Production of alkaline protease by Pseudomonas aeruginosa using proteinaceous solid waste generated from leather manufacturing industries

Animal fleshing (ANFL), the major proteinaceous solid waste discharged from leather manufacturing industries was used as the substrate for the production of alkaline protease by Pseudomonas aeruginosa. The strain isolated from the tannery wastewater was selected for its ability to produce protease of activity in the range 1160-1175 U ml(-1). The selective removal of non-fibrillar proteins such as albumin and globulin from ANFL by the protease enzyme during the progress of hydrolysis was confirmed using scanning electron microscopy (SEM). The breakdown of ANFL was also confirmed from the amino acid release into the fermentation medium by P. aeruginosa using high performance liquid chromatography (HPLC).     

Acidogenic fermentation of proteinaceous solid waste and characterization of different bioconversion stages and extracellular products

The purpose of this study was to investigate hydrolysis of animal fleshing (ANFL), a predominant tannery solid waste and to characterize the acetogenic fermentation products of anaerobic digestion. The acidogenic digestibility of the tannery solid wastes were evaluated up to 120 h using batch anaerobic digestion tests performed under mesophilic condition at 37 degrees C. The degradation of ANFL starts with non-fibrillar proteins and proceeds with fibrillar proteins. The release of aliphatic amino acid in the early stages of hydrolysis (24 h) and followed by aromatic amino acids (24-72 h) were evidenced by HPLC analysis. The maximum production of propionic and valeric acid were observed in 72 h followed by rapid increase in acetic acid in 96 h using GC-MS. Breakdown of ANFL and formations of other metabolites were evidenced by FT-IR and (1)H-NMR spectroscopy.     

Effects of Tween 80 and rhamnolipid on the extracellular enzymes of Penicillium simplicissimum isolated from compost

Extracellular enzymes of microorganisms play an important role in the decomposition of macromolecules in the composting process. In this study, the effects of Tween 80 and rhamnolipid on the extracellular amylase, carboxymethyl cellulose enzyme (CMCase), xylanase and protease of Penicillium simplicissimum isolated from compost were investigated during solid-state fermentation. The results showed that the enzyme activities of amylase, CMCase and xylanase were increased by Tween 80 and rhamnolipid, which, however, had a negative effect on the protease production. The stimulative effects on the three enzymes were quite different during the whole fermentation process. Tween 80 and rhamnolipid also increased the fungal biomass slightly. As a result of the enhanced enzyme activities, the organic matter were also improved to different extents by both surfactants, and the decomposition rates of hemicellulose and cellulose were increased about 8.0% and 11.6% by Tween 80 at best, respectively, as well as 5% and 5.5% by rhamnolipid.     

Improvement of direct ethanol fermentation from woody biomasses by the Antarctic basidiomycetous yeast, Mrakia blollopis, under a low temperature condition

The Antarctic basidiomycetous yeast Mrakia blollopis SK-4 can quite uniquely ferment various sugars under low temperature conditions. When strain SK-4 fermented lignocellulosic biomass using the direct ethanol fermentation (DEF) technique, approximately 30% to 65% of the theoretical ethanol yield was obtained without and with the addition of the non-ionic surfactant Tween 80, respectively. Therefore, DEF from lignocellulosic biomass with M. blollopis SK-4 requires the addition of a non-ionic surfactant to improve fermentation efficiency. DEF with lipase converted Eucalyptus and Japanese cedar to 12.6 g/l, and 14.6 g/l ethanol, respectively. In the presence of 1% (v/v) Tween 80 and 5 U/g-dry substrate lipase, ethanol concentration increased about 1.4- to 2.4-fold compared to that without Tween 80 and lipase. We therefore consider that the combination of M. blollopis SK-4 and DEF with Tween 80 and lipase has good potential for ethanol fermentation in cold environments.     

Direct ethanol fermentation from lignocellulosic biomass by Antarctic basidiomycetous yeast Mrakia blollopis under a low temperature condition

Antarctic basidiomycetous yeast Mrakia blollopis SK-4 has unique fermentability for various sugars under a low temperature condition. Hence, this yeast was used for ethanol fermentation from glucose and also for direct ethanol fermentation (DEF) from cellulosic biomass without/with Tween 80 at 10 °C. Maximally, 48.2 g/l ethanol was formed from 12% (w/v) glucose. DEF converted filter paper, Japanese cedar and Eucalyptus to 12.2 g/l, 12.5 g/l and 7.2 g/l ethanol, respectively. In the presence of 1% (v/v) Tween 80, ethanol concentration increased by about 1.1–1.6-fold compared to that without Tween 80. This is the first report on DEF using cryophilic fungi under a low temperature condition. We consider that M. blollopis SK-4 has a good potential for ethanol fermentation in cold environments.     

Fermentation of acid hydrolysates from olive-tree pruning debris by Pachysolen tannophilus

The influence of the type and concentration of acid in the hydrolysis process and its effect on the subsequent fermentation by Pachysolen tannophilus (ATCC 32691) to produce ethanol and xylitol was studied. The hydrolysis experiments were performed using hydrochloric, sulphuric and trifluoroacetic acids in concentrations ranging from 0.1 to 1.0 N, a temperature of 90 degrees C, and a time of 240 min. The fermentation experiments were conducted on a laboratory scale in a batch-culture reactor at pH 4.5 and 30 degrees C. The hydrolysis with the highest acid concentration produced the complete solubilization of hemicellulose to monosaccharides. The highest values for the specific rate of ethanol production were registered in cultures hydrolyzed with trifluoroacetic acid, and values were found to decrease as the acid concentration increased. The highest values of overall ethanol yields ( [Formula: see text] = 0.37 kg kg(-1)) were also found in the fermentation of the hydrolysates of trifluoroacetic acid.     

Quantification of intra- and extra-cellular thermophilic lipase/esterase production by Thermus sp.

Four Thermus strains produced lipolytic activity when grown in liquid medium for 30 h at 70 degrees C. The highest total lipase/esterase activity (57 U l(-1)) was in Thermus aquaticus YT-1, followed by Thermus thermophilus HB27 and HB8 (33 and 25 U l(-1), respectively), and finally by Thermus sp. (16 U l(-1)). Extra-cellular activity was detected in T. aquaticus YT-1 and T. thermophilus HB27 (33 and 17 U l(-1)). All enzymes were stable at 80 degrees C over 30 min, and their activity towards fatty acid esters increased as substrate chain-length diminished (i.e. hydrolysis rate was up to 6-fold higher on p-nitrophenyl caproate than on laurate).     

Quantitative characterisation of the morphology of Trichoderma harzianum cultured in shake-flasks and containing Tween 40

Image analysis was used to measure the effect of Tween 40 on the morphology of Trichoderma harzianum. The percentage of pellets was maximal (93%) with Tween at 0.2 ml l(-1), whereas the maximal proportion of dispersed mycelia (40%) was with Tween at 1.6 ml l(-1). The particle median diameter decreased from 2 to 0.5 mm as Tween concentration was increased and was inversely correlated to the biomass dry weight. Adding pre-determined aliquots of Tween 40 to the culture medium can be used to define the morphological characteristics of Trichoderma harzianum in submerged shake-flask culture.     

Surfactant-enhanced anaerobic acidogenesis of Canna indica L. by rumen cultures

Polyoxyethylene sorbitan monoolate (Tween 80) was used to enhance the anaerobic acidogenesis of Canna indica L. (canna) by rumen culture in this study. Dose of Tween 80 at 1 ml/l enhanced the volatile fatty acids (VFA) production from the acidogenesis of canna compared to the control. However, Tween 80 at higher dosages than 5 ml/l inhibited the rumen microbial activity and reduced the VFA yield. Response surface methodology was successfully used to optimize the VFA yield. A maximum of VFA yield of 0.147 g/g total solids (TS) added was obtained at canna and Tween 80 concentrations of 6.3g TS/l and 2.0 ml/l, respectively. Dosage of Tween 80 at 1-3.75 ml/l reduced the unproductive adsorption of microbes or enzymes on the lignin part in canna and increased microbial activity. A high VFA production was achieved from canna presoaked with Tween 80, suggesting that the structure of canna was disrupted by Tween 80.     

Benefits from tween during enzymic hydrolysis of corn stover

Corn stover is a potential substrate for fermentation processes. Previous work with corn stover demonstrated that lime pretreatment rendered it digestible by cellulase; however, high sugar yields required very high enzyme loadings. Because cellulase is a significant cost in biomass conversion processes, the present study focused on improving the enzyme efficiency using Tween 20 and Tween 80; Tween 20 is slightly more effective than Tween 80. The recommended pretreatment conditions for the biomass remained unchanged regardless of whether Tween was added during the hydrolysis. The recommended Tween loading was 0.15 g Tween/g dry biomass. (The critical relationship was the Tween loading on the biomass, not the Tween concentration in solution.) The 72-h enzymic conversion of pretreated corn stover using 5 FPU cellulase/g dry biomass at 50 degrees C with Tween 20 as part of the medium was 0.85 g/g for cellulose, 0.66 g/g for xylan, and 0.75 for total polysaccharide; addition of Tween improved the cellulose, xylan, and total polysaccharide conversions by 42, 40, and 42%, respectively. Kinetic analyses showed that Tween improved the enzymic absorption constants, which increased the effective hydrolysis rate compared to hydrolysis without Tween. Furthermore, Tween prevented thermal deactivation of the enzymes, which allows for the kinetic advantage of higher temperature hydrolysis. Ultimate digestion studies showed higher conversions for samples containing Tween, indicating a substrate effect. It appears that Tween improves corn stover hydrolysis through three effects: enzyme stabilizer, lignocellulose disrupter, and enzyme effector. Copyright 1998 John Wiley & Sons, Inc.     

Rhizoctonia solani, an elicitor of 6-pentyl-α-pyrone production by Trichoderma harzianum in a two liquid phases, extractive fermentation system

6-pentyl-alpha-pyrone (6PP) production by Trichoderma harzianum, in an extractive fermentation system, was elicitated by Rhizoctonia solani. The extent of 6PP elicitation was related to the state of Rhizoctonia and to the Trichoderma inoculum type. The use of non-viable Rhizoctonia solani mycelium in mycelium-inoculated Trichoderma harzianum culture, yielded the maximal 6PP production (474 mg l(-1)) compared to control cultures (147 mg l(-1)) and decreased the process time from 192 to 96 h.     

Thermophilic protease production by Streptomyces sp. 594 in submerged and solid-state fermentations using feather meal

AIMS: Protease production by Streptomyces sp. 594 in submerged (SF) and solid-state fermentation (SSF) using feather meal, an industrial poultry residue, and partial characterization of the crude enzyme. METHODS AND RESULTS: Streptomyces sp. 594 produced proteases in SF (7.2 +/- 0.2 U ml(-1)) and SSF (15.5 +/- 0.41 U g(-1)), with pH increase in both media. Considering protease activity, values obtained in the liquid extract after SSF (6.3 +/- 0.17 U ml(-1)) were lower than those from SF. The proteases, which belong to serine and metalloproteinase classes, were active over a wide range of pH (5.0-10.0) and high temperatures (55-80 degrees C). Strain 594 was also able to degrade feather in agar and liquid media. Keratinase activity (80 U l(-1)) also confirmed the keratin degrading capacity of this streptomycete. CONCLUSIONS: Proteases produced using residues from poultry industry have shown interesting properties for industrial purposes. SIGNIFICANCE AND IMPACT OF THE STUDY: As far as we are concerned, this is the first contribution towards the production of thermophilic protease by a streptomycete in SSF using a keratinous waste.     

A mechanistic study of the enhancing effect of Tween 80 on the mycelial growth and exopolysaccharide production by Pleurotus tuber-regium

Several new observations related to the enhancement effect of Tween 80 on the mycelial growth and exopolysaccharide production by the submerged fermentation of Pleurotus tuber-regium were reported in the present study. Firstly, it was found that the addition of Tween 80 on the 5th day could significantly increase the glucose consumption rate at the later stage of the fermentation compared to the control. Secondly, addition of Tween 80 could maintain the intact structure of the mycelial pellets of P. tuber-regium with little signs of disintegration as observed under microscope and kept the pH value of the fermentation broth at an acidic level lower than that of the control. Thirdly, the oleic acid (C18:1) composition in the mycelial cell membrane was significantly increased from 2.6% (in the control) to 18.5% (with addition of Tween 80) coincided with a decrease in the concentration of Tween 80 in the culture medium. These new findings provide some important insight to the elucidation of the detailed mechanism by which Tween 80 is used as a stimulatory agent in the submerged fermentation of mushroom mycelium.     

Identification and characterization of fermentation inhibitors formed during hydrothermal treatment and following SSF of wheat straw

A pilot plant for hydrothermal treatment of wheat straw was compared in reactor systems of two steps (first, 80°C; second, 190–205°C) and of three steps (first, 80°C; second, 170–180°C; third, 195°C). Fermentation (SSF) with Sacharomyces cerevisiae of the pretreated fibers and hydrolysate from the two-step system gave higher ethanol yield (64–75%) than that obtained from the three-step system (61–65%), due to higher enzymatic cellulose convertibility. At the optimal conditions (two steps, 195°C for 6 min), 69% of available C6-sugar could be fermented into ethanol with a high hemicellulose recovery (65%). The concentration of furfural obtained during the pretreatment process increased versus temperature from 50 mg/l at 190°C to 1,200 mg/l at 205°C as a result of xylose degradation. S. cerevisiae detoxified the hydrolysates by degradation of several toxic compounds such as 90–99% furfural and 80–100% phenolic aldehydes, which extended the lag phase to 5 h. Acetic acid concentration increased by 0.2–1 g/l during enzymatic hydrolysis and 0–3.4 g/l during fermentation due to hydrolysis of acetyl groups and minor xylose degradation. Formic acid concentration increased by 0.5–1.5 g/l probably due to degradation of furfural. Phenolic aldehydes were oxidized to the corresponding acids during fermentation reducing the inhibition level.     

Purification of extracellular acid protease and analysis of fermentation metabolites by Synergistes sp. utilizing proteinaceous solid waste from tanneries

The untanned proteinaceous tannery solid waste, animal fleshing (ANFL), was used as a substrate for acid protease production by Synergistes sp. The strain was isolated from an anaerobic digester used for the treatment of tannery solid waste and was selected for its enhanced protease production at activity 350-420 U/ml. The optimum pH was in the acidic range of 5.5-6.5 and optimum temperature was in mesophilic range of 25-35 degrees C. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and the zymogram analyses of the purified protein indicated an estimated molecular mass of 60 kDa. This protease could be classified as aspartic protease based on its inhibition by aspartate type protease inhibitor pepstatin and on non-inhibition by 1,10-phenanthroline, EDTA, EGTA and phenylmethylsulfonyl fluoride. The degradation of ANFL was confirmed by Gas Chromatography-Mass Spectroscopy (GC-MS), Proton Nuclear Magnetic Resonance Spectroscopy (H1 NMR) and Scanning Electron Microscopy (SEM) analyses. In this study we found that the activity of acid protease depended on factors such as calcium concentration, pH and temperature. Based on these lines of evidence, we postulate that this protease is a highly catalytic novel protease of its type.     

Optimisation of media and cultivation conditions for L(+)(S)-lactic acid production by Lactobacillus casei NRRL B-441

Process variables and concentration of carbon in media were optimised for lactic acid production by Lactobacillus casei NRRL B-441. Lactic acid yield was inversely proportional to initial glucose concentration within the experimental area (80-160 g l(-1)). The highest lactic acid concentration in batch fermentation, 118.6 g l(-1), was obtained with 160 g 1(-1) glucose. The maximum volumetric productivity, 4.4 g 1(-1) h(-1) at 15 h, was achieved at an initial glucose concentration of 100 g l(-1). Similar lactic acid concentrations were reached with a fedbatch approach using growing cells, in which case the fermentation time was much shorter. Statistical experimental design and response surface methodology were used for optimising the process variables. The temperature and pH optima for lactic acid production were 35 degrees C, pH 6.3. Malt sprout extract supplemented with yeast extract (4 g l(-1)) appeared to be an economical alternative to yeast extract alone (22 g l(-1)) although the fermentation time was a little longer. The results demonstrated both the separation of the growth and lactic acid production phases and lactic acid production by non-growing cells without any nutrient supplements. Resting L. casei cells converted 120 g l(-1) glucose to lactic acid with 100% yield and a maximum volumetric productivity of 3.5 g l(-1) h(-1).     

Entomotoxicity,protease and chitinase activity of Bacillus thuringiensis fermented wastewater sludge with a high solids content

This study investigated the production of biopesticides, protease and chitinase activity by Bacillus thuringiensis grown in raw wastewater sludge at high solids concentration (30 g/L). The rheology of wastewater sludge was modified with addition of Tween-80 (0.2% v/v). This addition resulted in 1.6 and 1.3-fold increase in cell and spore count, respectively. The maximum specific growth rate (μ_max) augmented from 0.17 to 0.22 h⁻¹ and entomotoxicity (Tx) increased by 29.7%. Meanwhile, volumetric mass transfer coefficient (k_La) showed marked variations during fermentation, and oxygen uptake rate (OUR) increased 2-fold. The proteolytic activity increased while chitinase decreased for Tween amended wastewater sludge, but the entomotoxicity increased. The specific entomotoxicity followed power law when plotted against spore concentration and the relation between Tx and protease activity was linear. The viscosity varied and volume percent of particles increased in Tween-80 amended wastewater sludge and particle size (D₅₀) decreased at the end of fermentation. Thus, there was an increase in entomotoxicity at higher suspended solids (30 g/L) as Tween addition improved rheology (viscosity, particle size, surface tension); enhanced maximum growth rate and OUR.     

The stimulatory effects of surfactants on composting of waste rich in cellulose

The chemical surfactant Tween 80 and biosurfactant rhamnolipid were respectively added to the composting substrate, a mixture of rice straw and bran, and their effects on the composting process were investigated. Samples were analysed for microbial communities of bacteria, actinomycetes and fungi, carboxymethylcellulose hydrolysis (CMCase) and xylanase activities, cellulose and hemicellulose fractions, water-soluble carbon (WSC) contents in the substrates, organic matter contents and pH values during the composting process. The results showed that both Tween 80 and rhamnolipid had slight stimulatory effects on the microbial populations of bacteria, actinomycetes and fungi. In addition, rhamnolipid increased the peak xylanase activity 15% higher than that of the control, while Tween 80 increased the maximum CMCase activity 35% higher than that of the control. As a result of the increased enzyme activities, treatments with Tween 80 and rhamnolipid were of higher WSC contents than the control during the whole composting process. Accordingly, the composting process was accelerated by the surfactants, since the organic matter was decomposed more quickly and the breakdown of cellulose and hemicellulose was better in the treatments with Tween 80 and rhamnolipid.     

Influence of amino nitrogen in the culture medium enhances the production of δ-endotoxin and biomass of <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> var. <Emphasis Type="Italic">israelensis</Emphasis> for the large-scale production of the mosquito control agent

We reported here the role of amino nitrogen in the commercial production of Bacillus thuringiensis var. israelensis media design. The insect pathogen B. thuringiensis var. israelensis was cultured in different media containing varying initial levels of amino nitrogen sources obtained from three different commercial venders. The biomass, mosquito larval toxicity and spore count produced were measured during the fermentation process. The results showed that the higher level of initial amino nitrogen concentrations in the medium led to higher yield of biomass (dry weight 4.78 g l(-1)), larvicidal activity (LC(50) 18.52 ng ml(-1)) and spore count (3.24 x 10(11) CFU ml(-1)). Similarly decreasing the initial amino nitrogen concentration in the medium led to a decreased biomass (dry weight 1.64 g l(-1)), larvicidal activity (LC(50) 27.01 ng ml(-1)) and spore count (3.7 x 10(10) CFUml(-1)).     

Biotechnological production of l(+)-lactic acid from wood hydrolyzate by batch fermentation of Enterococcus faecalis

The inhibition of lactic acid fermentation by wood hydrolyzate was decreased (approx. 20%) by adaptation of Enterococcus faecalis RKY1 to wood hydrolyzate-based medium whereby lactic acid productivity and cell growth were enhanced by 0.5 g l(-1) h(-1) and 2.1 g l(-1), respectively. When the diluted or concentrated wood hydrolyzate (equivalent to 25-100 g glucose l(-1)) was supplemented with 15 g yeast extract l(-1), 24-93 g lactic acid l(-1) was produced at a rate between 1.7 g l(-1) h(-1) and 3.2 g l(-1) h(-1).