Selection and characterization of probiotic lactic acid bacteria with heterocyclic amine binding and nitrosamine degradation properties.

Potential probiotic strain for being health protectant especially intestinal illness is strain specific. This study investigated the selection of a new strain of probiotic of non-human origin and of human origin with the properties of intestinal protection against cancer. From the primary screening results, the human feces origin strains showed more bile salt tolerance than the fermented food origin strains. Whereas none of the human feces origin isolates could grow well in the acid condition. Lactobacillus plantarum CM4 was the new probiotic of non-human origin strain for this study. CM4 cells is said to tolerate and grow in 0.3% bile salt after 5 hours of incubation, at pH3 after 6 hours of incubation. This is in agreement with in vivo study for intestinal adherence ability of probiotic, a live CM4 cells was able to persist in mice small intestine and colon for 5 days. Live CM4 cells showed most effectiveness to bind 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) mutagen after 24 hours of incubation with 46.32% of binding ability while 144 hours of incubation with 85.34% of binding ability was the most effective for 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) mutagen. The significant difference (p<0.05) was found at all those time points. Moreover, the CM4 strain could degrade diphenylnitrosamine (DPN) better than 1-nitrosopyrrolidine (NPR) with dose response relationship activity. These imply that the CM4 strain could be the value added for the consuming pharmaceutical probiotic product based on scientific proof of its role in intestinal survival properties and cancer prevention through binding PhIP and IQ mutagen as well as degrading nitrosamine.

Bioaugmentation of latex rubber sheet wastewater treatment with stimulated indigenous purple nonsulfur bacteria by fermented pineapple extract

Background Treating latex rubber sheet wastewater often leads to the generation of a rotten-egg odor from toxic H2S. To increase the treatment efficiency and eliminate H2S, purple nonsulfur bacteria (PNSB), prepared by supplementing non-sterile rubber sheet wastewater (RAW) with fermented pineapple extract (FPE), were used to treat this wastewater under microaerobic light conditions. The following 3 independent variables: chemical oxygen demand (COD), initial pH and FPE dose were investigated using the Box-Behnken design to find optimal conditions for stimulating the growth of indigenous PNSB (PNSBsi). Results The addition of 2.0% FPE into RAW, which had a COD of 2000 mg L- 1 and an initial pH of 7.0, significantly decreased oxidation reduction potential (ORP) value and stimulated PNSBsi to reach a maximum of 7.8 log cfu mL- 1 within 2 d. Consequently, these PNSBsi, used as inoculants, were investigated for their ability to treat the wastewater under microaerobic light conditions. A central composite design was used to determine the optimal conditions for the wastewater treatment. These proved to be 7% PNSBsi, 0.8% FPE and 4 d retention time and this combination resulted in a reduction of 91% for COD, 75% for suspended solids, 61% for total sulfide while H2S was not detected. Results of abiotic control and treatment sets indicated that H2S was produced by heterotrophic bacteria and it was then effectively deactivated by PNSBsi. Conclusions The stimulation of PNSB growth by FPE under light condition was to lower ORP, and PNSBsi proved to be effective for treating the wastewater.

Effects of 5-aminolevulinic acid (ALA)-containing supernatants from selected Rhodopseudomonas palustris strains on rice growth under NaCl stress, with mediating effects on chlorophyll, photosynthetic electron transport and antioxidative enzymes

Background: Rice is globally one of the most important food crops, and NaCl stress is a key factor reducing rice yield. Amelioration of NaCl stress was assessed by determining the growth of rice seedlings treated with culture supernatants containing 5-aminolevulinic acid (ALA) secreted by strains of Rhodopseudomonas palustris (TN114 and PP803) and compared to the effects of synthetic ALA (positive control) and no ALA content (negative control). Results: The relative root growth of rice seedlings was determined under NaCl stress (50 mM NaCl), after 21 d of pretreatment. Pretreatments with 1 μM commercial ALA and 10X diluted culture supernatant of strain TN114 (2.57 μM ALA) gave significantly better growth than 10X diluted PP803 supernatant (2.11 μM ALA). Rice growth measured by dry weight under NaCl stress ordered the pretreatments as: commercial ALA N TN114 N PP803 N negative control. NaCl stress strongly decreased total chlorophyll of the plants that correlated with non-photochemical quenching of fluorescence (NPQ). The salt stress also strongly increased hydrogen peroxide (H2O2) concentration in NaCl-stressed plants. The pretreatments were ordered by reduction in H2O2 content under NaCl stress as: commercial ALA N TN114 N PP803 N negative control. The ALA pretreatments incurred remarkable increases of total chlorophyll and antioxidative activities of catalase (CAT), ascorbate peroxide (APx), glutathione reductase (GR) and superoxide dismutase (SOD); under NaCl stress commercial ALA and TN114 had generally stronger effects than PP803. Conclusions: The strain TN114 has potential as a plant growth stimulating bacterium that might enhance rice growth in saline paddy fields at a lower cost than commercial ALA.

The use of rice straw broth as an appropriate medium to isolate purple nonsulfur bacteria from paddy fields

The aims were to explore an appropriate isolating medium for obtaining purple nonsulfur bacteria (PNSB) for use as biofertilizers in saline paddy fields and to obtain pure cultures. We therefore chose a defined isolating medium containing 0.25 percent NaCl, (Glutamate-Acetate broth, GA) and a rice straw broth to compare them for numbers of PNSB obtained, time to obtain pure cultures, diversity and costs. A total of 30 water and 30 sediment samples were collected from saline paddy fields in southern Thailand and used to isolate PNSB in both the isolating media. Based on 60 samples and a period of 13 days incubation under anaerobic light conditions, a greater number of samples produced PNSB growth in GA broth after only day 3; however, after that the rice straw broth provided about a 2 fold increase in the number of samples that produced PNSB growth. Colonies isolated from GA broth required a significantly higher number of repeated streaking to obtain a pure culture (average 3.5) than those from rice straw broth (average 2.7) and the latter medium also produced significantly (P < 0.05) more isolates per sample. Sixty samples of water and sediment, from rice paddies with salinity (average, 3.43 +/- 0.67 mS/cm) and slight acidity (average, pH 5.84 +/- 0.42) provided 62 PNSB isolates by GA broth and 210 isolates by rice straw broth, and rice straw broth also produced a greater prevalence of PNSB. Estimates of the costs based on current prices of media, Gas Pak and electricity to obtain PNSB with the use of GA broth was roughly 6 times higher than for the rice straw broth.

Removal of heavy metals by exopolymeric substances produced by resistant purple nonsulfur bacteria isolated from contaminated shrimp ponds

Two purple nonsulfur bacteria (PNSB) strains, Rhodobium marinum NW16 and Rhodobacter sphaeroides KMS24 were investigated for their potential to remove heavy metals (HMs) from contaminated shrimp pond water. Tolerance of both PNSB strains growing with both microaerobic-light and aerobic-dark conditions, based on their minimum inhibitory concentrations, was in the order of Cu2+ > Zn2+ > Cd2+ (Pb precipitation occurred at 0.34 mM). Results from a scanning electron microscope equipped with an energy dispersive X-ray spectrometer (SEM-EDX) indicated that Cu2+ and Zn2+ altered the cellular morphology of both strains and accumulated HMs were found in their cells. The highest amounts of both cations were found in their cell walls followed by the cytoplasm and cell membrane. Using the highest concentrations (mM) of HMs found in shrimp pond of 0.0067 Cd2+, 0.54 Cu2+, 0.30 Pb2+, 0.89 Zn2+ and 3 percent NaCl under both incubating conditions exopolymeric substances (EPS) produced by both strains showed a greater removal of all HMs (average percentages; 90.52-97.29) than their cells (average percentages; 14.02-75.03).

Enhancement of gamma-aminobutyric acid in a fermented red seaweed beverage by starter culture Lactobacillus plantarum DW12

Lactobacillus plantarum DW12, a gamma-aminobutyric acid (GABA) producing strain, was used as a starter culture to produce a functional fermented red seaweed beverage (FSB). Optimal conditions for producing FSB were established using Central Composite Design by varying the amounts of monosodium glutamate (MSG), sucrose and the initial pH in MRS medium. After a verification test, 1 percent MSG, 6 percent sucrose and an initial pH of 6 were selected. Four treatments were tested: traditional formula (A), red seaweed-cane sugar-potable water = 3:1:10, w/w/v, initial pH 6; the traditional formula with a 5 percent starter culture consisting of 4.1 x 10(9) CFU of DW 12/ml (B); formula A modified by changing the amounts of cane sugar and MSG to 6 percent and 1 percent, respectively (C); formula C with a 5 percent starter culture added (D). Comparison among the 4 treatments showed that the treatment D produced the highest amount of GABA (4000 mg/L) during days 45-60 while the GABA content of A, B and C treatments was 340, 730 and 1690 mg/L, respectively. However, the results of the sensory test for the treatments C and D showed that the presence of MSG produced an unsatisfactory salty taste. All finished products from the 4 treatments met Thai standard guidelines for chemical and microbiological qualities after 120 days. The results indicated that enrichment of the GABA content in FSB is possible by adding MSG and the GABA producing strain DW12; however, the appropriate amount of MSG addition should be further studied.

Characterization of the antiyeast compound and probiotic properties of a starter lactobacillus plantarum DW3 for possible use in fermented plant beverages

Lactobacillus plantarum DW3 produced antifungal compounds that inhibited the growth of Rhodotorula mucilaginosa DKA, contaminating yeast in fermented plant beverages (FPBs) and various potential human pathogens. Phenyllactic acid (PLA) identified by gas chromatography- mass spectrometry (GC-MS) was produced at 31 mg/L PLA in MRS medium and 5 mg/ml inhibited growth of the target yeast in vitro by 90 percent. Other inhibitors were also present but not specifically identified. Results of in vitro tests showed that DW3 also had probiotic properties as it survived various human biological barriers resistance to pH 3, bile salts, growth without vitamin B12 and the presence and absence of oxygen. Its inhibitory effect against food borne pathogenic bacteria and spoilage organisms was higher than that found for a commercial strain Lactobacillus casei R. An acute oral toxicity test on ICR mice at a high single dose of either 10(9) and 10(12) cells per mouse for 14 days showed that DW3 had no adverse effect on the general health status and there was no evidence of bacteremia. Mice fed DW3 had a reduced weight gain compared to the control. No significant difference (p > 0.05) was found for the spleen weight index (SWI) among the treatment and control groups whereas there was a significant difference (p < 0.05) for the liver weight ratio (LWR) in a group fed with 10(12) cells per mouse when compared with the control group.

Isolation of purple nonsulfur bacteria for the removal of heavy metals and sodium from contaminated shrimp ponds

In order to determine whether waters used for the shrimp cultivation contained toxic levels of heavy metals (HMs) and sodium (Na), analysis was carried out on 31 shrimp ponds in areas of southern Thailand. Purple nonsulfur bacteria (PNB) were also isolated from the same ponds to investigate if they could be used for bioremediation of the above contaminants. The highest HMs concentrations of the sediment samples in mg/kg dry weight were found as follows: 0.75 cadmium (Cd), 62.63 lead (Pb), 34.60 copper (Cu) and 58.50 zinc (Zn). However, all sediment samples met Hong Kong standards for dredged sediment. In contrast, contamination of Cu (9-30 ug/L) and Zn (140-530 ug/L) exceeding the standard guidelines for marine aquatic animal set by the Pollution Control Department, Thailand, were found in 32 and 61 percent of water samples, respectively. Two metal resistant PNB isolates, NW16 and KMS24, were selected from the 120 PNB isolates obtained. Both isolates reduced the levels of HMs by up to 39 percent for Pb, 20 percent for Cu, 7 percent for Cd, 5 percent for Zn and 31 percent for Na from water that contained the highest levels of HMs found and 3 percent NaCl when cultured with either microaerobic-light or aerobic-dark conditions. The isolate NW16 removed a greater percentage of the HMs than the isolate KMS24, but the isolate KMS24 was able to survive better under a greater variety of environmental conditions. Both strains were therefore suitable to use for further investigating their abilities to remediate water contaminated with HMs and Na.

Inhibition of shrimp pathogenic vibrios by extracellular compounds from a proteolytic bacterium Pseudomonas sp. W3 / Inhibición de vibrios patógenos del camarón por compuestos extracelulares de una bacterias proteolítica Pseudomonas sp.W3

Pseudomonas sp. W3, a bacterium known to produce an extracellular alkaline protease, secreted secondary metabolites that inhibited pathogenic bacteria responsible for shrimp luminous vibriosis disease. Antivibrio compounds in the culture supernatant or culture filtrates (0.45 um and 0.22 um) of the isolate W3 were tested using an agar well diffusion method on a number of pathogenic vibrios. Vibrio harveyi PSU 2015 a pathogenic isolate was the most sensitive strain. The effectiveness of preparations from the isolate W3 against V. harveyi PSU 2015, and V. cholerae PSSCMI 0062 was in the order of culture supernatant > 0.45 um culture filtrate > 0.22 um culture filtrate. These extracellular antivibrio compounds also lysed both dead and living cells of V. harveyi PSU 2015. Results of the partial characterization tests indicated that there was some particulate antivibrio compound that was destroyed by treatment with enzymes particularly alpha-chymotrypsin, autoclaving at 121ºC for 15 min and was mostly removed by filtration through a 0.22 µm filter. Most of the inhibitory compounds were of small molecular weight able to pass through a 0.22 um filter and were resistant to treatment with various enzymes, pH values between 4-8 and temperatures up to 121ºC for 30 min. The optimum pH for the antivibrio activity in the 0.45 um culture filtrate was between pH 6-7.

Microbial succession in a fermenting of wild forest noni (Morinda coreia Ham) fruit plus molasses and its role in producing a liquid fertilizer

The numbers of lactic acid bacteria (LAB) and yeasts that were present during a wild forest noni (Morinda coreia Ham) fermentation, the changes in its physico-chemical properties and levels of plant nutrients were investigated. LAB increased rapidly during the first 7 days and were the dominant population until after day 21 when the LAB were declining and the yeasts began to dominate. Identification of the LAB and yeasts to species level showed that the dominant LAB throughout was Lactobacillus plantarum while Lactobacillus pentosus was found but only at day 21. Saccharomyces cerevisiae was the most dominant species of yeast throughout but was slowly replaced by Pichia membranifaciens and then Pichia anomala. Rhodotolura mucilaginosa, an aerobic yeast, was only detected at the beginning of the fermentation process. It is suggested that the Pichia spp. were responsible for consuming lactic acid. After 56 days, the values of pH, acetic acid, ethanol and electrical conductivity in the fermented product were 3.66, 3.34 g L-1, 16.98 g L-1 and 14.47 mS cm-1, respectively. Increased amounts of plant nutrients were present at day 56 mostly derived from the degradation of plant material. At day 56 the amounts were as follows (in mg L-1): N 633, P 1210, K 4356, Ca 693, Mg 536, Mn 7, B 51, Zn 169, and total carbon/total nitrogen ratio (C/N ratio) 18. Based on the seed germination index (GI) of cherry tomato (Lycopersicon esculentum Mill), the extract diluted 256-fold gave the best GI of 157 percent.

Selection of sulfur oxidizing bacterium for sulfide removal in sulfate rich wastewater to enhance biogas production

Sulfur oxidizing bacteria (SOB) were isolated and tested in order to remove sulfide from high sulfate wastewater to reduce the amount of hydrogen sulfide (H2S) in the produced biogas. A promising SOB isolate, designated as isolate T307, was selected due to its best sulfide removal (86.7 percent) in the effluent of a sulfate reduction reactor (SRR) over a 24 hrs incubation. The bacterium was able to grow better as a mixotroph (yeast extract as a carbon source) than as a chemolithoautotroph. In addition, as a heterotroph, the bacterium grew well with yeast extract and peptone. Based on partial 16S rRNA gene sequence, the isolated T307 was an Alcaligenes sp. and was able to convert most of sulfide species (total sulfide: TS; dissolved sulfide: DS and H2S) into elemental sulfur or sulfate over a 20 hrs period of cultivation by controlling the speed of shaking. In a biogas reactor set, after pre-treating a sulfide medium with Alcaligenes sp. T307 there was a much higher specific yield of CH4 (238 ml CH4 g-1COD removed) and more biogas (154 ml L-1 d-1) was produced with the biogas containing more methane (48.1 percent CH4, 51.5 percent CO2 and 0.41 percent H2S) in comparison to a control with a specific yield of CH4, (72 ml CH4 g-1COD removed) 86 ml L-1 d-1 biogas produced with a composition of 35.5 percent CH4, 63.7 percent CO2 and 0.86 percent H2S.

The potential use of anoxygenic phototrophic bacteria for treating latex rubber sheet wastewater

A total of 92 isolates of the purple non sulphur photosynthetic bacteria (PNSB) were isolated from 23 samples of wastewater obtained from rubber sheet manufacturing processes from various places of southern, Thailand. The isolate DK6 had the best potential for use in wastewater treatment as it can out-compete indigenous strains of PNSB when grown with them under conditions of microaerobic-light conditions. The isolate DK6 was identified as being most closely allied to Rhodopseudomonas blastica. The optimal pH and temperature for cell growth were between 6.5-7.5 and 30ºC, respectively. Optimum growth of DK6 was obtained after supplementing the wastewater from a latex rubber sheet processing plant with 0.50 percent (NH4)2SO4 and 1 mg/L nicotinic acid under conditions of microaerobic-light (3000 lux). Using these optimum conditions for growth, indigenous microorganisms reduced the initial chemical oxygen demand (COD) of the wastewater from 7,328 to 3371 mg/L a reduction of 54 percent and the biochemical oxygen demand (BOD) (initial BOD 4967 mg/L) by 70 percent. Using the same conditions and either a pure culture of DK6 or a mixed culture (DK6 plus indigenous microorganisms) a reduction of 90 percent of both COD and BOD was achieved. Chemical analysis of the cultures after treatment of the enriched wastewater shows that the protein content of the pure DK6 was 65.2 percent of the dry weight, and in mixed culture the protein content was 66.7 percent. Hence, single cell protein (SCP) may be a possible bi- product of the treatment process.