Effects of carrier, sterilisation method, and incubation on survival of Bradyrhizobium japonicum in soybean (Glycine max L.) inoculants

The production and quality of rhizobial inoculants in many developing countries is limited by the availability of suitable carriers or technological limitations. Experiments were conducted to evaluate the potential of various inexpensive and widely available carrier materials. The carriers, evaluated, were: perlite with pH adjusted with calcium carbonate or charcoal, 1:4 mixtures of perlite and malt residue, sugarcane bagasse, coal, and rice husk. We also contrasted sterilisation procedures (autoclaving or gamma irradiation) and incubation after injection (with or without initial two weeks incubation at 28 °C) for these various carriers. Survival of Bradyrhizobium japonicum strain CB1809 was monitored over a period of 6 months upon storage at 4 °C. Most carriers evaluated, were able to maintain rhizobial populations of more than 1 × 10⁹ rhizobia per gram of inoculant over that time period, with mixtures of perlite with either sugarcane bagasse or malt residue supporting the largest rhizobial populations and a mixture of perlite and rice husk the lowest. All carriers supported rhizobial growth over the 6 months period. Initially, rhizobial populations were greater with gamma irradiation than autoclaving, however after 6 months, this response was significant only with the perlite and sugarcane bagasse mixture. The incubation of the inoculant after injection also ultimately did not benefit rhizobial levels for any of the carriers, tested. Using simple sterilisation procedures and without incubating after injection, perlite based carriers can produce high quality inexpensive inoculants, maintaining bacterial populations of more than 1 × 10⁹/g rhizobia for at least 6 months.     

Production of surfactin by Bacillus pumilus UFPEDA 448 in solid-state fermentation using a medium based on okara with sugarcane bagasse as a bulking agent

We studied the production of surfactin by Bacillus pumilus UFPEDA 448 in solid-state fermentation (SSF), using a medium based on okara with the addition of sugarcane bagasse as a bulking agent. The optimum proportions of okara and sugarcane bagasse were 50% each, by mass. Due to the relatively high production of proteases during SSF, pre-hydrolysis of the okara with a protease did not improve surfactin levels. The optimum temperature for surfactin production was 37 °C, with the incubation temperature affecting the ratios of the various surfactin homologues produced. Cultivation in column bioreactors with forced aeration under optimized conditions gave a surfactin level of 809 mg L^?1 of impregnating solution, which corresponds to 3.3 g kg-dry-solids^?1. This is the highest surfactin level that has been produced to date in SSF with a non-recombinant microorganism. The peak O₂ uptake rate was 20 mmol min^?1 kg-initial-dry-solids^?1, corresponding to metabolic waste heat production rate of 182 W kg-initial-dry-solids^?1. The tensioactive properties of the surfactin were similar to those reported in the literature for surfactin produced by submerged fermentation. These results suggest that it might be feasible to use SSF to produce surfactin but at large scale special attention will need to be given to heat removal.     

Adsorption of Cu(II), Cd(II), and Pb(II) from aqueous single metal solutions by mercerized cellulose and mercerized sugarcane bagasse chemically modified with EDTA dianhydride (EDTAD)

This work describes the preparation of new chelating materials derived from cellulose and sugarcane bagasse for adsorption of Cu²⁺, Cd²⁺, and Pb²⁺ ions from aqueous solutions. The first part involved the mercerization treatment of cellulose and sugarcane bagasse with NaOH 5 mol/L. Non- and mercerized cellulose and sugarcane bagasse were then reacted with ethylenediaminetetraacetic dianhydride (EDTAD) in order to prepare different chelating materials. These materials were characterized by mass percent gain, X-ray diffraction, FTIR, and elemental analysis. The second part consisted of evaluating the adsorption capacity of these modified materials for Cu²⁺, Cd²⁺, and Pb²⁺ ions from aqueous single metal solutions, whose concentration was determined by atomic absorption spectroscopy. These materials showed maximum adsorption capacities for Cu²⁺, Cd²⁺, and Pb²⁺ ions ranging from 38.8 to 92.6 mg/g, 87.7 to 149.0 mg/g, and 192.0 to 333.0 mg/g, respectively. The modified mercerized materials showed larger maximum adsorption capacities than modified non-mercerized materials.     

Bioaugmentation of carbofuran residues in soil using Burkholderia cepacia PCL3 adsorbed on agricultural residues

Burkholderia cepacia PCL3 (GenBank accession number of EF990634) is a carbofuran degrader isolated from phytoremediated rhizosphere soil in our laboratory. Free and the immobilized PCL3 on corncob and sugarcane bagasse were investigated for their abilities to degrade carbofuran in Basal Salt Medium (BSM) and soil microcosm. The reusability and survival of immobilized PCL3 in comparison to free cells were also examined. Short half-lives (t_1/2) of carbofuran of 3–4 d in BSM were obtained using the isolate PCL3 in both free and immobilized cell forms. Immobilized cells could survive (10⁶–10⁷ cfu ml^?1) through 30 d of incubation, while the number of free cells decreased continuously after 10 d. Immobilized B. cepacia PCL3 could be reused twice without loss in their abilities to degrade carbofuran in BSM, which suggested an advantage of using immobilized cell over free cell. Free and immobilized cells were augmented into soil and showed an effective capability to remediate carbofuran residues, both of which indicated by 5-folds decrease in carbofuran half-lives in augmented soil. Immobilization of PCL3 on corncob and sugarcane bagasse provided the possibilities of reusing the cells as well as improving the cell survival without decreasing carbofuran degradation activity.     

Evaluating agro-industrial by-products as dietary roughage source on growth performance of fattening steers

Silages from pineapple peel, sweet corn husk and cob mixed with bagasse and vinasse were evaluated to determine their chemical composition and fermentation characteristics as well as feeding performance in fattening steers. The experiment, which lasted 90 days, involved 48 fattening steers (264 ± 37.4 kg BW) randomly allocated to three diets. Treatments included: a control diet containing rice straw and molasses (T1); diet containing bagasse–vinasse mixture including sweet corn husk and cob silage (BS; T2); and diet containing bagasse–vinasse mixture including pineapple peel silage (BP; T3). All treatments included a commercial concentrate feed (13% CP) and ad libitum rice straw throughout the experiment. Results from chemical analysis showed that dry matter (DM) of BS was higher than BP (P < 0.05), whereas the protein content of BS and BP was similar (P > 0.05). For fermentation characteristics, pH in BP was lower than BS (P < 0.05); in addition, acetic and butyric acids in BS were higher than BP (P < 0.05). Findings from growth trial showed that total DM intake in steers fed T1 was higher compared to the other dietary treatments (P < 0.05), whereas the average BW gain was found to be grater in T3 steers (P < 0.05). As result from our findings, bagasse–vinasse mixture with pineapple peel silage appeared to be a viable feed ingredient in fattening steer diet and moreover it could become an economically feasible agro-industrial by-product for farmers.     

Xylitol production in a bubble column bioreactor: Influence of the aeration rate and immobilized system concentration

This work evaluated the xylitol production from sugarcane bagasse hemicellulosic hydrolysate in a bubble column bioreactor using cells of the yeast Candida guilliermondii immobilized in calcium-alginate. The fermentation runs were performed according to a 2² full factorial design with three replicates at the center point in order to determine the effect of the variables: aeration rate (0.66–1.33 vvm) and immobilized system concentration (20–40% v/v), on the efficiency of xylose-to-xylitol conversion and on the xylitol volumetric productivity. The results indicated a significant influence of both variables on xylitol production. The highest conversion efficiency (41%) was attained using 1.33 vvm aeration rate and 40% immobilized system. Under these conditions, the volumetric productivity was 0.21 g l⁻¹ h⁻¹.     

Enhancement of the enzymatic digestibility of sugarcane bagasse by alkali–peracetic acid pretreatment

The enzymatic digestibility of sugarcane bagasse was greatly increased by alkali (NaOH)–peracetic acid (PAA) pretreatment under mild conditions. The effects of several factors affecting the pretreatment were investigated. It was found that when bagasse was pre-pretreated by 10% (based on initial dry materials) NaOH with 3:1 liquid-to-solid ratio at 90 °C for 1.5 h and further delignified by 10% peracetic acid (based on initial dry materials) at 75 °C for 2.5 h, the yield of reducing sugars reached 92.04% by enzymatic hydrolysis for 120 h with cellulase loading of 15 FPU/g solid. Compared with acid and alkali pretreatment, alkali–PAA pretreatment could be conducted under milder conditions and was more effective for delignification with less carbohydrates being degraded in the pretreatment process. Alkaline stage played an important role for partial delignification, swelling fibers and subsequently reducing PAA loading. No loss of cellulase activity (FPA) was observed in the liquid phase for alkali–PAA pretreated bagasse after enzymatic hydrolysis for 120 h.     

Pretreatment of sugarcane bagasse using supercritical carbon dioxide combined with ultrasound to improve the enzymatic hydrolysis

This work evaluates the pretreatment of sugarcane bagasse combining supercritical carbon dioxide (SC-CO₂) and ultrasound to enhance the enzymatic hydrolysis of pretreated bagasse. In a first step the influence of process variables on the SC-CO₂ pretreatment to enhance the enzymatic hydrolysis was evaluated by mean of a Plackett–Burmann design. Then, the sequential treatment combining ultrasound + SC-CO₂ was evaluated. Results show that treatment using SC-CO₂ increased the amount of fermentable sugar obtained of about 280% compared with the non-treated bagasse, leading to a hydrolysis efficiency (based on the amount of cellulose) as high as 74.2%. Combining ultrasound + SC-CO₂ treatment increased about 16% the amount of fermentable sugar obtained by enzymatic hydrolysis in comparison with the treatment using only ultrasound. From the results presented in this work it can be concluded that the combined ultrasound + SC-CO₂ treatment is an efficient and promising alternative to carry out the pretreatment of lignocellulosic feedstock at relatively low temperatures without the use of hazardous solvents.     

The effects of irradiation dose and storage time following treatment on the viscoelastic properties of decellularised porcine super flexor tendon

Decellularised porcine super flexor tendon (pSFT) offers a promising solution to the replacement of damaged anterior cruciate ligament. It is desirable to package and terminally sterilise the acellular grafts to eliminate any possible harmful pathogens. However, irradiation techniques can damage the collagen structure and consequently reduce the mechanical properties. The aims of this study were to investigate the effects of irradiation sterilisation of varying dosages on the viscoelastic properties of the decellularised pSFT.Decellularised pSFT tendons were subjected to irradiation sterilisation using either 30 kGy gamma, 55 kGy gamma, 34 kGy E-beam, 15 kGy gamma, 15 kGy E-beam and (15 + 15) kGy E-beam (fractionated dose). Specimens then underwent stress relaxation testing at 0 and 12 months post sterilisation to determine whether any effect on the viscoelastic properties was progressive.Significant differences were found which demonstrated that all irradiation treatments had an effect on the time-independent and time-dependent viscoelastic properties of irradiated tendons compared to peracetic acid only treated controls. No significant differences were found between the irradiated groups and no significant differences were found between groups at 0 and 12 months. These results indicate the decellularised pSFT graft has a stable shelf-life.     

Efficient isolation of anthraquinone-derivatives from Trichoderma harzianum ETS 323

Anthraquinone-derivatives, chrysophanol and pachybasin, were purified by a silica column chromatography with two different solvent systems from Trichoderma harzianum ETS 323. The fungus was incubated in sugarcane bagasse solid medium at room temperature without rotation. Structure of chrysophanol was solved by X-ray diffraction and pachybasin by NMR spectra. About 233 ± 13 mg of pure chrysophanol and 773 ± 40 mg of pure pachybasin were recovered per kg of solid cultural medium, with yields 1.7 ± 0.2% and 5.6 ± 0.5%, respectively.     

Optimization studies to develop a low-cost medium for production of the lipases of Rhizopus microsporus by solid-state fermentation and scale-up of the process to a pilot packed-bed bioreactor

A low-cost lipase preparation is required for enzymatic biodiesel synthesis. One possibility is to produce the lipase in solid-state fermentation (SSF) and then add the fermented solids (FS) directly to the reaction medium for biodiesel synthesis. In the current work, we scaled up the production of FS containing the lipases of Rhizopus microsporus. Initial experiments in flasks led to a low-cost medium containing wheat bran and sugarcane bagasse (50:50 w/w, dry basis), supplemented only with urea. We used this medium to scale-up production of FS, from 10 g in a laboratory column bioreactor to 15 kg in a pilot packed-bed bioreactor. This is the largest scale yet reported for lipase production in SSF. During scale-up, the hydrolytic activity of the FS decreased 57%: from 265 U g⁻¹ at 18 h in the laboratory bioreactor to 113 U g⁻¹ at 20 h in the pilot bioreactor. However, the esterification activity decreased by only 14%: from 12.1 U g⁻¹ to 10.4 U g⁻¹. When the FS produced in the laboratory and pilot bioreactors were dried and added directly to a solvent-free reaction medium to catalyze the esterification of oleic acid with ethanol, both gave the same ester content, 69% in 48 h.     

Two-step acid and alkaline ethanolysis/alkaline peroxide fractionation of sugarcane bagasse and rice straw for production of polylactic acid precursor

Sugarcane bagasse and rice straw were subjected to acid and alkaline ethanolysis and sequential enzymatic hydrolysis to produce glucose for lactic acid production. Influence of physico-chemical treatments using ultrasonic bath and ultrasonic probe was studied compared with mechanical stirring. The results showed that the highest glucose yield with least contamination of xylose was obtained from acid ethanolysis fractionation (5 N H₂SO₄ + 50%, v/v ethanol) when stirred at 90 °C for 4 h. Alkaline ethanolysis accomplished high amount of both glucose and xylose released, however it was not favorable substrate for homofermentative lactic acid bacteria. In order to enhance enzymatic hydrolysis of acid ethanolysis fractionated samples, lignin was subsequently removed by the second step alkaline/peroxide delignification. The maximum lactic acid was obtained at 23.6 ± 0.2 g/L from Lactobacillus casei fermentation after 72 h when hydrolysate from two-step acid hydrolysis and alkaline/peroxide fractionated sugarcane bagasse containing 24.6 g/L initial glucose concentration was used as substrate.     

Rhizobial counts in peat inoculants vary amongst legume inoculant groups at manufacture and with storage: implications for quality standards

Background and aims

Inoculation of legumes at sowing with rhizobia has arguably been one of the most cost-effective practices in modern agriculture. Critical aspects of inoculant quality are rhizobial counts at manufacture/registration and shelf (product) life.

Methods

In order to re-evaluate the Australian standards for peat-based inoculants, we assessed numbers of rhizobia (rhizobial counts) and presence of contaminants in 1,234 individual packets of peat–based inoculants from 13 different inoculant groups that were either freshly manufactured or had been stored at 4 °C for up to 38 months to determine (a) rates of decline of rhizobial populations, and (b) effects of presence of contaminants on rhizobial populations. We also assessed effects of inoculant age on survival of the rhizobia during and immediately after inoculation of polyethylene beads.

Results

Rhizobial populations in the peat inoculants at manufacture and decline rates varied substantially amongst the 13 inoculant groups. The most stable were Sinorhizobium, Bradyrhizobium and Mesorhizobium with Rhizobium, particularly R. leguminosarum bv. trifolii the least stable. The presence of contaminants at the 10^?6 level of dilution, i.e. >log 6.7 g^?1 peat, reduced rhizobial numbers in the stored inoculants by an average of 37 %. Survival on beads following inoculation improved 2–3 fold with increasing age of inoculant.

Conclusions

We concluded that the Australian standards for peat-based rhizobial inoculants should be reassessed to account for the large differences amongst the groups in counts at manufacture and survival rates during storage. Key recommendations are to increase expiry counts from log 8.0 to log 8.7 rhizobia g^?1 peat and to have four levels of inoculant shelf life ranging from 12 months to 3 years.     

In vitro evaluation of starch degradation from feeds with or without various heat treatments

An in vitro method was used to evaluate starch degradation from various feeds with or without heat treatments in four studies. The method was based on incubation of feed samples with a buffered rumen fluid solution and subsequent enzymatic analysis of the remaining starch. In all studies, heat treatment of the feed samples increased rate or extent of starch degradation to glucose. In Study 1, measurements of remaining starch, after 5 h in vitro incubations, demonstrated substantial effects of cooking on starch degradation in potatoes, and a trend to faster degradation from autoclaving peas. Up to 0.60 of the starch remaining after a 5 h of incubation was not recovered by centrifugation at 3000 × g for 10 min. In Study 2, cooking increased in vitro starch degradation rate from isolated potato starch (from 0.038 to 0.197/h). Intact starch in barley and wheat grain had similar rates of degradation (0.117 and 0.109/h, respectively). In Study 3, both autoclaving time (15, 30, 60 min) and temperature (115, 130 and 145°C) affected in vitro starch degradation rates in peas, and, in no case did autoclaving for only 15 min increase degradation rates. For the 30 min autoclaving time, only the highest temperature (145°C) increased the degradation rate of the pea starch compared to the untreated peas (0.175 versus 0.110/h). When autoclaving for 60 min, both 130 and 145°C resulted in a considerable increase in starch degradation rate (0.211 and 0.193/h, compared to 0.110/h for the untreated peas). In Study 4, the proportion of starch degraded at 8 h of in vitro incubation was increased by heat treatment of pure potato starch (0.155 versus 0.870), peas (0.491 versus 0.815), barley (0.686 versus 0.913) and maize (0.351 versus 0.498). Measurements of volatile fatty acid production in the fermentation tubes showed a lower acetate:propionate ratio for the faster fermenting heat-treated feeds. Heat treatment generally increased starch degradation in vitro.     

Effect of antioxidant extraction on the enzymatic hydrolysis and bioethanol production of the extracted steam-exploded sugarcane bagasse

Ethyl acetate extraction (EAE) of the steam exploded sugarcane bagasse may be an effective and economic way to extract antioxidants as well as enhance the enzymatic hydrolysis and bioethanol yield from the extracted residue. Therefore, the effects of EAE on steam-exploded sugarcane bagasse (SESB) were studied. Under boiling solvent extraction (BSE), the efficiency of EAE for obtaining phenolics from SESB was approximately 20%. EA extracts obtained under BSE showed an H₂O₂ scavenging activity (210 μL) of 99%. The IC₅₀ values for 2,2-diphenyl-1-picrylhydrazyl radical scavenging activity and reducing power of BSE40 were 50.89 and 256.38 μg/mL, respectively, while those of vitamin C were 24 and 112 μg/mL, respectively. EAE improved the glucose yield by 30% but had no significant effect on the xylose yield during the enzymatic hydrolysis obtained using Celluclast 1.5L and Novozym 188. EAE also increased the ethanol yield by 8.78% by employing simultaneous saccharification and fermentation. The present study may be of great importance in industrial bioethanol production from steam-exploded biomass environmentally friendly and economically.     

Isolation of novel chitinolytic bacteria and production optimization of extracellular chitinase

Chitin is one of the most abundant biopolymers widely distributed in the marine and terrestrial environments. Chitinase enzyme has received increased attention due to its wide range of biotechnological applications, especially in agriculture for biocontrol of phytopathogenic fungi and harmful insects. In the present study, 58 bacterial isolates were screened for chitinolytic activity and on the basis of chitin hydrolysis zone 6 isolates were selected for chitinase production in broth media. Based on enzyme production, two most potent isolates identified as Aeromonas hydrophila HS4 and Aeromonas punctata HS6 were selected for further study. The effects of media composition and various fermentation conditions for optimization of chitinase production were studied. The maximum chitinase production was obtained at 37 °C and pH 8.0 after 24–48 h of incubation by HS4; and at 37 °C and pH 7 after 48 h incubation by HS6. Among the substrates colloidal chitin was the best for both the strains. Regarding carbon sources, starch (1%) was the best for both strains; while malt and yeast extract (1%) was found as the best nitrogen source for HS4 and HS6, respectively. Out of metal ions Mn²⁺ and Cu²⁺ enhanced enzyme production in the case of HS6. However, Co²⁺ was the most appropriate for HS4.     

The effect of forest fragments on abundance,diversity and species composition of predatory ants in sugarcane fields

Habitat loss and fragmentation have gradually caused loss of diversity and consequently the decline of ecological services. This study aimed to evaluate the effect of tropical forest fragments as natural habitats (river valley fragments and plateau fragments) on the community of predatory and omnivorous ants in nearby sugarcane fields. Twenty fields adjacent to these fragments were selected and evaluated one (dry season) and four months (rainy season) after harvest. In each field, ants were sampled in five linear plots (10 m inside the fragment, 0 m (field path between field and fragment), 5 m, 50 m and 100 m inside the crop fields). Each plot comprised ten sardine baits in a row parallel to the field edge. Species richness and frequency of ant species decreased with increasing distance from the forest fragments. Inside fields, species richness and frequency were higher during the period of vegetative growth (rainy season) than after harvest (dry season). Ant communities of sugarcane fields and forest fragments were more similar later in the season than directly after sugarcane harvest suggesting recolonization of the fields from the fragments. Several ant species were limited to forest fragments after harvest but occurred later in the season also in sugarcane fields confirming the potential contribution of fragments to the recolonization process and therefore to biological control of sugarcane-dominated pest insects.     

Manganese species from human serum,cerebrospinal fluid analyzed by size exclusion chromatography-, capillary electrophoresis coupled to inductively coupled plasma mass spectrometry

Manganese (Mn) at high concentrations can have adverse effects on health, mainly because of its toxicity to the central nervous system. Health impacts of Mn are known mostly from occupational health studies, but the exact mechanisms how Mn, being bound to transferrin (TF) in the blood, enters the brain – are unknown. Mn speciation at the neural barriers can help to obtain more information about the pathways and carriers. This paper summarizes investigations on the size distribution of Mn carriers (e.g. proteins, peptides, carbonic acids) in serum before the neural barriers and in cerebrospinal fluid (CSF) behind them as a first characterization step of the Mn carriers being involved in moving Mn across the neural barriers. Further identification of Mn-species in CSF was successfully achieved by CZE–inductively coupled plasma (ICP)–dynamic reaction cell (DRC)–mass spectrometry (MS). Serum samples showed Mn mean concentrations of 1.7±0.8 μg L⁻¹. The size distribution of Mn-carriers showed a main peak in the TF/albumin size fitting to the known physiological ligands. However, also an increasing Mn peak at 700 Da with increasing total Mn concentration was seen. Samples of CSF showed Mn mean concentrations of 2.6 μg L⁻¹=48 nM. In CSF Mn was found to be mostly bound to low-molecular-mass (LMM)-Mn carriers in the range of 640–680 Da. This is similar to the LMM compound in serum and to Mn–citrate complexes suggested to be present in body fluids. Citrate concentration was 573 μM, thus being in huge excess compared to Mn. CSF was further analyzed by CZE–ICP–DRC–MS. Several Mn-species were monitored and mostly identified. The most abundant Mn-species was Mn–citrate at a concentration of around 0.7 μg Mn L⁻¹.     

Production of xylanase by Thermoascus aurantiacus from sugar cane bagasse in an aerated growth fermentor

In recent years, the use of xylanases has been adopted by many processing industries, such as pulp and paper, food and textile factories. This study demonstrates that Thermoascus aurantiacus ATCC 204492 is able to produce a high level of thermostable xylanase when sugar cane bagasse is used as a substrate. Fermentations were performed in a glass-column reactor with forced aeration. A xylanase activity of 1597 U/g was attained after 10 days of solid-state fermentation. The effects of different airflow rates (0, 3.0, 6.0 l/(h g) bagasse) and initial mass of bagasse (8, 12.5, 17 g) on the production of xylanase were investigated using a statistical experimental design. The airflow rates had a significant effect on enzyme activity, whereas initial mass of bagasse had no significant effect on enzyme activity. 6 l/(h g) airflow rate and 8 g substrate resulted in the highest yields of xylanase (1597 U/g).     

Thermal stability of starch degrading enzymes of teff (Eragrostis tef) malt during isothermal mashing

Thermal stability of starch degrading enzymes varies from one source to another. This research was aimed to study thermal stability of starch degrading enzymes of teff malt. Isothermal mashing at temperatures ranging between 40 and 75 °C with sampling in 15 min interval for a total of 90 min was conducted. The study showed that deactivation rate constants of alpha- and beta-amylases ranged from 0.0003 to 0.0409 min^?1, and 0.002 to 0.032 min^?1, respectively. Rate of deactivation of limit dextrinase was not significant at temperatures lower than 60 °C but showed high deactivation at higher temperatures with rate constants ranging from 0.02 to 0.1 min^?1. The thermal deactivation energies of alpha-amylase, beta-amylase, and limit dextrinase were found to be 148, 82, and 144 kJ/mol, respectively. The present findings have significant applications in commercial processes where determination of the upper temperature limits for these enzymes is required.