A new practical approach for the biological treatment of a mixture of cheese whey and white wastewaters using Kefir grains.

Kefir grains are a microbial consortium of different genera of bacteria and yeasts. In this study, the performance of Tunisian Kefir grains during the biological treatment of a mixture of Gouda cheese whey and white wastewaters (GCW) in ratio 1:1 with very high organic matter concentration is investigated. The biological process was evaluated and optimized through the response surface methodology. Under the optimum conditions, Kefir grains concentration of 1.02%, temperature at 36.68 °C, and incubation time of 5.14 days, the removal efficiencies of COD, PO43-, and NO3- were 87, 37.48, and 39.5%, respectively. Interestingly, the reusability tests of the grains proved not only their high resistance to harsh environmental conditions but also their great potential for more practical applications. Particularly, different strains were isolated from the grains and identified as Kluyveromyces marxianus, Lactoccocus lactis, Lactobacillus kefiri, and Bacillus spp. using 16S rDNA sequence analysis and rep-PCR fingerprinting. At the biological level, the raw GCW (RGCW) has a negative impact on the Hordeum vulgare both on seed germination, and on the growth parameters of seedlings. Interestingly, after Kefir grains treatment, the treated GCW (TGCW) allow a seedlings growth and germination rate similar to those soaked in water.

Mixed culture of Lactococcus lactis and Kluyveromyces marxianus isolated from kefir grains for pollutants load removal from Jebel Chakir leachate.

The wastewater from the dumping site usually contains high pollutant levels. Biological process and physico-chemical treatments are among several technologies for wastewater treatment. Using microorganisms in the treatment of landfill leachate is an emerging research issue. Furthermore, bioremediation is a feasible approach for pollutants removal from landfill leachate which would provide an efficient way to resolve the issue of landfill leachate. In this study, the performance of yeast and bacteria isolated from kefir grains was assessed for landfill leachate treatment. Kefir grains microbial composition was evaluated by molecular approaches (Rep-PCR and 16S rRNA gene sequencing). The obtained outcomes denoted that high concentrations of lactic acid bacteria and yeast populations (over 107  CFU/ml) were found in the kefir grains and were essentially composed of Lactococcus lactis, Lactobaccillus kefirien, bacillus sp., L. lactis, and Kluyveromyces marxianus. The co-culture with 1% of inoculum size was demonstrated as the most efficient in the degradation of different contaminants. The overall abatement rate of chemical oxygen demand (COD), ammonium nitrogen ( NH 4 + - N ), and salinity were 75.8%, 85.9%, and 75.13%, respectively. The bioremediation process resulted in up of 75% removal efficiency of Ni and Cd, and a 73.45%, 68.53%, and a 58.17% removal rates of Cu, Pb, and Fe, respectively. The research findings indicate the performance of L. lactis and K. marxianus co-culture isolated from kefir grains for the bioremediation of LFL. PRACTITIONER POINTS: Isolation and identification of microorganisms from kefir grains was carried out. Biological treatment of LFL using monoculture of (Lactoccocus lactis; Kluyveromyces marxianus) and co-culture (5% of L. lactis and 5% K. marxianus) has been performed. Biological treatment using co-culture strain is an effective approach to remove organic matter, NH 4 + - N and heavy metals.

Purification and characterization of seven bioactive compounds from the newly isolated Streptomyces cavourensis TN638 strain via solid-state fermentation.

The strain TN638 was isolated from Tunisian soil contaminated with industrial wastewater and selected for its potent antimicrobial activity against the tested Gram positive bacteria: Staphylococcus aureus (S. aureus) ATCC 6538 and Listeria monocytogenes (L. monocytogenes) ATCCC 19117, and Gram negative bacteria: Agrobacterium tumefaciens (A. tumefaciens) ATCC 23308 and Salmonella typhimurium (S. typhimurium) ATCC 14028 and fungi: Candida albicans (C. albicans) ATCC 10231, Rhizoctonia solani (R. solani) ATCC 58938 and Fusarium sp. Solide-state fermentation (SSF) dry crude extract of the TN638 strain presents a strong inhibitory activity notably against the phytopathogenic microorganism A. tumefaciens ATCC 23308 and the two pathogenic bacteria S. aureus ATCC 6538 and L. monocytogenes ATCCC 19117 with a zone of inhibition of 48, 34 and 34 mm respectively. According to the morphological characteristic, the complete 16S rRNA gene nucleotide sequence determination [1492 bp deposited in National Center of Biotechnology Information (NCBI) database under the accession no. LN854629.1; https://www.ncbi.nlm.nih.gov/nuccore/LN854629.1/], and the phylogenetic analysis, we can deduce that our isolate is an actinomycete bacterium belonging to the genus Streptomyces and the most closely related strain was Streptomyces cavourensis (S. cavourensis) NRRL 2740T (99.9%). We propose the assignment of our strain as Streptomyces cavourensis (S. cavourensis) TN638 strain. Work-up and purification of the strain extract using different chromatographic techniques afforded seven bio-compounds namely: Cyclo-(Leu-Pro) (1), Cyclo-(Val-Pro) (2), Cyclo-(Phe-Pro) (3), nonactin (4), monactin (5), dinactin (6) and trinactin (7). The chemical structures of compounds 1-7 were confirmed by nuclear magnetic resonance (NMR) 1D and 2D spectroscopy, mass spectrometry, and comparison with literature data. The three purified diketopiperazine (DKP) derivatives (1-3), demonstrated significant antibacterial activity against A. tumefaciens ATCC 23308 and S. typhimurium ATCC 14028. The four pure macrotetrolides (4-7), exhibited strong inhibitory effect against all tested Gram positive and Gram negative bacteria notably against A. tumefaciens ATCC 23308 and S. typhimurium ATCC 14028 with a minimum inhibitory concentration (MIC) around 8 µg/mL quite similar to that of ampicillin. Thus, we propose the use of the (SSF) active extract of the S. cavourensis TN638 strain as safe biological product to control disease caused by plant pathogen A. tumefaciens. Also, the purified active molecules produced by this strain could be used in pharmaceutical field.

A new insight into highly contaminated landfill leachate treatment using Kefir grains pre-treatment combined with Ag-doped TiO2 photocatalytic process.

This study investigates the performance of the combination of biological pre-treatment with Kefir grains (KGs) and photocatalytic process using Ag-doped TiO2 nanoparticles (NPs) for the simultaneous removal of toxic pollutants from landfill leachate (LFL). After 5 days of 1% (w/v) KGs pre-treatment at 37 °C, TOC, COD, NH4+-N, and PO43- removal rates were 93, 83.33, 70 and 88.25%, respectively. The removal efficiencies were found to be 100, 94, 62.5, 53.16 and 47.52 % for Cd, Ni, Zn, Mn and Cu, respectively. The optimal conditions of Ag-doped TiO2 photocatalytic process were optimized using Box-Behnken design and response surface methodology (BBD-RSM) to enhance the quality of pre-treated LFL. Interestingly, Ag-doped TiO2 photocatalytic process increases the overall removal efficiencies to 98, 96, 85 and 93% of TOC, COD, NH4+-N, and PO43-, respectively. Furthermore, the removal efficiency of toxic heavy metals was gradually improved. In addition, KGs and Ag-doped TiO2 exhibited excellent recyclability showing the potential of combined biological/photocatalytic process to treat hazardous LFL.

Valorization of residual soft drinks by baker's yeast production and insight for dairy wastewater whey incorporation.

Residuals are responsible for the polluting load increase of soft drink industry wastewater due to their high sugar contents. The present work proposes an upstream segregation of residuals to be biologically treated by the bioconversion of their carbohydrates content into baker's yeast biomass. Carbonated soft drinks (CSD) and nectars and juices (NJ) ranges were considered. Different incorporation ratios of NJ in the CSD (0-75%) have been investigated for balanced growth medium. Despite the nitrogen deficiency of media, results showed that NJ incorporation promoted the microbial growth. Media containing more than 50% of NJ exhibited ∼25% sugar-biomass conversion rates. The chemical oxygen demand (COD) of the media exceeded 70% at the end of fermentation. Moreover, valuable components were recovered by yeast production. Nutrient consumption rates varied from 65.4% for sugar and calcium content to in excess of 99% for protein and other minerals. In order to investigate an available and low-cost source of nitrogen for yeast production, partial substitution of the soft drink growth medium by bactofugate whey was evaluated. The soft drink-whey mixture medium fermentation resulted in 63% COD removal rate after 28 h. Meanwhile, the biomass production yield revealed an improvement of about 25% compared to the balanced soft drink medium (NJ50).

Novel approach for the use of dairy industry wastes for bacterial growth media production.

This work proposes a novel approach for the reuse and the recovery of dairy wastes valuable components. Thermal coagulation was performed for dairy effluents and the main responsible fraction for the organic matter content (protein and fat) was separated. Dairy curds were prepared for the formulation of bacterial growth media. Protein, sugar, fat and fatty acids contents have been assessed. Samples treated at 100 °C exhibited marked improvement in terms of protein (25-50%) recovery compared to those treated at 80 °C. Fatty acid analysis revealed the presence of unsaturated fatty acids (mainly oleic acid) that are essential to promote Lactobacillus growth. Previously isolated and identified bacterial strains from dairy wastes (Lactobacillus paracasei, Lactobacillus plantarum, Lactococcus lactis and Lactobacillus brevis) were investigated for their ability to grow on the formulated media. All the tested lactic acid bacteria exhibited greater bacterial growth on the formulated media supplemented with glucose only or with both glucose and yeast extract compared to the control media. By reference to the commercial growth medium, the productivity ratio of the supplemented bactofugate (B) and decreaming (D) formulated media exceeded 0.6 for L. paracasei culture. Whereas, the productivity ratio of the supplemented B medium was greater than 1 compared to the control medium for all the tested strains. As for the supplemented D medium, its productivity ratio was greater than 1 compared to the control medium for both L. paracasei and L. plantarum strains.

The use of newly isolated Streptomyces strain TN258 as potential biocontrol agent of potato tubers leak caused by Pythium ultimum.

An actinomycete strain designated TN258, was isolated from Tunisian Sahara soil and selected for its antagonistic activity, especially against Pythium ultimum (P. ultimum) causing potato tubers leak. Based on the results of cultural characteristic of TN258 strain, the 16S rRNA gene nucleotide sequence (1433 bp, accession n° HE600071) and the phylogenetic analysis, we propose the assignment of our new isolate bacterium as Streptomyces TN258 strain. After culture optimization, the inhibitory effect of TN258 free cell supernatant against P. ultimum was evaluated. As result, by application of 50% (v/v) from 25 mg ml-1 of concentration, mycelial growth was totally inhibited with hyphal destruction. At the same concentration, the oospores were distorted and the germination was completely stopped. In potato tubers, Streptomyces TN258 filtrated supernatant, applied 24 h before inoculation by P. ultimum (preventive treatment group) was able to significantly decrease pathogen penetration by 62% and to reduce the percentage of weight loss by 59.43%, in comparison with non-treated group.

Genetic Characterization of Lactic Acid Bacteria Isolated from Tunisian Milk Waste and their Antimicrobial Activity Against some Bacteria Implicated in Nosocomial Infections.

BACKGROUND: A total of 94 lactic acid bacteria (LAB) were isolated from Tunisian artisanal (Ricotta cheese's whey) and industrial (bactofugate) milk waste, identified and then screened for their antimicrobial activity against some bacteria implicated on nosocomial infections. OBJECTIVE: Bacterial genera and species identification was performed using molecular tools. The antimicrobial activity was tested against 7 strains of Gram-negative bacteria and 4 strains of Gram-positive bacteria as well as 6 yeasts. METHOD: The Crude extract was found to have a narrow antimicrobial spectrum on Gram-positive bacteria mainly Listeria monocytogenes. Among the strains which showed antibacterial activity, four were determined to be bacteriocins-producers. They were identified as Lactococcus lactis. RESULTS: Brain Heart Infusion (BHI) Agar was found more adapted than Man, Rogosa and Sharpe (MRS) to investigate the antimicrobial activity of L. actococcus lactis against L. isteria monocytogenes. The genetic determinants encoding the antimicrobial peptides were targeted by specific PCR. CONCLUSION: All L. lactis bacteriocin producing strains possessed the Nisine Z gene (nisZ) except for one, which contained both Nisine A and Nisine Z genes (nisA and nisZ). They have been identified as antilisterial agentS.

An antilisterial bacteriocin BacFL31 produced by Enterococcus faecium FL31 with a novel structure containing hydroxyproline residues.

A new bacterium called FL31, which was selected for its antimicrobial activity against the pathogenic bacterium Listeria monocytogenes, was identified as Enterococcus faecium and noted to produce an antibacterial proteinaceous substance (BacFL31). The active peptide from the cell-free supernatant of E. faecium FL31 was purified in four steps and the results revealed a single band with an estimated molecular mass of approximately 3.5 kDa. The N-terminal amino acid sequence of the purified BacFL31 "GLEESXGHXGQXGPXGPXGAXGP" (X = hydroxyproline) showed the presence of six hydroxyproline residues. It displayed a bactericidal mode of action against L. monocytogenes. Its application at 400 AU/g was also noted to constitute an effective approach for preventing the contamination and growth of the pathogenic bacterium L. monocytogenes during the storage of minced beef meat at 4 °C.

Characterization of the bacteriocin BacJ1 and its effectiveness for the inactivation of Salmonella typhimurium during turkey escalope storage.

During a screening programme for bacteriocin producers, a new lactic acid bacterium called J1, isolated from chicken gizzard, was noted to produce a bacteriocin (BacJ1) that inhibited Gram-positive and Gram-negative food-borne pathogens, especially Salmonella typhimurium, and it was, therefore, selected for identification and further study. The results, from 16S rRNA gene nucleotide sequencing (1583 pb accession no HE861352) and phylogenetic analysis, suggested that the new isolate be assigned as Weissella paramesenteroides J1. BacJ1 was purified from the culture supernatant of the J1 strain using heat treatment (15 min at 90 °C), 80% ammonium sulfate precipitation, and gel filtration (Sephadex G25). MALDI-TOF analyses revealed that BacJ1 had an exact molecular mass of 1881.036 Da. The analysis of the N-terminal sequence (GPAGPFGKLY) of this active peptide revealed no significant similarity to currently available antimicrobial peptides. The addition of pre-purified BacJ1, at a final concentration of 400 AU per gramme, may open new promising opportunities for the prevention of contamination from and growth of pathogenic bacteria, particularly S. typhimurium, during turkey escalope storage at 4 °C.

Purification and characterization of two polyhydroxyalcanoates from Bacillus cereus.

This work aimed to study the potential of 155 strains of Bacillus sp., isolated from a collection of Tunisian microorganisms, for polyhydroxyalcanoates production. The strains were submitted to a battery of standard tests commonly used for determining bioplastic properties. The findings revealed that two of the isolates, namely Bacillus US 163 and US 177, provided red excitations at a wavelength of approximately 543 nm. The polyhydroxyalcanoates produced by the two strains were purified. Gas chromatography-mass spectroscopy (GC-MS), Fourier transformed infrared spectroscopy (FTIR), and gel permeation chromatography (GPC) were used to characterize the two biopolymers. Bacillus US 163 was noted to produce a poly methyl-3-hydroxy tetradecanoic acid (P-3HTD) with an average molecular weight of 455 kDa, a completely amorphous homopolymer without crystallinity. The US 177 strain produced a homopolymer of methyl-3-hydroxy octadecanoic acid (P3-HOD) with an average molecular weight of 555 kDa. Exhibiting the highest performance, US 163 and US 177 were submitted to 16S rRNA gene sequencing, and the results revealed that they belonged to the Bacillus cereus species. Overall, the findings indicated that the Bacilli from petroleum soil have a number of promising properties that make them promising candidates for bioplastic production.

Bioactive secondary metabolites from a new terrestrial Streptomyces sp. TN262.

During our search for Streptomyces spp. as new producers of bioactive secondary metabolites, the ethyl acetate extract of the new terrestrial Streptomyces isolate TN262 delivered eight antimicrobially active compounds. They were identified as 1-acetyl-beta-carboline (1), tryptophol (2), cineromycin B (3), 2,3-dihydrocineromycin B (4), cyclo-(tyrosylprolyl) (5), 3-(hydroxyacetyl)-indole (6), brevianamide F (7), and cis-cyclo-(L-prolyl-L-leucyl) (8). Three further metabolites were detected in the unpolar fractions using GC-MS and tentatively assigned as benzophenone (9), N-butyl-benzenesulfonamide (10), and hexanedioic acid-bis-(2-ethylhexyl) ester (11). This last compound is known as plasticizer derivatives, but it has never been described from natural sources. In this article, we describe the identification of the new Streptomyces sp. isolate TN262 using its cultural characteristics, the nucleotide sequence of the corresponding 16S rRNA gene and the phylogenetic analysis, followed by optimization, large-scale fermentation, isolation of the bioactive constituents, and determination of their structures. The biological activity of compounds (2), (3), (4), and those of the unpolar fractions was addressed as well.

Inhibition of fungi and gram-negative bacteria by bacteriocin BacTN635 produced by Lactobacillus plantarum sp. TN635.

The aim of this study was to evaluate 54 lactic acid bacteria (LAB) strains isolated from meat, fermented vegetables and dairy products for their capacity to produce antimicrobial activities against several bacteria and fungi. The strain designed TN635 has been selected for advanced studies. The supernatant culture of this strain inhibits the growth of all tested pathogenic including the four Gram-negative bacteria (Salmonella enterica ATCC43972, Pseudomonas aeruginosa ATCC 49189, Hafnia sp. and Serratia sp.) and the pathogenic fungus Candida tropicalis R2 CIP203. Based on the nucleotide sequence of the 16S rRNA gene of the strain TN635 (1,540 pb accession no FN252881) and the phylogenetic analysis, we propose the assignment of our new isolate bacterium as Lactobacillus plantarum sp. TN635 strain. Its antimicrobial compound was determined as a proteinaceous substance, stable to heat and to treatment with surfactants and organic solvents. Highest antimicrobial activity was found between pH 3 and 11 with an optimum at pH = 7. The BacTN635 was purified to homogeneity by a four-step protocol involving ammonium sulfate precipitation, centrifugal microconcentrators with a 10-kDa membrane cutoff, gel filtration Sephadex G-25, and C18 reverse-phase HPLC. SDS-PAGE analysis of the purified BacTN635, revealed a single band with an estimated molecular mass of approximately 4 kDa. The maximum bacteriocin production (5,000 AU/ml) was recorded after a 16-h incubation in Man, Rogosa, and Sharpe (MRS) medium at 30 degrees C. The mode of action of the partial purified BacTN635 was identified as bactericidal against Listeria ivanovii BUG 496 and as fungistatic against C. tropicalis R2 CIP203.