Value-added Bioproducts Research Articles

Oxygen-responsive genetic circuits constructed in Synechocystis sp. PCC 6803.

As photoautotrophic prokaryotes, cyanobacteria are promising platforms for producing value-added bioproducts. However, few regulatory genetic parts and devices (e.g., inducible promoters and regulatory circuits) have been developed for these potential hosts. Furthermore, the devices that have been created respond only to a single input. To address these issues, we developed an inducible genetic circuit that generates heterologous proteins in response to oxygen, an environmental signal. To test its performance and utility in Synechocystis sp. PCC 6803, a model cyanobacterial strain, we connected this circuit to either heterologous nifHDK genes, which encode oxygen-sensitive nitrogenase's structural proteins, or a fluorescent protein gene. The circuit was transcriptionally activated to generate nifHDK transcripts or fluorescent output only in low oxygen conditions. We expanded the oxygen-responsive circuit into a more complex circuit by building a two-input AND gate, which allows Synechocystis to specifically control expression of the fluorescent reporter in response to two signals, low oxygen and high anhydrotetracycline. To our knowledge, the AND gate is the first complex logic circuit built in a cyanobacterial strain. This work expands the synthetic biology tools available for complex gene expression in cyanobacteria, increasing their potential as biotechnology platforms.

Influence of Light Intensity on Growth and Pigment Production by Monascus ruber in Submerged Fermentation.

To reduce environmental problems caused by glycerine accumulation and to make the production of biodiesel more profitable, crude glycerin without treatment was used as substrate for obtaining higher value-added bioproducts. Monascus ruber is a filamentous fungus that produces pigments, particularly red ones, which are used for coloring foods (rice wine and meat products). The interest in developing pigments from natural sources is increasing due to the restriction of using synthetic dyes. The effects of temperature, pH, microorganism morphology, aeration, nitrogen source, and substrates have been studied in the cultivation of M. ruber. In this work, it was observed that light intensity is also an important factor that should be considered for understanding the metabolism of the fungus. In M. ruber cultivation, inhibition of growth and pigment production was observed in Petri dishes and blaffed flasks exposed to direct illumination. Growth and pigment production were higher in Petri dishes and flasks exposed to red light and in the absence of light. Radial growth rate of M. ruber in plates in darkness was 1.50 mm day(-1) and in plates exposed to direct illumination was 0.59 mm day(-1). Maximum production of red pigments (8.32 UA) and biomass (8.82 g L(-1)) were obtained in baffled flasks covered with red film and 7.17 UA of red pigments, and 7.40 g L(-1) of biomass was obtained in flasks incubated in darkness. Under conditions of 1248 lux of luminance, the maximum pigment production was 4.48 UA, with production of 6.94 g L(-1) of biomass, indicating that the fungus has photoreceptors which influence the physiological responses.

Dehydrated olive-waste cake as a source of high value-added bioproduct: Drying kinetics, physicochemical properties, and bioactive compounds

Olive (Olea europaea L.) oil processing produces significant amount of waste that can be utilized for the production of high value-added ingredients for various industrial applications. In this work, the effects of temperature on drying kinetics and quality indexes of the olive-waste cake during convective dehydration (40-90 °C) were investigated. Results on effective moisture diffusivity, physicochemical parameters, fatty acid profile, total phenolic, flavonoid, and flavanol contents as well as antioxidant capacity are also reported. Most of the fatty acids increased their content with respect to control sample with a temperature increase, i.e. oleic and linoleic acids increased 48% and 43% at 70 and 40 °C, respectively. Total flavanol content increased with temperature (48-62 mg catechin equivalents [CTE] 100 g-1 DM) except for 80 °C. Total phenolic and total flavonoid contents were highly correlated to antioxidant capacity (0.923 < r < 0.992), except for 70 and 80 °C, the rest of the samples maintained their initial antioxidant capacity by ORAC analysis. Thus, these parameters show that dried olive-waste cake has a high bioactive compounds with potential use as additives for the food or other industries.

Metabolic engineering of Escherichia coli for efficient free fatty acid production from glycerol

Crude glycerol, generated as waste by-product in biodiesel production process, has been considered as an important carbon source for converting to value-added bioproducts recently. Free fatty acids (FFAs) can be used as precursors for the production of biofuels or biochemicals. Microbial biosynthesis of FFAs can be achieved by introducing an acyl–acyl carrier protein thioesterase into Escherichia coli. In this study, the effect of metabolic manipulation of FFAs synthesis cycle, host genetic background and cofactor engineering on FFAs production using glycerol as feed stocks was investigated. The highest concentration of FFAs produced by the engineered stain reached 4.82g/L with the yield of 29.55% (g FFAs/g glycerol), about 83% of the maximum theoretical pathway value by the type II fatty acid synthesis pathway. In addition, crude glycerol from biodiesel plant was also used as feedstock in this study. The FFA production was 3.53g/L with a yield of 24.13%. The yield dropped slightly when crude glycerol was used as a carbon source instead of pure glycerol, while it still can reach about 68% of the maximum theoretical pathway yield.

Particle size and hydration medium effects on hydration properties and sugar release of wheat straw fibers

Wheat straw is gaining importance as a feedstock for the production of biofuels and high value-added bioproducts. Several pretreatments recover the fermentable fraction involving the use of water or aqueous solutions. Therefore, hydration properties of wheat straw fibers play an important role in improving pretreatment performance. In this study, the water retention capacity (WRC) and swelling of wheat straw fibers were studied using water, propylene glycol (PPG) and an effluent from a H2-producing reactor as the hydration media with three particle sizes (3.35, 2.00 and 0.212 mm). The effects of swelling were analyzed by optical and confocal laser scanning microscopy (CLSM). The highest WRC was reached with the effluent medium (9.84 ± 0.87 g g−1 in 4 h), followed by PPG (8.52 ± 0.18 g g−1 in 1 h) and water (8.74 ± 0.76 g g−1 in 10 h). The effluent hydration treatment had a synergic effect between the enzymes present and the water. The particle size had a significant effect on the WRC (P < 0.01), the highest values were reached with 3.35 mm fibers. The CLSM images showed that finer fibers were subjected to a shaving effect due to the grinding affecting its capacity to absorb the hydration medium. The microscopic analysis showed the increase in the width of the epidermal cells after the hydration and a more undulating cell wall likely due to the hydration of the amorphous regions in the cellulose microfibrils. The sugar release was determined, achieving the highest glucose content with the effluent hydration treatment.

Isolation and characterization of Bacillus subtilis strain BY-3, a thermophilic and efficient cellulase-producing bacterium on untreated plant biomass.

Bioconversion of biomass, particularly crop wastes, into biofuels is being developed as an alternative approach in meeting the high energy demand. In this study, a thermophilic bacterial strain BY-3 that exhibits cellulolytic potential was isolated from faecal samples of Tibetan pigs; this strain was identified as Bacillus subtilis. The strain can produce cellulase when grown on various substrates, including carboxymethyl cellulose, rice straw, corn stover, soluble starch and wheat bran. The maximum cellulase activity of the strain was up to 4·323±0·065Uml(-1) when cultivated in the medium containing corn stover (30gl(-1) ) for 24h. The results demonstrated that corn stover is the most suitable substrate for cellulase production by the strain BY-3. The crude cellulase of strain BY-3 was most active at pH5·5 and 60°C, and the enzyme in acetate buffer (50mmoll(-1) ) demonstrated a good stability at 60°C for at least 1h. The crude cellulase exhibited a strong antibacterial activity against Staphylococcus aureus. The strain can be used in cost-efficient cellulase production for bioconversion of agricultural residual biomass into biofuels. The increased consumption of fossil fuels has caused serious energy crisis and environmental problem. Thus, an alternative energy source is necessary. Bioconversion of biomass, particularly agricultural residuals, into value-added bioproducts, such as biofuels and chemical solvents, has received considerable attention. In this study, the newly isolated thermophilic Bacillus subtilis strain BY-3 produces cellulase efficiently with the use of untreated corn stover as a sole carbon source. This strain possesses the thermostable cellulase that is active with diverse crop wastes with a broad pH range and is a highly promising candidate for agricultural waste management.

Cost Analysis and Life-Cycle Environmental Impacts of Three Value-Added Novel Bioproducts: Processing and Production

A life-cycle assessment framework was used to evaluate the environmental impacts associated with processing, producing, and marketing three novel value-added bioproducts, intended as substitutes for peat growing media and soil amendments, namely: dried composted forestry bark (DCFB); dehydrated topdressing (DTD); and organic bio-fertilizer (OBF). Total cost of processing and producing the final bioproducts ($ t−1) was highest for OBF and lowest for DCFB. Environmental and ecological impacts were quantified and compared using indicators commonly used in the scholarly literature to capture global warming, and human and eco-toxicity impacts. Global warming impacts (measured in terms of total CO2-equivalent of dehydrated final bioproduct) were highest for OBF, followed by DTD, and then DCFB. Among the human and eco-toxicity indicators considered, NO x emissions were highest, followed by SO2, and lowest for CO emissions, for all three bioproducts.

Bacterial consortia constructed for the decomposition of Agave biomass

Research has shown that a greater variety of enzymes, as well as variety of microorganisms producing enzymes, can have an overall synergistic effect on the decomposition of lignocellulosic biomass for the production of value-added bio-products. Here, 8 cellulase-degrading bacterial isolates were selected to develop co-, tri-, and tetra-cultures for the decomposition of lignocellulosic biomass. Glucose and xylose equivalents released from imitation biomass media containing 0.5% (w/v) beechwood xylan and 0.5% (w/v) Avicel was measured using di-nitrosalicylic acid for all consortia, along with cell growth and survival. Thereafter, 6 co- and 2 tri-cultures with greatest decomposition were examined for ability to degrade Agave americana fiber. Interestingly, when strains were paired up in co-culture, four pairs: G+5, G+A, C+A1, and G+A1 produced high reducing sugars in 24 h: 6 µM, 8 µM, 8 µM, and finally, 6 µM, respectively. From 4 co-cultures with highest reducing sugar equivalents, tri- and tetra-cultures were produced. The bacterial consortia which had the highest reducing sugars detected were 2 tri-cultures: G + A1 + A4 and G + A1 + 5, displaying levels as high as 9 µM and 5 µM in day 1, respectively. All co- and tri-cultures maintained high cell survival for 14 days with 0.5 g ground Agave. Upon evaluating Agave dry weight after treatment, it was evident that almost half the biomass could be decomposed in 14 days. Scanning electron microscopy of treated Agave supported decomposition when compared with the control. These bacterial consortia have potential for further study of value-added by-product production during metabolism of lignocellulosic biomasses.

Enhancing the Halal Food Industry by Utilizing Food Wastes to Produce Value-added Bioproducts

Each year, millions of tons of halal food wastes are being disposed into the environment through land-filling or illegal dumping activities. Currently, the government has no other cheaper and easier options than landfill. Dumping of organic waste materials into the environment will partly contribute to the global warming phenomenon due to methane gas generation through anaerobic process, occurred inside the landfill or river bed. Methane gas has 21 times higher global warming potential (GWP) than carbon dioxide and can severely affect the environment if not properly managed. Therefore this paper will highlight the concept of biorefinery to enhance the current state of halal food waste handling which include kitchen waste, waste cooking oil, food waste, landscaping and garden waste, wet market waste, night market waste, halal slaughterhouse waste, food processing facility waste and other types of halal waste. Through biorefinery complex these waste materials can be converted into value-added bioproducts such as biodiesel, biogas, bioethanol, animal feed, biofertilizer, bioplastic, biomaterial and others. This will enhance the food halal industry and also support the government aspiration towards achieving high income country.

Performance of an enzymatic extract in Botrycoccus braunii cell wall disruption

Microalgae can produce and contain lipids, proteins and carbohydrates, which can be extracted and marketed as potential novel added-value bio-products. However, microalgae cell wall disruption is one of the most important challenges involved while processing this type of biomass. In this context, white-rot fungi, responsible for the biodegradation of lignin present in wood due to non-specific extracellular enzymes, could be applied for promoting microalgae cell wall degradation. Therefore, the aim of this study was to evaluate the use of an enzymatic extract produced by the white-rot fungi Anthracophyllum discolor as a biotechnological tool for Botryococcus braunii cell wall disruption. The fungus was inoculated in wheat grains and manganese peroxidase (MnP) activity was monitored while obtaining the enzymatic extract. Then, cell wall disruption trials with different MnP activity were evaluated by the biochemical methane potential (BMP). In relation to cell wall disruption, it was observed that the optimal value was obtained with enzymatic concentration of 1000 U/L with a BMP of 521 mL CH4/g VS. Under these conditions almost 90% of biomass biodegradability was observed, increasing in 62% compared to the microalgae without treatment. Therefore, the results indicate that enzymes secreted by A. discolor promoted the attack of the different cell wall components finally weakening it. Therefore, the application of this treatment could be a promissory biotechnological approach to decrease the energetic input required for the cell wall disruption step.

Development of an electrotransformation protocol for genetic manipulation of Clostridium pasteurianum.

BackgroundReducing the production cost of, and increasing revenues from, industrial biofuels will greatly facilitate their proliferation and co-integration with fossil fuels. The cost of feedstock is the largest cost in most fermentation bioprocesses and therefore represents an important target for cost reduction. Meanwhile, the biorefinery concept advocates revenue growth through complete utilization of by-products generated during biofuel production. Taken together, the production of biofuels from low-cost crude glycerol, available in oversupply as a by-product of bioethanol production, in the form of thin stillage, and biodiesel production, embodies a remarkable opportunity to advance affordable biofuel development. However, few bacterial species possess the natural capacity to convert glycerol as a sole source of carbon and energy into value-added bioproducts. Of particular interest is the anaerobe Clostridium pasteurianum, the only microorganism known to convert glycerol alone directly into butanol, which currently holds immense promise as a high-energy biofuel and bulk chemical. Unfortunately, genetic and metabolic engineering of C. pasteurianum has been fundamentally impeded due to lack of an efficient method for deoxyribonucleic acid (DNA) transfer.ResultsThis work reports the development of an electrotransformation protocol permitting high-level DNA transfer to C. pasteurianum ATCC 6013 together with accompanying selection markers and vector components. The CpaAI restriction-modification system was found to be a major barrier to DNA delivery into C. pasteurianum which we overcame by in vivo methylation of the recognition site (5’-CGCG-3’) using the M.FnuDII methyltransferase. With proper selection of the replication origin and antibiotic-resistance marker, we initially electroporated methylated DNA into C. pasteurianum at a low efficiency of 2.4 × 101 transformants μg-1 DNA by utilizing conditions common to other clostridial electroporations. Systematic investigation of various parameters involved in the cell growth, washing and pulse delivery, and outgrowth phases of the electrotransformation procedure significantly elevated the electrotransformation efficiency, up to 7.5 × 104 transformants μg-1 DNA, an increase of approximately three order of magnitude. Key factors affecting the electrotransformation efficiency include cell-wall-weakening using glycine, ethanol-mediated membrane solubilization, field strength of the electric pulse, and sucrose osmoprotection.ConclusionsC. pasteurianum ATCC 6013 can be electrotransformed at a high efficiency using appropriately methylated plasmid DNA. The electrotransformation method and tools reported here should promote extensive genetic manipulation and metabolic engineering of this biotechnologically important bacterium.

A full-scale house fly (Diptera: Muscidae) larvae bioconversion system for value-added swine manure reduction

Manure produced from confined animal farms can threaten public and environmental health if not managed properly. Herein, a full-scale commercial bioconversion operation in DeQing County, China for value-added swine manure reduction using house fly, Musca domestica L., larvae is reported. The greenhouse-assisted larvae bioreactor had a maximum daily treatment capacity of 35 m(3) fresh raw manure per day. The bioconversion process produced a fresh larvae yield of 95-120 kg m(3) fresh raw manure. This process provided an alternative animal foodstuff (having 56.9 and 23.8% protein and total fat as dry matter, respectively), as well as captured nutrients for agricultural re-utilization. Bioconversion reduced odour emission (characterized by 3-methylindole) and the Escherichia coli (E. coli) index by 94.5 and 92.0%, respectively, and reductions in total weight, moisture and total Kjeldahl nitrogen in solids were over 67.2, 80.0 and 76.0%, respectively. Yearly profit under this trial period ranged from US$33.4-46.1 per m(3). It is concluded that swine manure larvae bioconversion technology with subsequent production of value-added bio-products can be a promising avenue when considering a programme to reduce waste products in an intensive animal production system.

Cellulase Production by Bacteria: A Review

Cellulose is an abundant natural biopolymer on earth and most dominating Agricultural waste. This cellulosic biomass is a renewable and abundant resource with great potential for bioconversion to value-added bioproducts. It can be degraded by cellulase produced by cellulolytic bacteria. This enzyme has various industrial applications and now considered as major group of industrial enzyme. The review discusses application of cellulase, classification of cellulase, quantification of cellulase, the types of cellulolytic bacteria and their screening. It describes the current knowledge of cellulase production by submerged fermentation and solid state fermentation, properties of cellulase and cloning and expression of cellulase gene. The biotechnological aspect of cellulase research and their future prospects are also discussed.

Utilization of agricultural biomass in the production of the biopolymer schizophyllan

Schizophyllan is a homoglucan produced by the fungus Schizophyllum commune, with a β-1,3-linked backbone and β-1,6-linked side chains of single glucose units at every other residue. Schizophyllan is commercially produced for pharmaceutical and cosmetics uses. However, the unique physical properties of schizophyllan suggest that it may have biomaterials applications. Schizophyllan is conventionally produced by submerged culture fermentation using glucose as a carbon source. This study demonstrates for the first time the efficient utilization of agricultural biomass substrates, particularly distiller's dried grains with solubles, for schizophyllan production. Sugar composition analysis, NMR, and permethylation linkage analysis confirmed that the recovered product was schizophyllan. Schizophyllan produced from agricultural residues was of a high molecular weight and exhibited solution viscosity properties similar to those of commercially produced material. Utilization of biomass substrates could reduce the cost of schizophyllan production and provide a new value-added bioproduct for integrated biorefineries of the future.

Intracellular polyhydroxyalkanoates recovery by cleaner halogen-free methods towards zero emission in the palm oil mill

Abstract In this study, the effect of extraction process, bacterial strain and cell PHA content on halogen-free recovery methods using sodium hydroxide (NaOH) and water as cleaner and more environmental friendly processes were examined for polyhydroxyalkanoates (PHA) recovery. The bacterial strains used were local isolate wild type Comamonas sp. EB172 and recombinant Cupriavidus necator ( C. necator ), with 30% and 50% of PHA content. It was found that the PHA was efficiently recovered from bacterial cells by the non-halogenated methods. Although the PHA purity obtained by water recovery method was lower than alkaline treatment, it does not have any adverse effect on the polymer molecular weight. The performance of the halogen-free methods depends on the microbial strain which is due to the difference in the thickness of the cell membranes. The intracellular PHA content also affected the effectiveness of the extraction methods for PHA recovery as higher amount of polymer in the cell increased the quantity of PHA recovered. The halogen-free methods using NaOH and water can serve as promising alternatives for conventional industrial halogenated solvent extraction process, since these new methods are environmentally more benign, effective and simple in operation. Finally, the potential of integrating PHA production and halogen free recovery system with the palm oil industry is discussed. It is expected that the proposed integrated system herein provides a more attractive proposition to utilize biomass as renewable resource for the production of value-added bioproducts like PHA, which will make the palm oil industry more sustainable and profitable, and promotes zero-emission at the mill.

Biochar as a Renewable Matrix for the Development of Encapsulated and Immobilized Novel Added-Value Bioproducts

The use of biochar has traditionally been focused on agronomic applications. Today it is possible to find a wide range of research devoted to study the use of biochar in the most varied fields. This is mainly due to its properties and the diversity of materials that can be used in their synthesis. Also, the conditions of operation in the process of synthesis (pyrolysis) can be easily modified to obtain a product with the desired features for a given application. Consequently, this review examines the biochar properties that are relevant to its applicability in three areas of high industrial interest; control release and immobilization, where it could serve as alternative, cheap and renewable support material to existing conventional types.

Extraction, Characterization of Components, and Potential Thermoplastic Applications of Camelina Meal Grafted with Vinyl Monomers

Camelina meal contains oil, proteins, and carbohydrates that can be used to develop value-added bioproducts. In addition to containing valuable polymers, coproducts generated during the production of biofuels are inexpensive and renewable. Camelina is a preferred oilseed crop for biodiesel production because camelina is easier to grow and provides better yields. In this research, the components in camelina meal were extracted and studied for their composition, structure, and properties. The potential of using the camelina meal to develop thermoplastics was also studied by grafting various vinyl monomers. Oil (19%) extracted from camelina meal could be useful for food and fuel applications, and proteins and cellulose in camelina meal could be useful in the development of films, fibers, and thermoplastics. Thermoplastic films developed from grafted camelina meal had excellent wet tensile properties, unlike thermoplastics developed from other biopolymers. Camelina meal grafted with butylmethacrylate (BMA) had high dry and wet tensile strengths of 53.7 and 17.3 MPa, respectively.

Dynamic flux balance modeling of microbial co‐cultures for efficient batch fermentation of glucose and xylose mixtures

Sequential uptake of pentose and hexose sugars that compose lignocellulosic biomass limits the ability of pure microbial cultures to efficiently produce value-added bioproducts. In this work, we used dynamic flux balance modeling to examine the capability of mixed cultures of substrate-selective microbes to improve the utilization of glucose/xylose mixtures and to convert these mixed substrates into products. Co-culture simulations of Escherichia coli strains ALS1008 and ZSC113, engineered for glucose and xylose only uptake respectively, indicated that improvements in batch substrate consumption observed in previous experimental studies resulted primarily from an increase in ZSC113 xylose uptake relative to wild-type E. coli. The E. coli strain ZSC113 engineered for the elimination of glucose uptake was computationally co-cultured with wild-type Saccharomyces cerevisiae, which can only metabolize glucose, to determine if the co-culture was capable of enhanced ethanol production compared to pure cultures of wild-type E. coli and the S. cerevisiae strain RWB218 engineered for combined glucose and xylose uptake. Under the simplifying assumption that both microbes grow optimally under common environmental conditions, optimization of the strain inoculum and the aerobic to anaerobic switching time produced an almost twofold increase in ethanol productivity over the pure cultures. To examine the effect of reduced strain growth rates at non-optimal pH and temperature values, a break even analysis was performed to determine possible reductions in individual strain substrate uptake rates that resulted in the same predicted ethanol productivity as the best pure culture.

Pretreatment of guayule biomass using supercritical carbon dioxide-based method

Guayule, a desert shrub harvested for commercial production of hypoallergenic latex and resins constitutes <20% of the biomass. Converting the remaining bagasse to biorefinery feedstock for value-added products is an optimal economic option. A supercritical CO(2)-based process had been developed previously for resin extraction. In this study, the feasibility of including a supercritical CO(2)-based bagasse pretreatment method was evaluated. The pretreatment involved: adding water to the bagasse, raising system temperature, pressurizing using supercritical CO(2), holding the system for a period of time, and exploding the bagasse. The pretreated biomass was subjected to enzyme hydrolysis. The yields of released sugars were used as pretreatment effectiveness indicators. Supercritical method outperformed other methods and gave much higher overall sugar yields for guayule (as high as 77% for glucose and 86% for total reducing sugars through both pretreatment and hydrolysis, as compared to 50% for glucose and 52% for total sugars with the dilute-acid pretreatment and 36% for glucose and 52% for total sugars with the delignification pretreatment). The enzymatic hydrolyzates were tested on the cellulase-producing fungus Trichoderma reesei Rut C-30. No inhibitory/toxic effects were apparent in terms of cell growth, sugar consumption, and cellulase and xylanase production. The supercritical CO(2)-based method was found to be very promising for pretreatment of waste biomass as the feedstock for subsequent enzymatic hydrolysis and fermentation to produce value-added bioproducts.

Increased selectivity in the formation of the phenoxy ether of methyl lesquerolate over chloroalkyl-modified SBA-15-SO 3H catalysts

A novel synthetic catalysis was developed for selective modification of useful bioproducts from the oil of Lesquerella fendlerii, an agricultural commodity of increasing importance. Modification of the surface of the solid acid catalyst SBA-15-SO 3H with the chloroalkyl groups CH 2CH 2Cl, has improved the selectivity in the synthesis of the phenoxy ether of methyl 14-hydroxy-11-eicosenoate (methyl lesquerolate). A series of catalysts, SBA-15(EtCl) x SO 3H where x = 0, 2, 4, 8, have been prepared and characterized by 13C CPNMR, surface properties, and elemental analyses. Selectivity increased with increased loading of the chloroalkyl group. This selectivity can be attributed to the decrease in the rate of conversion of the phenoxy ether to the Friedel–Crafts adducts. This study demonstrates synthetic approaches can develop novel catalysts to increase yields and selectivity for value-added bioproducts from new crops.