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CSTR anaerobic bioreactors fed UltraFiltered Milk Permeate (UFMP) and Cottage cheese Acid Whey (CAW) were inoculated with acid sludge microbial communities and monitored overtime for lactic acid production. Samples were taken for metagenomics analyses.
Porous biodegradable poly(DL-lactic-co-glycolic acid) foams were seeded with rat marrow stromal cells and implanted into the rat mesentery to investigate in vivo bone formation at an ectopic site. Cells were seeded at a density of 6.83 x 10(5) cells/cm2 onto polymer foams having pore sizes ranging from either 150 to 300 to 710 microns and cultured for 7 days in vitro prior to implantation. The polymer/cell constructs were harvested after 1, 7, 28, or 49 days in vivo and processed for histology and gel permeation chromatography. Visual observation of hematoxylin and eosin-stained sections and von Kossa-stained sections revealed the formation of mineralized bonelike tissue in the constructs within 7 days postimplantation. Ingrowth of vascular tissue was also found adjacent to the islands of bone, supplying the necessary metabolic requirements to the newly formed tissue. Mineralization and bone tissue formation were investigated by histomorphometry. The average penetration depth of mineralized tissue in the construct ranged from 190 +/- 50 microns for foams with 500-710-microns pores to 370 +/- 160 microns for foams with 150-300-microns pores after 49 days in vivo. The mineralized bone volume per surface area and total bone volume per surface area had maximal values of 0.28 +/- 0.21 mm (500-710-microns pore size, day 28) and 0.038 +/- 0.024 mm (150-300-microns, day 28), respectively. As much as 11% of the foam volume penetrated by bone tissue was filled with mineralized tissue. No significant trends over time were observed for any of the measured values (penetration depth, bone volume/surface area, or percent mineralized bone volume). These results suggest the feasibility of bone formation by osteoblast transplantation in an orthotopic site where not only bone formation from transplanted cells but also ingrowth from adjacent bone may occur.
The liquid residue resulting from various agroindustrial processes is both rich in organic material and an attractive source to produce a variety of chemicals. Using microbial communities to produce chemicals from these liquid residues is an active area of research, but it is unclear how to deploy microbial communities to produce specific products from the different agroindustrial residues. To address this, we fed anaerobic bioreactors one of several agroindustrial residues (carbohydrate-rich lignocellulosic fermentation conversion residue, xylose, dairy manure hydrolysate, ultra-filtered milk permeate, and thin stillage from a starch bioethanol plant) and inoculated them with a microbial community from an acid-phase digester operated at the wastewater treatment plant in Madison, WI, United States. The bioreactors were monitored over a period of months and sampled to assess microbial community composition and extracellular fermentation products. We obtained metagenome assembled genomes (MAGs) from the microbial communities in each bioreactor and performed comparative genomic analyses to identify common microorganisms, as well as any community members that were unique to each reactor. Collectively, we obtained a dataset of 217 non-redundant MAGs from these bioreactors. This metagenome assembled genome dataset was used to evaluate whether a specific microbial ecology model in which medium chain fatty acids (MCFAs) are simultaneously produced from intermediate products (e.g., lactic acid) and carbohydrates could be applicable to all fermentation systems, regardless of the feedstock. MAGs were classified using a multiclass classification machine learning algorithm into three groups, organisms fermenting the carbohydrates to intermediate products, organisms utilizing the intermediate products to produce MCFAs, and organisms producing MCFAs directly from carbohydrates. This analysis revealed common biological functions among the microbial communities in different bioreactors, and although different microorganisms were enriched depending on the agroindustrial residue tested, the results supported the conclusion that the microbial ecology model tested was appropriate to explain the MCFA production potential from all agricultural residues.
The composition of algal biomass is highly dynamic, with protein, lipid, and carbohydrate contents varying in response to nutrient and environmental conditions during cultivation. Because shifts in biomass composition are often associated with reduced biomass productivity, production costs can often increase if targeting higher biomass compositional quality (enriched in carbohydrates or lipids at reduced protein content) as input for the algal biorefinery. The optimal algal biorefinery configuration is thus a function of many factors. One of the key strengths of the Combined Algal Processing (CAP) process is the versatility of feedstocks and products produced. The concept has been demonstrated with ethanol and a variety of carboxylic acids (succinic, butyric, muconic) as coproducts along with lipid upgrading to biofuel. Modification of the approaches, processes and downstream upgrading to fuels has allowed the CAP process to reduce costs and improve efficiency. Muconic acid is a high-value, potential fermentation coproduct of interest because it can be easily converted to adipic acid, a high-volume monomer for the production of nylon and other valuable consumer plastics. As such, the production of muconic acid through CAP was explored to expand the suite of products from algal biomass and to begin exploring the valorization of high protein content biomass from rapidly grown algae biomass. We have established initial performance parameters and shown that the range of substrates consumed by the muconic acid-producing microbe, Pseudomonas putida, includes at least glucose, mannose, glycerol, and lactic acid. We achieved complete utilization of these four major hydrolysate substrates achieving productivities of 0.037 (g/L/h) from Scenedesmus obliquus and 0.029 (g/L/h) from Monoraphidium minutum hydrolysates. Final titer and process yield (mol of muconic acid per mol of substrate (molP/molS)) were 0.99 g/L and 0.42 molP/molS from S. obliquus hydrolysate and 0.75 g/L and 0.23 molP/molS from M. minutum hydrolysate, respectively.
The role of lipo-chitooligosaccharides (LCOs) as signaling molecules that mediate the establishment of symbiotic relationships between fungi and plants is being redefined. New evidence suggests that the production of these molecular signals may be more of a common trait in fungi than what was previously thought. LCOs affect different aspects of growth and development in fungi. For the ectomycorrhizal forming fungi, Laccaria bicolor, the production and effects of LCOs have always been studied with a symbiotic plant partner; however, there is still no scientific evidence describing the effects that these molecules have on this organism. Here, we explored the physiological, molecular, and metabolomic changes in L. bicolor when grown in the presence of exogenous sulfated and non-sulfated LCOs, as well as the chitooligomers, chitotetraose (CO 4 ), and chitooctaose (CO8). Physiological data from 21 days post-induction showed reduced fungal growth in response to CO and LCO treatments compared to solvent controls. The underlying molecular changes were interrogated by proteomics, which revealed substantial alterations to biological processes related to growth and development. Moreover, metabolite data showed that LCOs and COs caused a downregulation of organic acids, sugars, and fatty acids. At the same time, exposure to LCOs resulted in the overproduction of lactic acid in L. bicolor. Altogether, these results suggest that these signals might be fungistatic compounds and contribute to current research efforts investigating the emerging impacts of these molecules on fungal growth and development.
A fact-finding team of American scientists and engineers visited Japan to assess the status of research and development and applications in biodegradable polymers. The visit was sponsored by the National Science Foundation and industry. In Japan, the team met with representatives of 31 universities, government ministries and institutes, companies, and associations. Japan's national program on biodegradable polymers and plastics evaluates new technologies, testing methods, and potential markets for biodegradables. The program is coordinated by the Biodegradable Plastics Society of Japan, which seeks to achieve world leadership in biodegradable polymer technology and identify commercial opportunities for exploiting this technology. The team saw no major new technology breakthroughs. Japanese scientists and engineers are focusing on natural polymers from renewable resources, synthetic polymers, and bacterially-produced polymers such as polyhydroxyalkanoates, poly(amino acids), and polysaccharides. The major polymers receiving attention are the Zeneca PHBV copolymers, Biopol(registered trademark), poly(lactic acid) from several sources, polycaprolactone, and the new synthetic polyester, Bionolle(registered trademark), from Showa High Polymer. In their present state of development, these polymers all have major deficiencies that inhibit their acceptance for large-scale applications.
Recently, we proposed a new model of early glycolysis in which the oxidation of glyceraldehyde self-hemiacetals yielded energy rich polyglyceric acid instead of energy rich thioesters. In this model, polyglyceric acid not only acts as an energy source for phosphoanhydride synthesis, but also as an autocatalyst that can replicate the sequence of D and L residues in its structure. We began our investigation of this new hypothesis - the triose model - by developing a thermal method for the racemization-free synthesis of polyglyceric acid. The hydrolytic stability and the role of chirality in interactions of polyglyceric acid were studied using this thermal polymer. Next, we established that the 2- and 3-glycerol esters of polyglyceric acid are energy rich by measuring the Gibbs free energy change of hydrolysis of the 2- and 3-glycerol esters of 2 and 3-O-L glyceroyl-glyceric acid methyl ester - a model of polyglyceric acid. Recently, we discovered that glyceraldehyde is bound and oxidized to glyceric acid on the surface of ferric hydroxide and that soluble ferric ion catalyzes the rearrangement of glyceraldehyde to lactic acid. We are exploring the possibility that these reactions could yield polyglyceric acid and polylactic acid under plausible prebiotic conditions.
Nonmodel microbial hosts can efficiently biotransform unconventional organic feedstocks into advanced bioproducts, leveraging their superior metabolic capabilities and resilience to process-relevant physicochemical conditions. In this study, we domesticated Erwinia spp. strain. LJJL01 (Er LJJL01) as a potent microbial chassis to advance the emerging biorefinery strategy for the valorization of lignin-rich biomass and plastic, thereby enabling the circular economy. The strain exhibits remarkable chemical tolerance and can biofunnel various substrates, including sugars, acids, polyols, and aromatics, in minimal salt media to produce native fine chemicals such as acetoin, 2,3-butanediol, and lactic acid. As a proof of concept, engineering this strain with advanced genetic tools enables the production of tailored high-value chemicals, such as cis,cis-muconate from plastic-derived terephthalate and polyhydroxybutyrate from lignocellulosic hydrolysate. We established Er LJJL01 as a potent microbial chassis for the green synthesis of bioproducts from unconventional organic feedstocks.
A lignin-graft-poly(lactic- co -glycolic) acid (PLGA) biopolymer was synthesized with two types of lignin (LGN), alkaline lignin (ALGN) and sodium lignosulfonate (SLGN), at different (A/S)LGN/PLGA ratios (1:2, 1:4, and 1:6 w/w). 1 H NMR and Fourier-transform infrared spectroscopy (FT-IR) confirmed the conjugation of PLGA to LGN. The (A/S)LGN-graft-PLGA biopolymers were used to form nanodelivery systems suitable for entrapment and delivery of drugs for disease treatment. The LGN-graft-PLGA NPs were generally small (100–200 nm), increased in size with the amount of PLGA added, monodisperse, and negatively charged (-48 to -60 mV). Small-angle scattering data showed that particles feature a relatively smooth surface and a compact spherical structure with a distinct core and a shell. The core size and shell thickness varied with the LGN/PLGA ratio, and at a 1:6 ratio, the particles deviated from the core–shell structure to a complex internal structure. The newly developed (A/S)LGN-graft-PLGA NPs are proposed as a potential delivery system for applications in biopharmaceutical, food, and agricultural sectors.
The origins of the biosynthetic pathways for the branched-chain amino acids cannot be understood in terms of the backwards development of the present acetolactate pathway because it contains unstable intermediates. We propose that the first biosynthesis of the branched-chain amino acids was by the reductive carboxylation of short branched chain fatty acids giving keto acids which were then transaminated. Similar reaction sequences mediated by nonspecific enzymes would produce serine and threomine from the abundant prebiotic compounds glycolic and lactic acids. The aromatic amino acids may also have first been synthesized in this way, e.g. tryptophan from indole acetic acid. The next step would have been the biosynthesis of leucine from alpha-ketoisovalerc acid. The acetolactate pathway developed subsequently. The first version of the Krebs cycle, which was used for amino acid biosynthesis, would have been assembled by making use fo the reductive carboxylation and leucine biosynthesis enzymes, and completed with the development of a single new enzyme, succinate dehydrogenase. This evolutionary scheme suggests that there may be limitations to inferring the origins of metabolism by a simple back extrapolation of current pathways.
Aqueous surfactant free emulsion grafting-through polymerization produced poly(methyl methacrylate) coated cellulose nanofibrils that retained microstructure and strengthened poly(lactic acid) composites.
Bioproduction of medium chain carboxylic acids has recently emerged as an alternative strategy to valorize low-value organic waste and side-streams. Key to this route is chain elongation, an anaerobic microbial process driven by ethanol, lactic acid, or carbohydrates. Additionally, because these technologies use wastes as feedstocks, mixed microbial communities are often considered as biocatalysts. Understanding and steering these microbiomes is key to optimize bioprocess performance. From a meta-analysis of publicly available sequencing data, we (i) explore how the current collection of isolated chain elongators compares to microbiome members, (ii) discuss the main beneficial and antagonistic interactions with community partners, and (iii) identify the key research gaps and needs to help understand chain elongation microbiomes, and design/steer these novel bioproduction processes.
Dairy manure (DM) is an abundant agricultural residue that is largely composed of lignocellulosic biomass. The aim of this study was to investigate if carbon derived from DM fibers can be recovered as medium-chain fatty acids (MCFAs), which are mixed culture fermentation products of economic interest. DM fibers were subjected to combinations of physical, enzymatic, chemical, and thermochemical pretreatments to evaluate the possibility of producing carbohydrate-rich hydrolysates suitable for microbial fermentation by mixed cultures. Among the pretreatments tested, decrystalization dilute acid pretreatment (DCDA) produced the highest concentrations of glucose and xylose, and was selected for further experiments. Bioreactors fed DCDA hydrolysate were operated. Acetic acid and butyric acid comprised the majority of end products during operation of the bioreactors. MCFAs were transiently produced at a maximum concentration of 0.17 mg COD MCFAs /mg COD Total . Analyses of the microbial communities in the bioreactors suggest that lactic acid bacteria, Megasphaera , and Caproiciproducens were involved in MCFA and C4 production during DCDA hydrolysate metabolism.
The formation of alanine (ala) form C(14)-glyceraldehyde and ammonium phosphate in the presence or absence of a thiol is reported. At ambient temperature, ala synthesis was six times more rapid in the presence of 3-mercaptopropionic acid than in its absence (0.6 and 0.1 percent, respectively, after 60 days). Similarly, the presence of another thiol, N-acetylcysteinate, increased the production of ala, as well as of lactate. The reaction pathway of thiol-catalyzed synthesis of ala, with the lactic acid formed in a bypath, is suggested. In this, dehydration of glyceraldehyde is followed by the formation of hemithioacetal. In the presence of ammonia, an imine is formed, which eventually yields ala. This pathway is consistent with the observation that the rate ratio of ala/lactate remains constant throughout the process. The fact that the reaction takes place under anaerobic conditions in the presence of H2O and with the low concentrations of simple substrates and catalysts makes it an attractive model prebiotic reaction in the process of molecular evolution.
Arrested anaerobic digestion (AAD) offers a promising route for producing fatty acids (FAs) from organic residues, yet optimal conditions for selectively generating medium-chain fatty acids (MCFAs) remain poorly defined. Here, we systematically evaluated the main and interaction effects of pH (5, 7, 9), feedstock (food waste, manure), temperature (35 and 45 °C), and inoculum source on microbiome composition and FA production. Anaerobic digester sludge and a novel bison rumen inoculum were compared. Significant higher-order interactions among operating parameters governed FA profiles and microbiome structure. Butyric acid production was driven by a three-way interaction among pH, feedstock, and temperature (p < 0.001), with maximum concentrations achieved in food waste reactors at pH 5.0 and 35 °C (1.2 ± 0.1 g L −1 with sludge and 1.1 ± 0.3 g L −1 with rumen). MCFA production exhibited significant four-way interactions (p < 0.1 to p < 0.001). At 45 °C and pH 5.0, inoculum source tuned MCFA selectivity: sludge favored pentanoic acid (0.4 ± 0.1 g L −1 ), whereas rumen favored hexanoic and heptanoic acids (up to 0.4 ± 0.2 g L −1 ). Manure reactors produced < 0.2 g L −1 MCFAs under all conditions. Genera, including Megasphaera, Prevotella, and Lactobacillus, were associated with production of specific MCFAs. PICRUSt2-based pathway predictions were consistent with MCFA production patterns and suggested a potential role for lactic acid–driven chain elongation pathways. This study provides insights into how interacting operating conditions shape AAD microbiomes, their FA profiles, and advances the trajectory of research aimed at engineering robust and controllable microbiomes for waste valorization.
Three-dimensional (3D) printing is a rapidly growing technology that best fits for cell culture and other biological applications due to its potential to build immensely complex structures from customized designs. However, use of 3D printed structures for cell adhesion, proliferation, and activation requires tailoring of surface characteristics. In this context, this work investigates the use of two biocompatible 3D printable polymer materials, Poly-L-lactic Acid (PLA) and Acrylonitrile Butadiene Styrene (ABS) for bio applications, and the effect of their post-printing surface modification processes to achieve desired bio functionality. Three post-printing surface modification techniques, alkaline hydrolysis, ultraviolet ozone plasma irradiation and gold thin film deposition are performed with the objective of introducing useful levels of surface functionalities. Poly-L-lysine (PLL) labelled with FITC chromophore is immobilized on the surface modified samples following standard protocols. The effect of surface roughness and porosity of the 3D printed structures on the polypeptide immobilization is compared on as-printed versus mechanically polished surfaces. Different characterization methods, viz., Fluorescence microscopy, Raman Spectroscopy and UV–vis spectroscopy, are used to study the efficacy of the surface modification and PLL immobilization techniques. Our results demonstrated denser PLL attachment on polished hydrolyzed PLA and ABS surfaces which enables the use of these printable polymers for a range of applications such as tissue scaffolds and microfluidics.
The role of auxin in plant–microbe interaction has primarily been studied using indole-3-acetic acid (IAA)-producing pathogenic or plant-growth-promoting bacteria. However, the IAA biosynthesis pathway in bacteria involves indole-related compounds (IRCs) and intermediates with less known functions. Here, we seek to understand changes in plant response to multiple plant-associated bacteria taxa and strains that differ in their ability to produce IRCs. We had previously studied 47 bacterial strains isolated from several duckweed species and determined that 79% of these strains produced IRCs in culture, such as IAA, indole lactic acid (ILA), and indole. Using Arabidopsis thaliana as our model plant with excellent genetic tools, we performed binary association assays on a subset of these strains to evaluate morphological responses in the plant host and the mode of bacterial colonization. Of the 21 tested strains, only four high-quantity IAA-producing Microbacterium strains caused an auxin root phenotype. Compared to the commonly used colorimetric Salkowski assay, auxin concentration determined by LC–MS was a superior indicator of a bacteria’s ability to cause an auxin root phenotype. Studies with the auxin response mutant axr1-3 provided further genetic support for the role of auxin signaling in mediating the root morphology response to IAA-producing bacteria strains. Interestingly, our microscopy results also revealed new evidence for the role of the conserved AXR1 gene in endophytic colonization of IAA-producing Azospirillum baldaniorum Sp245 via the guard cells.
Combined substrate-product toxicity remains one of the main obstacles hampering the scale-up and cost-effectiveness of bioprocesses harnessing lignocellulosic feedstocks, the most abundant, renewable terrestrial resource. Hydrolytic pretreatments release numerous inhibitors impinging on cell viability: the three most acute to yeast S. cerevisiae (the industry dominant biocatalyst) — and universal to all plant sources — are furfural, hydroxymethylfurfural (HMF), and acetic acid. Likewise, desired fermentation products, such as fuel ethanol or commodity organic acids, are toxic to microbes, typically via unknown biological mechanisms. Here, we have engineered both hydrolysate and end-product tolerance in yeast by combining previously-shown alcohol protective modifications with evolved genetic activities targeting the major pretreatment inhibitors. When tested on a wide sampling of genuine lignocellulosic feedstocks, ethanol production increased by >30% on average to titers >100 g/L, achieving parity with clean-sugar equivalents where conditions permit. Furthermore, we designed the tolerance capability to be fully transferable to pre-existing metabolic chassis strains. As such, we “drop-in” hydrolysate competence into one producing lactic acid, demonstrating the first-ever production of a cellulosic plastic at industrial titers. Our advances thus renew the potential of cellulosic biomass utilization for sustainable fuel and non-fuel products at scale.