Non-aseptic Conditions Research Articles

Fungal treatment of corn processing wastewater in an attached growth system

Corn processing wastewater was treated in an attached growth system of Rhizopus oligosporus fungi. The effects of hydraulic retention time (HRT) and plant-based components in the support media were evaluated in 1 L reactors under non-aseptic conditions. Plastic composite support (PCS) tubes, composed of 50% (w/w) polypropylene (PP) and 50% (w/w) agricultural products were used as support media or, as a test, PP only. A maximum chemical oxygen demand (COD) removal of 78% was achieved at a 5 h HRT with a biomass yield of 0.44 gVSS/gCODremoved. The biomass yield increased to 0.48 gVSS/gCODremoved while COD removal reduced to 70% at a 2.5 h HRT. Competitive bacterial growth was reduced at the shorter HRT. A lower HRT of 1.25 h led to biomass wash out from the reactor. Significantly lower COD removal of 28% and biomass yield of 0.19 gVSS/gCODremoved were observed in a biofilm reactor with PP tubes alone, indicating that the agricultural components in PCS media were essential for better biofilm formation and organic removal.

Survival of Day 3 and Day 5 Embryos Following Vitrification in Aseptic and Non-Aseptic Conditions: A Prospective Randomized Analysis

The success rates of vitrification of embryos have been increased with the use of an ultra-rapid cooling rate that are based on direct contact between a reduced volume of cryoprotectant and liquid nitrogen (LN2). One of the disadvantages of such an approach is that an unprotected exposure to LN2 bears a certain degree of biohazard such as bacteriological and viral contamination of embryos. Therefore, to minimize such risks, aseptic technologies have to be introduced, shielding embryos from LN2 during the cooling process while maintaining cooling rates compatible with high survival rates. Vitrification using non-aseptic and aseptic vitrification methods: prospective randomized analysis on sibling embryos. Fifty three day 3 embryos originating from 1PN or 3PN zygotes and 158 supernumerary day 5 blastocysts not eligible in our ART cryopreservation program were vitrified after exposure for 1 to 2 min for day 3 embryos and 2 to 3 min for blastocysts to dehydration solutions containing a 10% of DMSO and ethylene glycol (EG). This step was followed by a 30 seconds exposure at room temperature (RT) to 20% EG, 20% DMSO, 25 μmol/l Ficoll and 0.75 M sucrose. During this short time period, a very small amount (< 1μl) of vitrification solution containing the embryos was deposited to the tip of the trough of a straw (termed: hemi-straw) using an attenuated pipette. Each vitrification assay contained at least 3 embryos, each of them were allocated randomly to 3 different vitrification protocols. In the control group, the hemi-straw (HS) was directly plunged into a Dewar of LN2 allowing contact of the embryos with LN2. In the experimental group, direct contact with LN2 was avoided. Before plunging into LN2, the HS was inserted inside a 250μl straw or inside a straw with a larger diameter (500 μl straw). Afterward, the straws were sealed and then plunged into LN2. The same warming procedure was applied for the three groups. The tip of the HS holding the embryos was immediately immersed into 0.5M, 0.25M and 0.125M sucrose bath at intervals of 2 minutes at RT. The embryos were then washed several times in PBS-HSA solution. Embryo survival was assessed after 24 hours culture in Global medium. Day 3 embryos had to cleave further to the morulae stage and day 5 embryos had to re-expand. Tabled 1 The results show that post-thaw survival rates of day 3 and day 5 embryos vitrified at a lower cooling rate inside an hermetic sealed straw are comparable to the control non-aseptic group. These two aseptic vitrification protocols provides a simple and effective strategy for eliminating the risk of contamination during vitrification and long term storage. However, blastocysts seems more sensitive to the cooling rate as compared to day 3 embryos when they were vitrified in a larger volume straw (500μl). Such observations support the idea that the cooling rate is critical for an optimal protection of blastocysts and more particularly for blastocysts with a larger blastocoele cavity.

Use of single-leaf cuttings of potato for efficient mass propagation

Potato stock plants of five commercially propagated varieties, which derived from tissue culture, were grown at a low light irradiance in non-aseptic conditions. The plants produced tiny-leaved etiolated stems, which were a good source of cuttings for further propagation. The method was tested by growing micropropagated and cutting propagated plants as three lines of stock plants, based on different clonal generations, and which were repeatedly harvested for cuttings. There was wide variation in the multiplication capacity among the varieties used. The most productive variety produced more cloned potato plants than when micropropagated in vitro. Stock plants derived from single-leaf cuttings produced more new cuttings than when derived from tissue culture. The growing facilities used were simple and inexpensive and could be set up anywhere. The method is economically competitive due to the need of only very simple and inexpensive growing rooms, and would be worth using particularly in developing countries and also in advanced potato production. All the cuttings of different harvests rooted well and the last propagated cuttings of the lines were still free of prevalent viruses although using simple growing facilities.

Aerobic Biodegradation of Mononitrotoluenes in Batch and Continuous Reactor Systems

Degradations of nitrotoluenes, individually and in a mixture, were carried out in batch and continuous aerobic reactors by a defined mixed microbial culture. The degradation rates and efficiencies of the isomers were evaluated in batch and continuous reactors. The results demonstrated that all the three-mononitrotoluene isomers were degraded simultaneously and completely in presence of excess oxygen. The nitro group position on the benzene ring influenced the degradation rates of the individual MNT isomers in batch systems. In the continuous biofilm reactor with a sufficient biocatalyst quantity, quality and excessive oxygen supply rate, the degradation rates of the mononitrotoluenes were almost identical as long as the compounds were present individually and their loading did not exceed the capacity of the catabolic master reaction. The microbial composition of the biofilm changed qualitatively and quantitatively during long-term continuous operation under aerobic non-aseptic conditions. This complex investigation resulted in data that can be applied for the scale-up procedure for field experiments.

The sourdough microflora: biodiversity and metabolic interactions

The production of sourdough bread can be traced back to ancient times. Sourdough is a mixture of flour and water that is fermented with lactic acid bacteria (LAB). Sourdough is an intermediate product and contains metabolically active yeast and LAB strains. The LAB that develop in the dough may originate from selected natural contaminants in the flour or from a starter culture containing one or more known species of LAB. Sourdough can be produced in bakeries or obtained from commercial suppliers. The microbial ecology of the sourdough fermentation is determined by ecological factors. Microbiological studies have revealed that more than 50 species of LAB, mostly species of the genus Lactobacillus, and more than 20 species of yeasts, especially species of the genera Saccharomyces and Candida, occur in this ecological niche. The sourdough microflora is composed of stable associations of lactobacilli and yeasts, in particular due to metabolic interactions. As shown for certain industrial sourdough processes, such microbial associations may endure for years, although the fermentation process runs under non-aseptic conditions. A reproducible and controlled composition and activity of the sourdough microflora is indispensable to achieve a constant quality of sourdough bread.

Ethanol fermentation of a diluted molasses medium by Saccharomyces cerevisiae immobilized on chrysotile

In this work, the catalytic role of chrysotile support on the acceleration of alcoholic fermentation under non-aseptic conditions by Saccharomyces cerevisiae was investigated. The fermentation medium employed consisted only of diluted sugar-cane molasses. In the batch fermentations process with immobilized yeasts, the initial rate of CO2 production increased roughly 27 % during the first 30 minutes, compared to systems containing no chrysotile. A study of continuous alcoholic fermentation with chrysotile in the reactor bed showed a higher ethanol production rate at the different dilution rates investigated compared to similar fermentations without chrysotile.

Somatic embryo culture for propagation, artificial seed production, and conservation of sawara cypress (Chamaecyparis pisifera Sieb. et Zucc.)

Somatic embryogenesis in Chamaecyparis pisifera Sieb. et Zucc. was initiated from immature seeds collected from the end of June to early July. Mass propagation through adventitious shoot bud production from somatic embryo culture on Woody Plant (WP) medium and artificial seed production using sodium alginate was achieved. A high bud forming index value (25.8) was obtained on medium supplemented with 1μM 6-benzylaminopurine. The conversion rates from artificial seeds under aseptic and nonaseptic conditions were 60%–100% and 10%–12%, respectively. For germplasm conservation, somatic embryos and embryogenic cells were successfully stored at 4°C (medium-term storage) and in liquid nitrogen for long-term storage.

Rabbit and pig ear skin sample cryobanking: effects of storage time and temperature of the whole ear extirpated immediately after death

The post-mortem temporal and thermal limits within which there will be ample guarantees of rescuing living skin cells from dead specimens of two species, rabbit and pig, were studied. Post-mortem extirpated whole ears were stored (in non-aseptic conditions) either at 4 °C or at room temperature (from 22 to 25 °C) or at 35 °C for different time lapses after animal death. In both species, the post-mortem maximum time lapses where cell viability was not significantly reduced were 240, 72, and 24 h post-mortem (hpm) for 4, 22–25 and 35 °C, respectively. Once the post-mortem temporal limits for each tested thermal level at which cells from skin samples are able to grow in culture were defined, the survival ability of skin samples submitted to these temporal limits and cryopreserved were tested. In the pig, skin samples stored at the three tested thermal levels survived after vitrification-warming, reaching confluence in culture. In rabbit, only tissue samples from ears stored at 35 °C for 24 hpm did not survive after vitrification-warming. In conclusion, we should remark that cell survival rates obtained according to the assayed post-mortem time lapses and thermal levels are sufficient to collect and to cryopreserve skin samples from the majority of dead specimens.

Production of canthaxanthin by Haloferax alexandrinus under non-aseptic conditions and a simple, rapid method for its extraction.

The production of carotenoids from Haloferax alexandrinus strain TM(T) was investigated at various concentrations of NaCl (10-25%) in culture media under non-aseptic conditions. PCR and dot blot hybridization assays were employed to monitor the growth of Hfx. alexandrinus in the culture under aseptic and non-aseptic conditions. The amplified PCR products of 16S rDNA from Hfx. alexandrinus grown under aseptic conditions were used as specific probes, which bound with amplified PCR products of 16S rDNA dots from both aseptic and non-aseptic conditions (20-25% NaCl). The results indicated that contamination of the culture was precluded at high NaCl concentrations (20-25%). Therefore, it is not necessary to perform asepsis during the biotechnological processes of carotenoid production by Hfx. alexandrinus. A 1-l-scale cultivation of the cells in flask cultures under non-aseptic conditions produced 3.12+/-0.5 g dry weight, 6.34+/-2.5 mg total carotenoids and 2,156.67+/-0.1 microg canthaxanthin. Further experiments in a batch fermenter, under non-aseptic conditions, also demonstrated increases in the biomass concentration and carotenoid production. When grown in a standard growth medium at 25% NaCl, the cells of Hfx. alexandrinus lysed spontaneously in fresh water and hence carotenoids could be extracted directly from the cells without any mechanical disintegration. These results demonstrate the feasibility and simplicity of commercial production of carotenoids using Hfx. alexandrinus.

Effects of double encapsulation and coating on synthetic seed conversion in M.26 apple rootstock

Encapsulated vitro -derived apical buds of M.26 apple rootstock (Malus pumila Mill) can be employed for the formation of the synthetic seed. Satisfactory levels of conversion (plantlets from synthetic seed) can be achieved if there are adequate (i) rooting induction treatment, (ii) protocol of encapsulation, and (iii) nutritive and environmental conditions. For capsule manufacturing, sodium alginate is largely used; however, this is excessively permeable with loss of the nutritive substances (artificial endosperm) and/or dehydration risks during conservation and transport causing detrimental effects on the synthetic seed conversion and on the plantlet's growth. In order to overcome these problems, two experiments were carried out comparing simple encapsulation in alginate with double encapsulation, and with encapsulation-coating procedures. The presence of a second layer of alginate (double encapsulation) and of a thin external coating layer over the alginate (encapsulation-coating) did not show any detrimental effects on viability, sprouting and regrowth of the encapsulated microcuttings. Satisfactory conversion (70%) was reached with the encapsulation-coating procedure, whereas the double and simple encapsulation converted less than 40% of the synthetic seed. The effect of the addition to the capsule of an anti-microbial substance (Plant Preservative Mixture - PPM#174;) was examined: it did not compromise the conversion of the encapsulated microcuttings sown in ex-vitro non-aseptic conditions.

Effects of experimental conditions on mycorrhizal relationships between Pinus sylvestris and Lactarius deliciosus and unprecedented fruit-body formation of the Saffron milk cap under controlled soilless conditions.

The mycorrhizal relationships between pines and two edible species of Lactarius sect. Dapetes were investigated by optimizing the experimental conditions of mycelial growth and of mycorrhizal colonization of pine seedlings. In vitro mycelial growth of Lactarius deliciosus and L. sanguifluus was improved on a buffered medium containing glucose, amino acids, and vitamins. Two methods of mycorrhization of pines with Lactarius deliciosus were tested. The mycorrhizal colonization was rapid and intense under non-aseptic conditions with a low nutrient supply and without exogenous glucose. A positive influence of mycorrhizal colonization on Pinus sylvestris growth was subsequently observed. Under axenic conditions and with a high nutrient supply, mycorrhization was stimulated at 10 g/L of exogenous glucose, irrespective of the phosphorus concentration. At high phosphorus level (1 mM) and 0.1, 1.0, or 10.0 g/L glucose, growth of Pinus sylvestris was reduced by inoculation. Stability and development of Pinus spp./Lactarius deliciosus symbioses were assayed in a climatic chamber using containers filled with a synthetic substrate. Over a 2-year culture period, the root systems of the pine seedlings were heavily colonized by Lactarius deliciosus. One year following inoculation, Lactarius deliciosus fruit-body primordia appeared associated with Pinus sylvestris seedlings. Six months later, two mature basidiomata were obtained. This is the first report of soilless fruit-body formation of this edible mushroom.

Biosurfactant-enhanced degradation of residual hydrocarbons from ship bilge wastes

The use of Bacillus subtilis O9 biosurfactant (surfactin) and of bioaugmentation to improve the treatment of residual hydrocarbons from ship bilge wastes was studied. A biodegradation experiment was conducted in aquaria placed outdoors under non-aseptic conditions. Three treatments were examined: culture medium plus bilge wastes, bioaugmentation with microorganisms from bilge wastes, and bioaugmentation plus biosurfactant. Samples were analyzed for viable counts, aliphatic and aromatic hydrocarbon concentrations, n-C17/pristane and n-C18/phytane ratios. While the addition of biosurfactant stimulated hydrocarbon degradation, bioaugmentation did not produce any remarkable effect. At day 10, the remaining percentages of aliphatic and aromatic hydrocarbons in aquaria, which received biosurfactant, were 6.8 and 7.2, respectively, while it took 20 days to reach comparable results with the other treatments. The biosurfactant did not affect the preferential biodegradation of n-C17/pristane and n-C18/phytane. This biosurfactant, which can be produced in a relatively simple and inexpensive process, is a promising alternative in the optimization of hydrocarbon waste treatment. Journal of Industrial Microbiology & Biotechnology (2000) 25, 70–73.

Sclerotium rolfsii ATCC 15205 in continuous culture: Economical aspects of scleroglucan production

Sclerotium rolfsii ATCC 15205 was cultivated in continuous culture (48 l reactor volume) for scleroglucan production. The maximum volumetric productivity (QP max) amounted to 7.2 g/ld and was more than twice as high as in comparable batchwise cultivations. In addition, the relation between (a) volumetric productivity (QP [g/ld]) and product yield (YGlucan/Glucose [1]), (b) volumetric productivity and product quality (MW [g/mol]), and (c) product quality and impeller tip speed (Nd [m/s]) was studied in continuous culture. It was found, that an increase in volumetric productivity led to a decline in product yield and product quality. Furthermore, an impeller tip speed of >0.7 m/s decreased the attainable product quality considerably. Based on these results, the impact of the operational setpoint of the process in terms of oxygen supply and reactor scale on the economics of scleroglucan production was discussed. In contrast to batchwise cultivation, scleroglucan production in continuous culture proved to be not feasible under non-aseptic conditions.

Microbial degradation of thermo‐oxidized low‐density polyethylene

Thermogravimetric analysis (TGA), differential scanning calorimetry (DSC), and Fourier transform infrared spectroscopy (FTIR) were used to determine the relative heat of fusion, crystallinity, and composition of thermo-oxidized low-density polyethylene (LDPE) inoculated with Phanerochaete chrysosporium and incubated for 3 months at 30°C under nonaseptic conditions. DSC analysis of thermo-oxidized samples (150°C, 120 h) followed by microbiological treatment showed a remarkable reduction on the heat of fusion (36%) compared to that of untreated LDPE. TGA allowed determination of changes due to the biological activity but not those that occurred by thermal treatment. A significant increase in the carbonyl index of the thermally treated (150°C, 120 h) LDPE samples was observed due to the increase in the oxidation level during this treatment. In contrast, the double-bond index (evaluated by FTIR) increased considerably (23%) as a result of the microbial treatment. The observed structural and morphological modifications of the biologically treated LDPE indicate that the reduction of the chain size may be carried out by scission mechanisms. © 1999 John Wiley & Sons, Inc. J Appl Polym Sci 73: 1435–1440, 1999

Shelf-life extension of fresh-like ready-to-use pear cubes

Minimally processed fruit and vegetables are generally more perishable than the original raw materials due to the injury stresses during preparation that greatly increase tissue respiration and lead to deterioration through various biochemical pathways. In this paper, the effects of low intensity blanching treatments on enzymatic and microbiological stability on the fresh-like quality of ready-to- use pear cubes has been evaluated. Blanching treatments as well as packaging were carried out both in aseptic and in non-aseptic conditions. The combination of low intensity blanching with superficial enzymatic treatments has been carried out. The results confirm that the combination of a mild heat treatment (3 min at 95 °C) under aseptic conditions is enough to enhance the stability of this minimally processed product with an acceptable reduction in the texture. The superficial treatment with lysozyme did not result in an improvement in the stability of the blanched pear cubes during storage under refrigerated conditions. # 1999 Society of Chemical Industry

Production of valuable products from organic waste streams

A process was developed at the University of Pretoria through which it became possible to simultaneously purify organic effluents and produce a constant quality Single Cell Protein (SCP) product under non-aseptic conditions. Performance of the process as applied to a sugar-furfural effluent is described. The organism selected was Aspergillus fumigatus, a thermotolerant filamentous fungus. Typical yield is 0.56 g biomass per g COD utilized. The amino acid profile of the biomass compares well with other edible protein sources. It was possible to reduce the COD concentration in the effluent from ± 8000 mg/L to ± 150 mg/L.

Production of valuable products from organic waste streams

A process was developed at the University of Pretoria through which it became possible to simultaneously purify organic effluents and produce a constant quality Single Cell Protein (SCP) product under non-aseptic conditions. Performance of the process as applied to a sugar-furfural effluent is described. The organism selected was Aspergillus furnigatus, a thermotolerant filamentous fungus. Typical yield is 0.56 g biomass per g COD utilized. The amino acid profile of the biomass compares well with other edible protein sources. It was possible to reduce the COD concentration in the effluent from ± 8000 mg/L to ± 150 mg/L.

A trickle bed reactor for ferrous sulphate oxidation using Thiobacillus ferrooxidans

A trickle bed reactor was used to improve ferrous sulphate oxidation rate with Thiobacillus ferrooxidans immobilised in BSPs (Biomass Support Particles). A maximum iron(II) oxidation rate of 4.4 gL−1h−1 was observed at a dilution rate D = 0.9 h−1. The ability of the reactor to operate under non-aseptic conditions due to the chemoautotrophy and acidophilia of the bacterium makes industrial application promising.

Biosurfactant Production Using Mixed Cultures Under Non-Aseptic Conditions

AbstractSurfactants increase the accessibility of adsorbed hydrocarbons and mobilize immiscible petroleum hydrocarbons for treatment. Biosurfactants have the advantage of biodegradability and non-toxicity over their synthetic counterparts, and can be produced from renewable sources. In this study the production of biosurfactant from molasses was investigated in continuously stirred batch reactors. The effects of substrate concentration, yeast extract and peptone on biomass accumulation and biosurfactant production were investigated. Biosurfactant production was quantified by surface tension reduction and critical micelle dilution (CMD). Biosurfactant production was directly correlated with biomass production, and was improved with the addition of yeast extract. Centrifugation of the whole broth reduced surface tension. The performance of the biosurfactant produced from molasses under non-aseptic condition is comparable to other published results.

Alcohol production in submerged cashew pomace

The variations in the total titratable acidity, hydrogen ion concentration, reducing sugar, soluble solids and alcohol contents of the supernatant of the submerged cashew pomace in a non-aseptic condition were investigated. The crude fibre content of the pomace was also determined during fermentation. At intervals, the changes in microbial load in both the pomace and its supernatant were examined during the 14-day study. Fifty four percent of the total soluble solids (TSS) was utilised within 7 days of degradation while an increase of about 91% in the total titratable acidity occurred in the cashew pomace within the same period bringing its pH to 3.24. Acid hydrolysis of the pomace accounted for the relative high value of 2.15% TSS on the 8th day. Isolates of the genera Leuconostoc, Lactobacillus, Pediococcus, Aspergillus, Rhizopus and some yeast strains were obtained from the fresh pomace. The fungal colonies constituted about 76% of the population in the cashew pomace. The 86% drop in microbial population of the 8th day biodegraded pomace could be attributed to a decrease in nutrients of the substrate and the inhibitory effect of the organic acids produced during fermentation.