Raw Substrate Research Articles

Weedy lignocellulosic feedstock and microbial metabolic engineering: advancing the generation of ‘Biofuel’

Lignocellulosic materials are the most abundant renewable organic resources (~200billion tons annually) on earth that are readily available for conversion to ethanol and other value-added products, but they have not yet been tapped for the commercial production of fuel ethanol. The lignocellulosic substrates include woody substrates such as hardwood (birch and aspen, etc.) and softwood (spruce and pine, etc.), agro residues (wheat straw, sugarcane bagasse, corn stover, etc.), dedicated energy crops (switch grass, and Miscanthus etc.), weedy materials (Eicchornia crassipes, Lantana camara etc.), and municipal solid waste (food and kitchen waste, etc.). Despite the success achieved in the laboratory, there are limitations to success with lignocellulosic substrates on a commercial scale. The future of lignocellulosics is expected to lie in improvements of plant biomass, metabolic engineering of ethanol, and cellulolytic enzyme-producing microorganisms, fullest exploitation of weed materials, and process integration of the individual steps involved in bioethanol production. Issues related to the chemical composition of various weedy raw substrates for bioethanol formation, including chemical composition-based structural hydrolysis of the substrate, need special attention. This area could be opened up further by exploring genetically modified metabolic engineering routes in weedy materials and in biocatalysts that would make the production of bioethanol more efficient.

Bioconversion of novel substrate Saccharum spontaneum, a weedy material, into ethanol by Pichia stipitis NCIM3498

Rising fuel prices and environmental issues have paved the way for the exploration of cellulosic ethanol. However, challenges involving substrate hydrolysis and cost-effectiveness still limit the efficient bioconversion and utilization of cellulosic ethanol. We aimed to evaluate a cheaper and abundantly available wild sugarcane variety, Saccharum spontaneum, as the raw substrate for bioconversion of ethanol by Pichia stipitis NCIM3498. Three different strategies for substrate hydrolysis using acid (dilute sulfuric acid) and alkali (dilute sodium hydroxide) and aqueous ammonia (AA) treatment followed by enzymatic hydrolysis were studied. A maximum of 631.5±3.25 mg/g sugars with 89.38% hydrolytic efficiency (HE) could be achieved after enzymatic hydrolysis of AA-pretreated S. spontaneum. All the substrate hydrolysates were evaluated for ethanol conversion in batches by P. stipitis. The microbial fermentation of released sugars into ethanol showed (g/g) 0.36±0.011, 0.384±0.022, 0.391±0.02, and 0.40±0.01 yield from detoxified acid hydrolysate and acid-, NaOH- and AA-pretreated substrate S. spontaneum enzymatic hydrolysates, respectively.

Simultaneous saccharification of cassava starch and fermentation of algae for biodiesel production

A simpler approach to produce biodiesel from cassava starch was established, which successfully integrates the simultaneous saccharification and heterotrophic algal fermentation in an identical system. Batch experiments were investigated to verify the feasibility of raw starchy substrates fermentation for microalgal oil. The highest cell density (49.34 g L−1) and oil content (54.60%) were obtained in 5-L fed-batch cultivation via simultaneous saccharification and fermentation (SSF). It is demonstrated that the previous multistep hydrolysis and fermentation for feedstock oil could be replaced by SSF with higher energy efficiency and lower facility costs.

Production of lipid from N‐acetylglucosamine by Cryptococcus curvatus

AbstractN‐Acetylglucosamine (GlcNAc), the monomeric constituent of chitin, is rarely used as a carbon source for fermentation technology. In this study, we demonstrate that the oleaginous yeast Cryptococcus curvatus ATCC 20509 can produce intracellular lipid during the cultivation process and total lipid content can reach 54% on a GlcNAc‐based medium. Culture of C. curvatus under various conditions indicated that lipid accumulation also occurred at a relatively broad range of temperatures as well as relatively high initial GlcNAc concentrations. Fatty acid analysis indicated that the product was rich in palmitic acid, stearic acid, and oleic acid, closely resembling the composition of palm oil. More importantly, the lipid sample produced at 22 °C had a total saturated fatty acid content of 54.2 wt%, suggesting that it may be explored as cocoa‐butter equivalent. Our data suggested that GlcNAc could be used as a feedstock for industrial biotechnology and that C. curvatus ATCC 20509 is a strain capable of accumulating high intracellular lipid using this nitrogen‐rich renewable material.Practical applications: Microbial lipid is a versatile material, especially for biodiesel production. Stable and abundant renewable raw substrates remain to be explored for large‐scale production of microbial lipid. The present work reports lipid production using N‐acetylglucosamine (GlcNAc) by the oleaginous yeast Cryptococcus curvatus ATCC 20509 to yield up to 54% intracellular lipid content. More significantly, the lipid sample produced at 22 °C had a total saturated fatty acid content of 54.2 wt%, suggesting that it may be explored as cocoa‐butter equivalent. Our technology provides the opportunity to effectively convert GlcNAc, available from one of the most abundant renewable materials chitin, into lipid. This procedure should prove valuable in terms of renewable energy production as well as environmental pollution control.

Simultaneous saccharification and fermentation (SSF) of aqueous ammonia pretreated Saccharum spontaneum (wild sugarcane) for second generation ethanol production

Ethanol has drawn attention worldwide to be a true alternative of fossil fuel due to its renewable nature and economics. In the present study, we aimed to evaluate a cheaper and abundantly available wild species of sugarcane, Saccharum spontaneum, as the raw substrate for ethanol production under simultaneous saccharification and fermentation (SSF) conditions. The substrate was first pretreated with aqueous ammonia followed by enzymatic hydrolysis coupled with the fermentation of released sugars into ethanol by monoculture as well as mixed cultures of thermotolerant S. cerevisiae VS3 and P. stipitis NCIM 3498. The cellulase enzyme used for saccharification was prepared from the culture supernatants of Aspergillus oryzae MTCC1846 grown under submerged fermentation conditions. A maximum of 0.85 ± 0.07 IU/mL of filter paperase (FPase), 1.25 ± 0.04 IU/mL of carboxy methyl cellulase (CMCase) and 55.56 ± 0.52 IU/mL of xylanase activity was obtained after 7 days of incubation using delignified S. spontaneum as carbon source under submerged fermentation conditions. During SSF, P. stipitis, VS3 and mixed culture showed ethanol production-15.73±0.44 g/l (productivity, 0.218±0.04 g/l/h), 14.22±0.15 g/l (productivity, 0.197±0.02 g/l/h) and 17.73 ± 0.25 g/l (productivity, 0.246 ± 0.02 g/l/h), respectively.

A Facile and Efficient Route for Coating Polyaniline onto Positively Charged Substrate

By means of the “swelling−diffusion−interfacial polymerization method” (SDIPM), we successfully coated polyaniline (PANi) onto the positively charged polystyrene (PS) particles, which electrostatically repulse each other. After initially forming aniline-swollen PS particles, diffusion of the monomer toward the aqueous phase was controlled through a slow addition of hydrochloric acid, eventually leading to its polymerization on the particle surface. It is an unique, facile, and efficient approach based on raw substrate particles with cationic surface, in comparison with the previous efforts focusing on laborious surface modification or customized design of the substrate particles. The synthesized composite particles have been extensively characterized using scanning electron microscope, transmission electron microscope, Fourier transform infrared, Raman spectroscopy, and thermogravimetry. The resultant PS/PANi core/shell conductive composites possessed a uniform, intact PANi overlayer, and furthermore, the...

Production of methane by co-digestion of cassava pulp with various concentrations of pig manure

Cassava pulp is a major by-product produced in a cassava starch factory, containing 50–60% of starch (dry basis). Therefore, in this study we are considering its potential as a raw material substrate for the production of methane. To ensure sufficient amounts of nutrients for the anaerobic digestion process, the potential of co-digestion of cassava pulp (CP) with pig manure (PM) was further examined. The effect of the co-substrate mixture ratio was carried out in a semi-continuously fed stirred tank reactor (CSTR) operated under mesophilic condition (37 °C) and at a constant OLR of 3.5 kg VS m −3 d −1 and a HRT of 15 days. The results showed that co-digestion resulted in higher methane production and reduction of volatile solids (VS) but lower buffering capacity. Compared to the digestion of PM alone, the specific methane yield increased 41% higher when co-digested with CP in concentrations up to 60% of the incoming VS. This was probably due to an increase in available easily degradable carbohydrates as the CP ratio in feedstock increased. The highest methane yield and VS removal of 306 mL g −1 VS added and 61%, respectively, were achieved with good process stability (VFA:Alkalinity ratio < 0.1) when CP accounted for 60% of the feedstock VS. A further increase of CP of the feedstock led to a decrease in methane yield and solid reductions. This appeared to be caused by an extremely high C:N ratio of the feedstock resulting in a deficiency of ammonium nitrogen for microbial growth and buffering capacity.

Biohydrogen production from poplar leaves pretreated by different methods using anaerobic mixed bacteria

Leaves are one of the main by-products of forestry. In this study, batch experiments were carried out to convert poplar leaves pretreated by different methods into hydrogen using anaerobic mixed bacteria at 35 °C. The effects of acid (HCl), alkaline (NaOH) and enzymatic (Viscozyme L, a mixture of arabanase, cellulase, β-glucanase, hemicellulase and xylanase) pretreatments on the saccharification of poplar leaves were studied. Furthermore, the effects of acid and enzymatic pretreatment on hydrogen production, together with their corresponding degradation efficiencies for the total reducing sugar (TRS) and metabolites were compared. A maximum cumulative hydrogen yield of 44.92 mL/g-dry poplar leaves was achieved from substrate pretreated with 2% Vicozyme L, which was approximately 3-fold greater than that in raw substrate and 1.34-fold greater than that from substrate pretreated with 4% HCl. The results show that enzymatic pretreatment is an effective method for enhancing the hydrogen yield from poplar leaves.

Fermentation of glucose and starch particles using an inexpensive medium

AbstractBACKGROUND: In North America, compared with sugar, starch is a more economical raw material to produce ethanol. Recently low temperature processing of starch granules directly to ethanol has been commercialized. With a view to minimizing process costs while maintaining a satisfactory ethanol yield, an inexpensive medium that does not require the addition of commercial α‐amylase has been developed for low temperature, direct fermentation of starch particles.RESULTS: Compared with an expensive medium that contained both yeast extract and α‐amylase, the inexpensive medium resulted in identical and 10% higher ethanol yields using glucose and starch granules as the raw substrates, respectively, but required longer processing times. Based on the different ingredient prices for expensive and inexpensive media, the operating cost to produce 10 million litres per year of ethanol utilizing simultaneous raw starch hydrolysis and fermentation is reduced over $ 2 million (Cdn) per year when using inexpensive medium.CONCLUSION: The new inexpensive medium and simultaneous raw starch hydrolysis and fermentation is considered the best strategy to produce ethanol directly from starch particles in North America. Copyright © 2009 Society of Chemical Industry

Performance of a pilot scale up-flow septic tank for on-site decentralized treatment of residential wastewater

The aim of this research was to study the on-site anaerobic treatment of a medium-strength residential wastewater in a pilot-scale up-flow septic tank (UST). The effects of three different hydraulic retention times (HRTs) of 24, 12 and 6h on the UST performance were investigated. The UST removed 85, 77, and 86% of biochemical oxygen demand (BOD5), chemical oxygen demand (COD) and total suspended solids (TSS), respectively, at steady state operation and with a 24h HRT. Decreasing the HRT to 12 and then 6h resulted in deteriorated effluent quality and significantly reduced reactor performance. The sludge showed a high specific methanogenic activity (SMA) of 15.2mLCH4g−1VSSd−1 with raw wastewater substrate. The solids accumulated in the tank by the end of the experiment had a VSS/TSS of 0.57, demonstrating significant stabilization. Overall, the UST is concluded to be a technically and economically promising alternative to conventional septic tanks for the on-site decentralized treatment of residential wastewater, particularly in the rural communities of developing countries.

L(+) Lactate production from carbohydrates and lignocellulosic materials by Rhizopus oryzae UMIP 4.77

The lactate excretion by Rhizopus oryzae on various carbohydrates was studied in order to assess the potential of lactate production from raw materials. Six collection strains were tested on ten commercial carbohydrates i.e. glucose, xylose, glycerol, sucrose, lactose, cellobiose, inulin, starch, cellulose and fructose in flask or stirred-tank bioreactor. On glucose and xylose, lactate was produced by R. oryzae UMIP 4.77 at 73 and 8 gL−1 respectively, indicating that lignocellulosic materials could be used as possible raw substrates. Two lignocellulosic substrates were therefore experimented in stirred-tank bioreactor with R. oryzae UMIP 4.77. The first one was hemicellulosic material, which is a concentrated C6 and C5 sugar solution resulting from washing of a steamed wheat straw sample. The second was cellulosic material, which is a partially-hydrolyzed unbleached pulp paper. In a simultaneous saccharification fermentation process, a final production of 24.1 gL−1 of lactate was obtained from cellulosic material.

Dangling Chain Elastomers as Repeatable Fibrillar Adhesives

This work reports on repeatable adhesive materials prepared by controlled grafting of dangling hetero chains from polymer elastomers. The dangling chain elastomer system was prepared by grafting poly(n-butyl acrylate) (PBA) chains from prefunctionalized polydimethylsiloxane (PDMS) elastomer networks using atom transfer radical polymerization. To study the effects of chain growth and network strain as they relate to network adhesion mechanics, various lengths of PBA chains with degree of polymerizations (DP) of 65, 281, 508, and 1200 were incorporated into the PDMS matrix. PBA chains with a DP value of 281 grafted from a flat PDMS substrate showed the highest (approximately 3.5-fold) enhancement of nano- and macroscale adhesion relative to a flat raw (ungrafted and not prefunctionalized) PDMS substrate. Moreover, to study the effect of PBA dangling chains on adhesion in fibrillar elastomer structures inspired by gecko foot hairs, a dip-transfer fabrication method was used to graft PBA chains with a DP value of 296 from the tip endings of mushroom-shaped PDMS micropillars. A PBA chain covered micropillar array showed macroscale adhesion enhancement up to approximately 7 times relative to the flat ungrafted prefunctionalized PDMS control substrate, showing additional nonoptimized approximately 2-fold adhesion enhancement due to fibrillar structuring and mushroom-shaped tip ending. These dangling hetero chains on elastomer micro-/nanofibrillar structures may provide a novel fabrication platform for multilength scale, repeatable, and high-strength fibrillar adhesives inspired by gecko foot hairs.

Enhanced diffuse reflection of light into the air using silver coating on periodically textured 430BA stainless steel substrate

The total reflection (TR) rate and the diffuse reflection (DR) rate of a textured substrate are important indexes when increasing the light trapping efficiency of a solar cell. In this study, we found that the smaller the interval or the larger the diameter of a concave periodically textured 430BA stainless steel (430BA SS) substrate the more the DR rate of light was increased. In addition, a 300 nm thick Ag film was deposited on the surface of the periodically textured 430BA SS substrates by e-beam evaporation. Experimental results showed that the DR rate of Ag film coated/textured 430BA SS substrates increased 2-fold and 14-fold, compared with textured 430BA SS and raw 430BA SS substrates, respectively. Moreover, the TR rate increased to more than 90% for all of the Ag film coated/textured 430BA SS substrates. It is our belief that the increase in the TR and DR rates was due to the high reflectance of the thick and continuous Ag film coating on a textured 430BA SS substrate.

Isolation of raw starch hydrolysing fungi and purification of α-amylase from Geotrichum candidum CMSS06

Three starch degrading fungal species were isolated by exposing raw cassava and potato substrates to air and soil solutions. These three fungi were identified as Aspergillus spp., Mucor spp. and Geotrichum candidum by slide culture method. The optimum culture conditions for the production of extra- cellular a-amylase from these three species were determined by measuring the a-amylase activity, protein content and the pH of the culture media at 12 h intervals. The highest raw starch hydrolysing a-amylase activity (6540 mU/mL) was shown by the species G.candidum 72 h after incubation. The culture supernatant containing the enzyme activity was concentrated by 50% ammonium sulphate precipitation and two peaks were identified to contain the high a-amylase activity following Dowex-cation exchange column chromatography. The two fractions collected from these two activity peaks had the specific activity of 136.3 mU/mL and 394.7 mU/ml for hydrolysing raw starch (RSHI) and soluble starch (SSI) respectively. The purification fold and recovery of the enzyme were 3.5 and 2 in RSHI and 11.2 and 10.9 in SSI. Further the maximum activity of the enzyme in RSHI fraction was detected at 40 o C and pH 7.

Feasibility of nutrient recovery from industrial sludge by vermicomposting technology

Transformation of industrial sludges into vermicompost is of double interest: on the one hand, a waste is converted into value added product, and, on the other, it controls a pollutant that is a consequence of increasing industrialization. This paper reports the feasibility of utilization of vermicomposting technology for nutrient recovery from industrial sludge in laboratory scale experiment employing Eisenia fetida earthworm. A total of nine vermireactors, having different percentage of wastewater treatment plant sludge of a food industry and cow dung, were established and monitored for fertilizer quality of vermicompost and growth and fecundity of the earthworms for 3 months. The earthworms were unable to survive in 100% FIS. There was a decrease in pH, organic carbon content, organic matter, C:N ratio, and increase in ash content, EC, nitrogen, potassium and phosphorus content in all the vermireactors. Total Kjeldhal nitrogen (TKN) content increased in the range of 12.2–28.7 g kg −1 in different vermireactors after vermicomposting. C:N ratio was 1.59–5.24 folds lesser in final vermicomposts than initial raw substrate. The heavy metals’ content in final vermicomposts was higher than initial feed mixtures. Maximum worm biomass was observed in control, i.e., 100% CD (836 mg earthworm −1) and the lowest in 30% CD + 70% FIS feed mixture (280 mg earthworm −1). Cocoon production was started during 6–7th week in all feed mixture except in feed mixture no. 9. After 12 weeks maximum cocoons (57) were counted in 100% CD and minimum (2) in 30% CD + 70% FIS feed. The results indicated that food industry sludge could be converted into good quality manure by vermicomposting if mixed up to 30% with cow dung.

Probiotic potential of noni juice fermented with lactic acid bacteria and bifidobacteria

The present study assesses the feasibility of noni as a raw substrate for the production of probiotic noni juice by lactic acid bacteria (Lactobacilluscasei and Lactobacillus plantarum) and bifidobacteria (Bifidobacteriumlongum). Changes in pH, acidity, sugar content, cell survival and antioxidant properties during fermentation were monitored. All tested strains grew well on noni juice, reaching nearly 109 colony-forming units/ml after 48 h fermentation. L.casei produced less lactic acid than B.longum and L. plantarum. After 4 weeks of cold storage at 4°C, B.longum and L. plantarum survived under low-pH conditions in fermented noni juice. In contrast, L.casei exhibited no cell viability after 3 weeks. Moreover, noni juice fermented with B.longum had a high antioxidant capacity that did not differ significantly (P <0.05) from that of lactic acid bacteria. Finally, we found that B.longum and L. plantarum are optimal probiotics for fermentation with noni juice.

Mechanical matching and microstructural evolution at the coating/substrate interfaces of cold-sprayed Ni, Al coatings

The effect of mechanical hard/soft matching of raw powder and substrate in the cold gas dynamic spraying process (CDSP) on the formation of intermetallic compounds was examined. Instead of pre-alloyed materials, pure Al and Ni were selected as a soft and a hard material, respectively, and post-annealing was used for compound formation. Most of the aluminide layers were observed in the coated layer, but not in the substrate, along with the entire original interface for both Al coating on a Ni substrate and vice versa. Thickening of the compound layer depended mainly on the creation of defects during spraying and intrinsic diffusivity of atoms moving toward the coating side. When Ni was coated, the compound layer was made thicker by fast diffusion of Al, while the thickness was limited in soft Al coating on hard Ni substrate. However, the composition of the compound can be affected by relative transfer of diffusing atoms toward both the coating and the substrate. So, for Ni coating on an Al substrate, most of the intermetallic compound formed was Ni-rich and conversion of the Al-rich compound was observed after post-annealing above 500 °C.

Iron oxide coating of geosynthetic fibers for water treatment applications

Polypropylene fibers present favorable physical, hydraulic and specific surface area properties for use as a raw material or substrate in developing a novel iron-oxide-coated medium for the adsorption of heavy metals from drinking water. However, polypropylene fibers have not been explored for their ability to retain iron oxide coating under realistic flow rates for water treatment. This study evaluated the ability of polypropylene to retain its coating under flow rates used in water treatment, through column studies. A full factorial experimental design was used to evaluate the effects of flow rate, packing scheme (in-plane or cross-plane) and location within the column on iron retention. The polypropylene mat achieved an iron density of 86 mg Fe/g medium of material and retained 73% of this coating after being subjected to hydraulic flows in the cross-plane direction several times those used in water treatment. This is substantially higher than the amount of iron retained on sand as reported by previous researchers. Further investigation should evaluate pre-treatment methods which may facilitate strong chemical bonds between the iron and fiber to create a more consistent coating.

Formation and evolution of phosphorus-containing species during high-temperature oxidation of TiAl dipped in a low-concentrated phosphoric acid solution

TiAl samples were dipped in a low-concentrated phosphoric acid solution, air dried, then heated to 800 °C under argon gas before oxidation under reconstituted air at this temperature. At the end of the heating ramp, sample surfaces were covered by a titanium pyrophosphate (TiP 2O 7) layer, well adhered to the substrate. This pyrophosphate was shown to be detectable from 400 °C. Chemical reactions have been proposed for the formation of this compound. TiP 2O 7 coating strongly increased the oxidation resistance of TiAl at 800 °C for 100 h. After 100 h, a transition period occurred leading to the change of TiP 2O 7 into TiO 2, which was achieved after 230 h, total mass gain always remaining below that of raw substrate. This evolution was demonstrated not resulting from a thermal decomposition of pyrophosphate compound.

Antibiotic resistance and microbial composition along the manufacturing process of Mozzarella di Bufala Campana

The use of antibiotics as growth promoters in livestock, banned in all EU member states in January 2006, has led to selection of antibiotic resistant strains within environmental bacteria, including gram-positive, non pathogenic bacteria that colonize the GI tract of humans and animals. In Italy and in other Mediterranean countries, fermented foods employing environmental bacteria pre-existing in the raw substrates, rather than industrial starters of defined genotype, represent a significant proportion of cheese and meat products carrying the official PDO designation (Protected Designation of Origin). Our study focused on the microbiological and molecular analysis of lactobacilli and of other lactic acid bacteria (LABs) isolated from the Italian PDO product water buffalo Mozzarella cheese, with the aim of identifying genes responsible for tetracycline, erythromycin and kanamycin resistance. We isolated over 500 LAB colonies from retail products, as well as from raw milk and natural whey starters employed in their production. Microbiological analysis showed that about 50% of these isolates were represented by lactobacilli, which were further characterized in terms of species and strain composition, as well as by determining phenotypic and genotypic antibiotic resistance. To overcome the limits of culture-dependent approaches that select only cultivable species, we have also extracted total DNA from the whole microbiome present in the cheese and investigated the presence of specific antibiotic resistance genes with molecular approaches. Genetic determinants of antibiotic resistance were identified almost exclusively in bacteria isolated from the raw, unprocessed substrates, while the final, marketed products did not contain phenotypically resistant lactobacilli, i.e. displaying MIC values above the microbiological breakpoint. Overall, our results suggest that the traditional procedures necessary for manufacturing of this typical cheese, such as high temperature treatments, lead to a final product with low bacterial counts, lower biodiversity and lack of significant presence of antibiotic resistant lactobacilli.