition of lipid feedstocks. This review thus addresses the intrinsic interactions between lipid feedstocks and lipid-based biofuels, including biodiesel, and renewable equivalents to conventional gasoline, diesel, and jet fuel. Advancements in lipid-associated biofuel technology, as well as the properties and applicability of various lipid sources in terms of biofuel production, are also discussed. Furthermore, current progress in lipid production and profile optimization in the context of plant lipids, microbial lipids, and animal fats are presented to provide a wider context of lipid-based biofuel technology.

Ruminant-Waste Protein Hydrolysates and Their Derivatives as a Bio-Flocculant for Oil Sands Tailing Management.

Reclamation of tailings ponds is a critical issue for the oil industry. After years of consolidation, the slurry in tailings ponds, also known as fluid fine tailings, is mainly comprised of residual bitumen, water, and fine clay particles. To reclaim the lands that these ponds occupy, separation of the solid particles from the liquid phase is necessary to facilitate water removal and recycling. Traditionally, synthetic polymers have been used as flocculants to facilitate this process, but they can have negative environmental consequences. The use of biological polymers may provide a more environmentally friendly approach to flocculation, and eventual soil remediation, due to their natural biodegradability. Peptides derived from specified risk materials (SRM), a proteinaceous waste stream derived from the rendering industry, were investigated to assess their viability for this application. While these peptides could achieve >50% settling within 3 h in bench-scale settling tests using kaolinite tailings, crosslinking peptides with glutaraldehyde greatly improved their flocculation performance, leading to a >50% settling in only 10 min. Settling experiments using materials obtained through different reactant ratios during crosslinking identified a local optimum molar reactant ratio of 1:32 (peptide amino groups to glutaraldehyde aldehyde groups), resulting in 81.6% settling after 48 h. Taken together, these data highlight the novelty of crosslinking waste-derived peptides with glutaraldehyde to generate a value-added bioflocculant with potential for tailings ponds consolidation.

Using Specified Risk Materials-Based Peptides for Oil Sands Fluid Fine Tailings Management.

Fluid fine tailings are produced in huge quantities by Canada's mined oil sands industry. Due to the high colloidal stability of the contained fine solids, settling of fluid fine tailings can take hundreds of years, making the entrapped water unavailable and posing challenges to public health and the environment. This study focuses on developing value-added aggregation agents from specified risk materials (SRM), a waste protein stream from slaughterhouse industries, to achieve an improved separation of fluid fine tailings into free water and solids. Settling results using synthetic kaolinite slurries demonstrated that, though not as effective as hydrolyzed polyacrylamide, a commercial flocculant, the use of SRM-derived peptides enabled a 2-3-fold faster initial settling rate than the blank control. The pH of synthetic kaolinite tailings was observed to be slightly reduced with increasing peptides dosage in the test range (10-50 kg/ton). The experiments on diluted fluid fine tailings (as a representation of real oil sands tailings) demonstrated an optimum peptides dosage of 14 kg/ton, which resulted in a 4-fold faster initial settling rate compared to the untreated tailings. Overall, this study demonstrates the novelty and feasibility of using SRM-peptides to address intractable oil sands fluid tailings.

Co-production of ethanol and cellulose nanocrystals through self-cycling fermentation of wood pulp hydrolysate.

A promising approach to help offset production costs for the cellulosic ethanol industry is to improve ethanol productivity while simultaneously generating value-added by-products. This study reports integration of an advanced fermentation approach (self-cycling fermentation) with the production of cellulose nanocrystals. Specifically, wood pulp was enzymatically hydrolyzed to yield dissolved sugars, which were fed to a self-cycling fermentation system for ethanol production, and residual solids were used for cellulose nanocrystals production via acid hydrolysis. Self-cycling fermentation achieved stable ethanol production for 10 cycles with significantly greater productivity than batch operation: ethanol volumetric productivity increased by 63-95% and annual ethanol productivity by 96 ± 5%. Additionally, the enzyme hydrolysis approach employed did not impede ethanol fermentation, and the cellulose nanocrystals generated displayed properties consistent with previous studies. Taken together, these results highlight the potential of this co-production strategy to produce both cellulosic ethanol and cellulose nanocrystals from a single feedstock.

Incorporation of Biosolids as Water Replacement in a Two-Step Renewable Hydrocarbon Process: Hydrolysis of Brown Grease with Biosolids.

ABSTRACT: The accumulating volumes of biosolids in lagoons worldwide have intensified the need to develop innovative wastewater treatment strategies. Here, we provide proof-of-concept for the incorporation of biosolids into the hydrolysis step of a two-step thermal conversion of lipids for production of renewable hydrocarbons, which can be utilized as renewable fuels. Brown grease was hydrolysed with biosolids or water at 260-280 °C for 60 min at a mass ratio of 1:1 feed to water or biosolids. The feedstock and products were characterized using various analytical techniques to compare the performance of biosolids to water. The results indicated that there was no significant difference in the degree of hydrolysis of brown grease when biosolids was used as water replacement. The fatty acids composition after hydrolysis when biosolids was used as a water replacement also remained largely unchanged. Hydrolysis of brown grease with biosolids could be achieved at pH ranging from 3.3 to 8.9, and at a lower than previously established temperature. Significantly, the rapid settling of solid material in biosolids observed after thermal hydrolysis of brown grease may reduce the necessity of biosolids settling lagoons. Thus, incorporation of biosolids into a lipid hydrolysis-pyrolysis process may simultaneously benefit the biofuel and waste management sectors.

A Biorefinery Strategy That Introduces Hydrothermal Treatment Prior to Acid Hydrolysis for Co-generation of Furfural and Cellulose Nanocrystals.

Hydrothermal treatment of wood pulp at 150-225°C prior to acid hydrolysis was investigated in the context of isolating cellulose nanocrystals (CNCs). The objective was 2-folds as follows: (a) generating furfural as a value-added co-product; and (b) concentrating and forming new CNC precursors through thermal re-orientation of para-crystalline cellulose chains that will in turn improve CNC recovery and yield. Furfural yields up to 19 and 21% xylan conversion were obtained at 200 and 225°C hydrothermal treatments, respectively. In addition, these hydrothermal treatment conditions increased the crystallinity index of the pulp (77%) to 84 and 80%, respectively. Consequently, the CNC yield from hydrothermally treated wood pulp, when compared to untreated wood pulp, improved by up to 4- and 2-folds, respectively. An efficient acid hydrolysis process with yield improvements can translate to reduced CNC isolation and purification costs and increased production capacity. The qualities of the CNCs in terms of particle size and crystallinity were not affected due to hydrothermal treatment. However, the zeta potential, sulfur, hydrogen, and oxygen content of the CNCs were significantly lower at 225°C while carbon composition increased, and dark brown coloration was observed that indicates caramelization. This study demonstrates for the first time a novel biorefinery strategy that introduces hydrothermal treatment prior to acid hydrolysis to co-generate furfural and CNC with improved efficiency.

Pyrolysis of fatty acids derived from hydrolysis of brown grease with biosolids.

The escalating generation of biosolids and increasing regulations regarding their safe handling and disposal have created a great environmental challenge. Recently, biosolids have been incorporated into the hydrolysis step of a two-step thermal lipid conversion process to act as water replacement in the production of renewable chemicals and fuels. Here, the hexane extract recovered from hydrolysis of biosolids, lipids from brown grease hydrolyzed using either water (control) or biosolids as a water replacement, was pyrolyzed at 410-450 °C for 2 h. The product distribution and composition were not significantly different when biosolids were used to hydrolyze brown grease instead of water. The liquid product consisted mainly of alkanes, alkenes, aromatics, and cyclic compounds similar to those in petroleum-derived liquid fuels. However, the use of biosolids as a water substitute resulted in a significant increase in sulphur content of the pyrolysate, which will necessitate processes to reduce the sulphur content before or after pyrolysis. Nevertheless, the pathways proposed in this paper are considered as potentially economically viable approaches to not only resolve the issues associated with disposal of biosolids but also to produce renewable hydrocarbons for fuel and chemical applications. Graphical abstract.

The potential of fiber-depleted starch concentrate produced through air currents assisted particle separation of barley flour in bio-ethanol production.

Isolation of fiber concentrate enriched in ß-glucan from barley flour via air currents assisted particle separation (ACAPS) generates an underutilized by-product stream, starch concentrate. Since barley starch concentrate (BSC) is depleted in soluble fibre, we examined the enzyme requirements for its hydrolysis and subsequent fermentation. Lower doses of a common raw starch hydrolyzing enzyme (STARGEN™ 002) effectively hydrolyzed BSC, achieving similar hydrolysis kinetics to the wheat benchmark. Hydrolysis of BSC did not require further enzyme supplementation, which is required for optimal wheat hydrolysis. This likely resulted from the smaller particle size of BSC relative to wheat feedstocks. Interestingly, simultaneous saccharification and fermentation of BSC using a 0.25X dose of STARGEN™ 002 alone enabled efficient ethanol production, though a requirement for phosphorus supplementation was identified. This study proposes a biorefining strategy that supports the generation of a value-added co-product, starch concentrate, while significantly reducing the enzyme requirements for bioethanol production.

Improved bioethanol productivity through gas flow rate-driven self-cycling fermentation.

BACKGROUND: The growth of the cellulosic ethanol industry is currently impeded by high production costs. One possible solution is to improve the performance of fermentation itself, which has great potential to improve the economics of the entire production process. Here, we demonstrated significantly improved productivity through application of an advanced fermentation approach, named self-cycling fermentation (SCF), for cellulosic ethanol production. RESULTS: The flow rate of outlet gas from the fermenter was used as a real-time monitoring parameter to drive the cycling of the ethanol fermentation process. Then, long-term operation of SCF under anaerobic conditions was improved by the addition of ergosterol and fatty acids, which stabilized operation and reduced fermentation time. Finally, an automated SCF system was successfully operated for 21 cycles, with robust behavior and stable ethanol production. SCF maintained similar ethanol titers to batch operation while significantly reducing fermentation and down times. This led to significant improvements in ethanol volumetric productivity (the amount of ethanol produced by a cycle per working volume per cycle time)-ranging from 37.5 to 75.3%, depending on the cycle number, and in annual ethanol productivity (the amount of ethanol that can be produced each year at large scale)-reaching 75.8 ± 2.9%. Improved flocculation, with potential advantages for biomass removal and reduction in downstream costs, was also observed. CONCLUSION: Our successful demonstration of SCF could help reduce production costs for the cellulosic ethanol industry through improved productivity and automated operation.

Co-Production of Cellulose Nanocrystals and Fermentable Sugars Assisted by Endoglucanase Treatment of Wood Pulp.

In this study, fermentable sugars and cellulose nanocrystals (CNCs) were co-produced from endoglucanase treatment of wood pulp, followed by acid hydrolysis. Enzymatic hydrolysis was performed using two endoglucanases differentiated by the presence or absence of a cellulose-binding domain (CBD). The enzyme with an intact CBD gave the higher glucan conversion (up to 14.1 ± 1.2 wt %) and improved the degree of crystallinity of the recovered wood pulp fiber (up to 83.0 ± 1.0%). Thus, this endoglucanase-assisted treatment successfully removed amorphous content from the original cellulosic feedstock. CNC recovery (16.9 ± 0.7 wt %) from the feedstock going into the acid hydrolysis was improved relative to untreated pulp (13.2 ± 0.6 wt %). The mass loss from enzymatic treatment did not cause a decrease in the CNC yield from the starting material. The characteristics of CNCs obtained through acid hydrolysis (with or without enzyme treatment of pulp) were analyzed using X-ray diffraction, transmission electron microscopy, dynamic light scattering, Fourier transform infrared spectroscopy, and differential scanning calorimetry as characterization techniques. The CNCs generated through acid hydrolysis of endoglucanase-treated wood pulp displayed comparable properties relative to those generated using untreated pulp. Thus, endoglucanase treatment can enable co-production of CNCs and sugars for biofuel fermentation.

Characterization of Cellulase-Treated Fibers and Resulting Cellulose Nanocrystals Generated through Acid Hydrolysis.

Integrating enzymatic treatment and acid hydrolysis potentially improves the economics of cellulose nanocrystal (CNC) production and demonstrates a sustainable cellulosic ethanol co-generation strategy. In this study, the effect of enzymatic treatment on filter paper and wood pulp fibers, and CNCs generated via subsequent acid hydrolysis were assessed. Characterization was performed using a pulp quality monitoring system, scanning and transmission electron microscopies, dynamic light scattering, X-ray diffraction, and thermogravimetric analysis. Enzymatic treatment partially reduced fiber length, but caused swelling, indicating simultaneous fragmentation and layer erosion. Preferential hydrolysis of less ordered cellulose by cellulases slightly improved the crystallinity index of filter paper fiber from 86% to 88%, though no change was observed for wood pulp fibre. All CNC colloids were stable with zeta potential values below -39 mV and hydrodynamic diameters ranging from 205 to 294 nm. Furthermore, the temperature for the peak rate of CNC thermal degradation was generally not affected by enzymatic treatment. These findings demonstrate that CNCs of comparable quality can be produced from an enzymatically-mediated acid hydrolysis biorefining strategy that co-generates fermentable sugars for biofuel production.

Accelerating settling rates of biosolids lagoons through thermal hydrolysis.

Although the improved dewaterability and digestibility of primary biosolids subjected to thermal hydrolysis has been studied for decades, there are a surprisingly small number of studies exploring the use of this thermal treatment for digested biosolids that are typically left to settle in large settling lagoons. This is likely because of the high capital and operating costs associated with thermal hydrolysis, coupled with the limited applications and value of the resulting products. However, due to the anticipated increases in the amount of generated biosolids combined with issues surrounding potential environmental release and the limited availability of land for additional lagoons, other biosolids management strategies are being explored. Here, we show that thermal hydrolysis at 280 °C for 1 h resulted in 78.2 ±â€¯0.8% settling after 2 h. Furthermore, addition of phosphoric acid to lower the pH of the hydrolysate to pH 3 resulted in increased settling rates, but the final volume of unsettled material after 2 h was statistically similar to the thermally hydrolyzed material without pH adjustment (75.7 ±â€¯2.3%). Remarkably, when the pH of the digested biosolids was adjusted to 3 prior to thermal hydrolysis, a settling rate of 87.3 ±â€¯1.1% was observed after just 15 min. Significantly, the dewaterability of thermally hydrolyzed biosolids was measured in our experiments through natural settling, without the use of external mechanics. Taken together, the data presented in this paper demonstrate that high temperature thermal hydrolysis is a promising method for accelerating the settling rates of digested biosolids and may represent a viable alternative to building and maintaining biosolids lagoons.

Development of hydrolysed protein-based plywood adhesive from slaughterhouse waste: effect of chemical modification of hydrolysed protein on moisture resistance of formulated adhesives.

Specified risk materials (SRM) constitute the proteinaceous waste of slaughterhouses and are currently being disposed off either by incineration or by land filling. Over the last few years, our efforts have focused on developing technology platforms for deployment of this renewable resource for various value-added industrial applications. This report describes a technology for utilization of SRM for the development of an environmentally friendly plywood adhesive with an improved water resistance property. The feedstock (SRM) was first thermally hydrolysed according to a standard protocol, and the hydrolysed protein fragments (peptides) were recovered from the hydrolysate. The recovered peptides were chemically modified through esterification reaction using ethanol, and then chemically crosslinked with polyamideamine-epichlorohydrin (PAE) resin to develop a wood adhesive system. Plywood specimens were then developed using the peptides-PAE resin-based adhesive. The effects of crosslinking time, solid content of the adhesive formulation, ratio of peptides and crosslinking agent in the formulation, and curing conditions of specimen preparation on lap shear strength of resulting plywood specimens were systematically evaluated. Despite the hydrophilic nature of hydrolysed protein fragments, the peptides-PAE resin formulations exhibited remarkable water resistance property after curing. Capping of polar carboxyl groups of peptides by converting them to esters further improved the water resistance property of this adhesive system. Under the optimum conditions of adhesive preparation and curing, the ethyl ester derivative of peptides and PAE resin-based formulations resulted in plywood specimens having comparable dry as well as soaked shear strengths to those of commercial phenol formaldehyde resin.

Utilization of Slaughterhouse Waste in Value-Added Applications: Recent Advances in the Development of Wood Adhesives.

Globally, slaughterhouses generate large volumes of animal byproducts. While these byproducts are an important resource of industrial protein that could potentially be utilized in various value-added applications, they are currently either underutilized in high-value applications or being used for production of relatively low-value products such as animal feed and pet food. Furthermore, some of the byproducts of animal slaughtering cannot enter food and feed chains and thus their disposal possesses a serious environmental concern. An innovative utilization of the proteinaceous waste generated by slaughterhouses comprises of waste processing to extract proteins, which are then incorporated into industrial processes to produce value-added bio-based products. In this report, we review the current processes for extraction of protein from proteinaceous waste of slaughterhouses, and utilization of the recovered protein in the development of protein-based wood adhesives.

Improving ethanol productivity through self-cycling fermentation of yeast: a proof of concept.

BACKGROUND: The cellulosic ethanol industry has developed efficient strategies for converting sugars obtained from various cellulosic feedstocks to bioethanol. However, any further major improvements in ethanol productivity will require development of novel and innovative fermentation strategies that enhance incumbent technologies in a cost-effective manner. The present study investigates the feasibility of applying self-cycling fermentation (SCF) to cellulosic ethanol production to elevate productivity. SCF is a semi-continuous cycling process that employs the following strategy: once the onset of stationary phase is detected, half of the broth volume is automatically harvested and replaced with fresh medium to initiate the next cycle. SCF has been shown to increase product yield and/or productivity in many types of microbial cultivation. To test whether this cycling process could increase productivity during ethanol fermentations, we mimicked the process by manually cycling the fermentation for five cycles in shake flasks, and then compared the results to batch operation. RESULTS: Mimicking SCF for five cycles resulted in regular patterns with regards to glucose consumption, ethanol titer, pH, and biomass production. Compared to batch fermentation, our cycling strategy displayed improved ethanol volumetric productivity (the titer of ethanol produced in a given cycle per corresponding cycle time) and specific productivity (the amount of ethanol produced per cellular biomass) by 43.1 ± 11.6 and 42.7 ± 9.8%, respectively. Five successive cycles contributed to an improvement of overall productivity (the aggregate amount of ethanol produced at the end of a given cycle per total processing time) and the estimated annual ethanol productivity (the amount of ethanol produced per year) by 64.4 ± 3.3 and 33.1 ± 7.2%, respectively. CONCLUSIONS: This study provides proof of concept that applying SCF to ethanol production could significantly increase productivities, which will help strengthen the cellulosic ethanol industry.

Simultaneous hydrolysis and co-fermentation of whey lactose with wheat for ethanol production.

Whey permeate was used as a co-substrate to replace part of the wheat for ethanol production by Saccharomyces cerevisiae. The simultaneous saccharification and fermentation was achieved with ß-galactosidase added at the onset of the fermentation to promote whey lactose hydrolysis. Aspergillus oryzae and Kluyveromyces lactis ß-galactosidases were two enzymes selected and used in the co-fermentation of wheat and whey permeate for the comparison of their effectiveness on lactose hydrolysis. The possibility of co-fermentations in both STARGEN and jet cooking systems was investigated in 5L bioreactors. Ethanol yields from the co-fermentations of wheat and whey permeate were evaluated. It was found that A. oryzae ß-galactosidase was more efficient for lactose hydrolysis during the co-fermentation and that whey permeate supplementation can contribute to ethanol yield in co-fermentations with wheat.

Surface and thermal enhancement of the cellulosic component of thermo mechanical pulp using a rapid method: Iodomethane modification.

The feasibility of employing chemical methods for enhancement of cellulose-based materials is dependent on the availability, price, and green index of the modifying agent. This study details the use of iodomethane, an inexpensive organo halide, to increase the hydrophobicity of thermo mechanical (TMP) samples, which renders them better structural elements for composite materials. For this system, we studied the influence of various concentration of iodomethane, concentration of caustic, and reaction time. Infrared spectroscopy suggested reaction of the organo halide with the hydroxyl groups of cellulose and lignin components of TMP. Pulp samples treated for 4 h or at low caustic concentration showed the least improvements plausibly due to pulp degradation or poor pulp swelling, respectively. On the other hand, pulp treated at 3 h using high concentrations of caustic were characterized with surfaces that were more hydrophobic. Thus, this study outlines a fast and organic solvent-free (clean up) method that can be used to enhance pulp samples for composite applications.