Fungal isolates from natural pectic substrates for polygalacturonase and multienzyme production

Pectin rich wastes and waste dump yard soils were screened and eighty pectinolytic fungal isolates were obtained by enrichment culturing and ruthenium red plate assay. Eight isolates with higher zones of pectin hydrolysis were selected and tested for polygalacturonase production. One isolate identified as Aspergillus awamori MTCC 9166 with highest polygalacturonase activity was tested for utilization of raw pectins for enzyme production. Polygalacturonase production was high in raw pectin sources like Orange peel (16.8 U/ml) Jack fruit rind (38 U/ml) Carrot peel (36U/ml) and Beet root peel (24U/ml). Selected Aspergillus awamori MTCC 9166 was found to be having good polygalacturonase, xylanase, cellulase and weak amylase and protease activities. This isolate with multi-enzyme production could have application for enzymes production and degradation of fruit and vegetable waste in the process of urban waste disposal.     

The fermentative production of oxytetracycline on industrial by-products byStreptomyces rimosus 12907

Three strains ofStreptomyces rimosus were grown on four different media. The one suitable for the production of oxytetracycline byStreptomyces rimosus 12907 was modified by black strap molasses, fodder yeast (40% total protein) and rice bran. The volume of the fermentation medium was scaled up in a 1200-litre fermentor aerated with sterile air obtained from a system used in the purification of air. 850 g crude oxytetracyeline was obtained when the fermented medium (700 litres) was extracted with 1-butanol. This work was carried out in the Laboratories of the Chemical Factories of the Egyptian Sugar anio Distillation Company with the cooperation of the National Research Centre, Dokki, Cairo, Arab Republd of Egypt (A.R.E.).     

Agro industrial by-products as flavinogenic stimulators for riboflavin production

Plate and frame filter used as a recirculated batch reactor for the production of 6-APA was studied. Penicillin-G solution was recirculated between the immobilized penicillin-G amidase (IPGA) loaded filter and the pH controlled neutralization tank. As penicillin-G solution flowed convectively through the frames loaded with IPGA particles, penicillin-G was hydrolyzed by IPGA and 6-APA was produced. Because of the large filtration area and short filtration depth of plate and frame filter, the batch hydrolysis reaction was operated at high recirculation flow rate without causing high pressure drop. The effect of recirculation flow rate on 2% penicillin-G hydrolysis was stronger than that on 8% penicillin-G hydrolysis. The amount of IPGA loaded in filter had no significant effect on the production yield of IPGA. The operational stability of IPGA in the filter was very satisfactory. There was no appreciable activity decrease after 25 batches of reaction.     

Studies on by-products from the industrial extraction of alginate

The content, the chemical composition and some physical-chemical properties of soluble and insoluble dietary fibres from an industrial alginate extraction by-product, flotation cellulose, were measured by two enzymatic gravimetric methods: an adaptation of the AOAC method (standard method) and a physiological method which used conditions closer to those prevailing in the digestive tract (pH, temperature, ionic strength and ionic composition). Total dietary fibres content obtained with the two methods were close (48.4–52.7; 56.3–59.8%) and about 68–95% of them were insoluble. Soluble fibre were essentially composed of uronic acids and were extracted under the simulated gastric conditions.Swelling in water and water absorption in NaCl (154 mM) of insoluble fibres with particle size between 250–500 µm were 21.9 g g^-1 and 3.6 gg^-1, respectively. The content and physical-chemical characteristics of fibres from flotation cellulose are close to those obtained from other plant and algal industrial by-products. Soluble fibre presented low intrinsic viscosity (152 ml g^-1).     

Studies on by-products from the industrial extraction of alginate

The chemical composition of fucans isolated from leach-water, an industrial alginate extraction by-product, was investigated. Several fractions were obtained by anion exchange and gel permeation chromatography. They all contained fucose, but differed in the uronic acid, sulfate, xylose and galactose contents. They distributed as a continuum between uronic acid rich and sulfate poor to sulfate rich and uronic acid poor molecules. Two highly sulfated fractions were studied in particularly by chemical means (methylation, carboxy reduction, desulfation, controlled acid hydrolysis) and by¹³C nuclear magnetic resonance spectroscopy. One fraction consisted of a highly branched fucan (43.8% fucose) composed mostly of 1,2,3,4- and 1,2,4-linked fucose with some 1,4-,1,3,4- and 1,3-linkages and sulfate (23.9%) occurring on O₂ and/or O₃ and/or O₄. The other was composed mainly of fucose (31.6%), galactose (24.7%) and sulfate (23.7%). It consisted primarily of 1,6-, 1,4,6-, 1,3- and 1,3,6-linked galactose 6-and/or 4- and/or 3-sulfate on which are linked essentially terminal fucose or 1,4-linked with sulfate on O₂ and/or O₃ and/or O₄. None of these highly branched fractions contained sufficiently regular segments to yield series of homologous oligosaccharides on partial acid hydrolysis or interpretable¹³C NMR spectra.author for correspondence     

Utilization of salt-rich by-products from the dairy industry as feedstock for recombinant protein production by Debaryomyces hansenii

The dairy industry processes vast amounts of milk and generates high amounts of secondary by-products, which are still rich in nutrients (high Chemical Oxygen Demand (COD) and Biochemical Oxygen Demand (BOD) levels) but contain high concentrations of salt. The current European legislation only allows disposing of these effluents directly into the waterways with previous treatment, which is laborious and expensive. Therefore, as much as possible, these by-products are reutilized as animal feed material and, if not applicable, used as fertilizers adding phosphorus, potassium, nitrogen, and other nutrients to the soil. Finding biological alternatives to revalue dairy by-products is of crucial interest in order to improve the utilization of dry dairy matter and reduce the environmental impact of every litre of milk produced. Debaryomyces hansenii is a halotolerant non-conventional yeast with high potential for this purpose. It presents some beneficial traits – capacity to metabolize a variety of sugars, tolerance to high osmotic environments, resistance to extreme temperatures and pHs – that make this yeast a well-suited option to grow using complex feedstock, such as industrial waste, instead of the traditional commercial media. In this work, we study for the first time D. hansenii's ability to grow and produce a recombinant protein (YFP) from dairy saline whey by-products. Cultivations at different scales (1.5, 100 and 500 ml) were performed without neither sterilizing the medium nor using pure water. Our results conclude that D. hansenii is able to perform well and produce YFP in the aforementioned salty substrate. Interestingly, it is able to outcompete other microorganisms present in the waste without altering its cell performance or protein production capacity.     

Production of lysozyme-enriched biomass from cheese industry by-products

Cheese whey and cottage cheese whey are by-products of the milk and cheese industry, resulting from the production of cheese and cottage cheese (ricotta) from milk. They are still rich in organic substances and cannot be discarded into the environment without proper treatment. Whey and cottage cheese whey were used as culture media for some strains of the yeast Kluyveromyces lactis, transformed with the human lysozyme gene. It was found that the yeast strains grew well in both media and produced a considerable amount of recombinant protein. Production kinetics showed that the human lysozyme was produced in a greater amount within 36 h of fermentation (125 micrograms ml-1 vs 25 micrograms ml-1 in the control) than in the synthetic commercial media used for strain preparation and characterization. The recombinant protein produced was actually shown to be the human lysozyme, using renaturing SDS-PAGE and Western blot techniques. While producing recombinant protein, the Kluyveromyces strain cleared the cottage cheese whey of most organic substances and produced a considerable amount (almost 3%) of lysozyme-enriched useful biomass.     

Recovery of phenolic antioxidants from wine industry by-products

The recovery process of the phenolic compounds contained in the wine industry by-products and their antioxidant activity were examined in this work. To this purpose, a combined process of liquid and supercritical solvent extraction was employed. At first the effect of various process parameters of the liquid solvent extraction--the type of solvent and the pretreatment of the raw material (composition (skins, seeds, stems) and crushing)--on the antioxidant activity of the extract was examined. It was shown that an extract of a higher antioxidant activity was obtained by using ethyl acetate as solvent and raw material free of stems. These extracts were further treated with supercritical carbon dioxide (SC CO(2)) at various extraction pressures, which resulted in their significant enrichment in phenolic compounds and the improvement of their antioxidant and organoleptic properties, especially at pressures higher than 100 bar. The antioxidant activity of the products was determined by using the Rancimat method, as well as a simple and not time-consuming free radical method. It was proved that both the ethyl acetate extracts and those treated with SC CO(2) had antioxidant activity comparable to that of antioxidants commonly used in industry, that is butylhydroxytoluene (BHT), a synthetic antioxidant, and Rosemary extract, a widely known natural one.     

Fungal production of citric acid

Citric acid is the principal organic acid found in citrus fruits. To meet increasing demands it is produced from carbohydrate feedstock by fermentation with the fungus Aspergillus niger and the yeasts of Candida spp. Effect of various fermentation conditions and the biochemistry of citric acid formation by A. niger have been discussed. Commercially citric acid is produced by surface, submerged and solid state fermentation techniques. Recovery of pure acid from fermentation broth is done primarily by precipitation with lime and also by solvent extraction.     

Utilization of by-products from ethanol production as substrate for biogas production

The aims of this work were to determine the specific biogas yields of steam-exploded sugarcane straw and bagasse as well as to estimate their energy potential under Brazilian conditions. Steam-explosion was carried out under different time and temperature conditions. The specific biogas yields were analyzed in batch-tests according to VDI 4630.Results have shown that steam-explosion pre-treatment increased the specific biogas yields of straw and bagasse significantly compared to the untreated material. The utilization of these by-products can contribute to 5% of the total energy consumption and thereby higher energy independence in Brazil. Further efforts in defining the optimum pretreatment conditions with steam-explosion as well as implementing this technology in large scale plants should be made.     

Biorefinery cascade processing for creating added value on tomato industrial by-products from Tunisia

Background

In today’s consumer perception of industrial processes and food production, aspects like food quality, human health, environmental safety, and energy security have become the keywords. Therefore, much effort has been extended toward adding value to biowastes of agri-food industries through biorefinery processing approaches. This study focused, for the first time, on the valorization of tomato by-products of a Tunisian industry for the recovery of value-added compounds using biorefinery cascade processing.

Results

The process integrated supercritical CO₂ extraction of carotenoids within the oil fractions from tomato seeds (TS) and tomato peels (TP), followed by a batch isolation of protein from the residues. The remaining lignocellulosic matter from both fractions was then submitted to a liquid hot water (LHW) hydrolysis. Supercritical CO₂ experiments extracted 5.79% oleoresin, 410.53 mg lycopene/kg, and 31.38 mg β-carotene/kg from TP and 26.29% oil, 27.84 mg lycopene/kg, and 5.25 mg β-carotene/kg from TS, on dry weights. Protein extraction yields, nearing 30% of the initial protein contents equal to 13.28% in TP and 39.26% in TS, revealed that TP and TS are a rich source of essential amino acids. LHW treatment run at 120–200 °C, 50 bar for 30 min showed that a temperature of 160 °C was the most convenient for cellulose and hemicellulose hydrolysis from TP and TS, while keeping the degradation products low.

Conclusions

Results indicated that tomato by-products are not only a green source of lycopene-rich oleoresin and tomato seed oil (TSO) and of protein with good nutritional quality but also a source of lignocellulosic matter with potential for bioethanol production. This study would provide an important reference for the concept and the feasibility of the cascade fractionation of valuable compounds from tomato industrial by-products.

Open image in new window

Graphical abstract Schema of biorefinery cascade processing of tomato industrial by-products toward isolation of valuable fractions.

     

Optimization of multienzyme production by two mixed strains in solid-state fermentation

F₃ and F₄ strains of Aspergillus niger were screened from five strains of fungi to produce multienzyme preparations (containing cellulase, hemicellulase, glucoamylase, pectinase, and acidic proteinase) as dietary supplementation. Enzyme activities indicated that 1:4 (F₃ to F₄) was the optimum mixture proportion, and 0.3% (W/W) was the preferable pitching rate. In bran mash containing 54.5% (W/W) water, F₃ and F₄ could produce the supplementation better when cultured 30 to 36 h at 30 °C. Monofactorial and orthogonal experiments were performed to optimize media. Results of the variance and range analysis showed that the optimum medium contained 80 g of bran, 20 g of cottonseed powder, 1 g of (NH₄)₂SO₄, and 0.1 g of KH₂PO₄. When F₃ and F₄ strains were cultured in the optimum medium containing 54.5% (W/W) water, the activity of cellulase, hemicellulase, glucoamylase, pectinase, and acidic proteinase reached 996; 15,863; 13,378; 7,621; and 5,583 U/g, respectively.     

Effect of ultrasound and addition of bacterial product on hydrolysis of by-products from the meat-processing industry

The hydrolysis efficiency of ultrasound (US) and bacterial product (BP; Liquid Certizyme 5™) was studied as a pre-treatment for anaerobic digestion (AD) of four animal by-products (ABP) from the meat-processing industry (digestive tract content + drumsieve waste, dissolved air flotation (DAF) sludge, grease trap sludge and the mixture of these). The change in volatile solids (VS) based hydrolysis parameters was screened using different specific US energy (Es) inputs (1000-14,000 kJ/kg of total solids (TS)) and durations of BP treatment (3-48 h). The Es of 6000-8500 kJ/kg TS increased most extensively the soluble chemical oxygen demand (COD_sol) and VS ratio, by 45-290%, and soluble nitrogen (N_sol) by 44-99%. Simultaneously, the average particle size (APS) decreased by 45-75%, from the original value. With BP, the highest increases in COD_sol/VS (29-130%), N_sol (35-63%) and decline in APS (40-70%) depended on the raw material (digestive tract content + drumsieve waste: 3 and 24 h; DAF sludge: 3 and 6 h, mixture: 12 and 48 h). BP apparently was more effective at hydrolyzing smaller cellulose particles, while US appeared to enhance the degradation of grease-containing cells.     

Fungal production of volatiles during growth on fiberglass.

Acoustic and thermal fiberglass insulation materials used in heating, ventilation, and air-conditioning systems were colonized with fungi in laboratory chambers. The mixed fungal population, principally Aspergillus versicolor, Acremonium obclavatum, and Cladosporium herbarum, produced odoriferous volatiles, including 2-ethyl hexanol, cyclohexane, and benzene. These volatiles may be related to poor indoor air quality and the sick building syndrome.     

Biopolymers production from mixed cultures and pyrolysis by-products

Polyhydroxyalkanoates (PHAs) production from low value substrates and/or byproducts represents an economical and environmental promising alternative to established industrial manufacture methods. Bio-oil resulting from the fast-pyrolysis of chicken beds was used as substrate to select a mixed microbial culture (MMC) able to produce PHA under feast/famine conditions. In this study a maximum PHA content of 9.2% (g/g cell dry weight) was achieved in a sequencing batch reactor (SBR) operated for culture selection. The PHA obtained with bio-oil as a carbon source was a copolymer composed by 70% of hydroxybutyrate (HB) and 30% of hydroxyvalerate (HV) monomers. Similar results have been reported by other studies that use real complex substrates for culture selection indicating that bio-oil can be a promising feedstock to produce PHAs using MMC. To the best of our knowledge this is the first study that demonstrated the use of bio-oil resulting from fast pyrolysis as a possibly feedstock to produce short chain length polyhydroxyalkanoates.     

Induction of in vivo DNA adducts by 4 industrial by-products in the rat-lung-cell system

Benz[a]anthracene (BA), dibenz[a,h]anthracene (DBA), dibenzo[a,i]pyrene (DBP), and dibenz[a,h]acridine (DBAC) are by-products found in many industrial wastes and emissions. Workers in the related occupational settings are potentially exposed to these substances through inhalation. In the present study, induction of DNA adducts in vivo by these chemicals was investigated using ³²P-postlabeling analysis in the rat-lung-cell system. The potency of DNA-adduct inducing activity was also compared to that of two cytogenetic endpoints i.e., sister-chromatid exchange (SCE) and micronucleus formation. Via intratracheal instillation, male CD rats (6/group) were dosed 3 times with BA, DBA, DBP or DBAC in a 24-h interval. Lung cells were enzymatically separated and used to determine the frequency of DNA adducts, SCE and micronuclei. Results show that all 4 test compounds induced DNA adducts, SCEs, and micronuclei in the rat-lung cell in vivo and that the postlabeling DNA adduct assay detected genotoxic activity at lower dose levels than the two cytogenetic assays. These findings suggest that BA, DBA, DBP or DBAC are rat pulmonary genetoxicants and the DNA-adduct assay is more sensitive than SCE or micronucleus assays for detecting the pulmonary genotoxicity of these industrial PAHs in the in vivo rat-lung-cell system.     

Fungal fermentation on anaerobic digestate for lipid-based biofuel production

Background

Anaerobic digestate is the effluent from anaerobic digestion of organic wastes. It contains a significant amount of nutrients and lignocellulosic materials, even though anaerobic digestion consumed a large portion of organic matters in the wastes. Utilizing the nutrients and lignocellulosic materials in the digestate is critical to significantly improve efficiency of anaerobic digestion technology and generate value-added chemical and fuel products from the organic wastes. Therefore, this study focused on developing an integrated process that uses biogas energy to power fungal fermentation and converts remaining carbon sources, nutrients, and water in the digestate into biofuel precursor-lipid.

Results

The process contains two unit operations of anaerobic digestion and digestate utilization. The digestate utilization includes alkali treatment of the mixture feed of solid and liquid digestates, enzymatic hydrolysis for mono-sugar release, overliming detoxification, and fungal fermentation for lipid accumulation. The experimental results conclude that 5 h and 30 °C were the preferred conditions for the overliming detoxification regarding lipid accumulation of the following fungal cultivation. The repeated-batch fungal fermentation enhanced lipid accumulation, which led to a final lipid concentration of 3.16 g/L on the digestate with 10% dry matter. The mass and energy balance analysis further indicates that the digestate had enough water for the process uses and the biogas energy was able to balance the needs of individual unit operations.

Conclusions

A fresh-water-free and energy-positive process of lipid production from anaerobic digestate was achieved by integrating anaerobic digestion and fungal fermentation. The integration addresses the issues that both biofuel industry and waste management encounter—high water and energy demand of biofuel precursor production and few digestate utilization approaches of organic waste treatment.