Extracellular protease from Bacillus coagulans,a psychrotrophic,Antarctic bacterium

Bacillus coagulans, when grown on casein at 20°C, produced an inducible, metalloprotease of 28 kDa at 1.6 U/mg cell protein. (NH₄)₂SO₄ at 2 g/l decreased enzyme production irrespective of carbon source.The authors are with the Defence R & D Establishment, Tansen Road, Gwalior-474 002, India.     

Pyridine degradation characteristics of a newly isolated bacterial strain and its application with a novel reactor for the further treatment in pyridine wastewater

A pyridine-degrading strain Gemmobacter sp. ZP-12, isolated from an activated sludge, was able to use pyridine as the sole carbon and nitrogen source for the growth. The strain could effectively degrade pyridine and remove TOC over a wide range of initial pyridine concentrations. The pyridine degradation rate for 100, 500, 1000, 1500 and 2000 mg/L was 2.90 ± 0.17; 13.72 ± 0.21, 20.40 ± 0.24, 31.09 ± 0.26, 27.63 ± 0.17 mg/L/h, respectively. During the pyridine degraded, a large amount of NH₄⁺-N was released and accumulated. The accumulation of NH₄⁺-N increased with the increase of pyridine concentration. For further removing the NH₄⁺-N producing in pyridine degradation, an aerobic-moving bed biofilm reactor coupled with intermittent-aeration membrane biological reactor (a-MBBR-IMBR) was constructed, in which the strain and the aerobic / anoxic mixed sludge combined to remove the pollutants in the wastewater containing 500 mg/L pyridine. After 96 h of operation, the final TOC removal efficiency was 96.5 ± 1.05 %. The average residual concentration of NO₃⁻-N and NH₄⁺-N was respectively 9.09 ± 4.13 mg/L and 7.85 ± 3.88 mg/L. The study provides a viable option for treating pyridine wastewater.     

Influence of COD/NH3–N ratio on organic removal and nitrification using a modified RBC

Synthetic wastewaters were prepared with different influent concentrations of ammonia nitrogen (NH₃–N) and COD and the treatment studies were conducted using a rotating biological contactor (RBC). If organic removal and nitrification can be simultaneously effected in one process, it will be an ideal solution to water pollution control. The RBC used in the present study was a four stage laboratory model and the discs were modified by attaching porous netlon sheets to enhance biofilm area. The COD loads (S ₀) used were about 1000 and 1500?mg/l whereas NH₃–N concentrations used were in the range of 20 to 185?mg/l. Hydraulic load (q) of 0.03?m³?.?m^-2?.?d^-1 and ammonia nitrogen loadings in the range of 0.66 to 5.5?g NH₃–N?.?m^-2?.?d^-1 were used. The RBC was operated at two different rotating speeds of 6 and 12?rpm. The results showed that the nitrification and percentage of COD removal were not affected up to the value of the COD/NH₃–N in the range from 47 to 23 at w=6?rpm and for an average influent COD of 1003?mg/l. Beyond that range only the nitrification rate decreased much whereas the percentage of COD removal was not affected. Similarly, at an influent COD load of 1557?mg/l, the nitrification and percentage COD removal were not affected for the value of the COD/NH₃–N in the range from 44 to 23 but beyond that range only the nitrification rate decreased while the percentage of COD removal was approximately constant and still high. A correlation plot between the NH₃–N removed and NH₃–N applied was presented at a rotating speed of 6?rpm and it was found that the nitrification rate of 3.93?g NH₃–N?.?m^-2?.?d^-1 was achieved at ammonia loading of 5.55?g NH₃–N?.?m^-2?.?d^-1. Also the results at w=12?rpm showed improvement of nitrification rate over those at 6?rpm.     

In vitro plant regeneration through direct organogenesis in Populus deltoides clone G48 from petiole explants

A rapid and efficient protocol is developed for in vitro plantlet regeneration of Populus deltoides clone G48 using petiole explants. The highest frequency of shoot regeneration (74.75%) from petiole was obtained on MS medium supplemented with 0.50?mg/l BAP and 0.20?mg/l IAA. The regenerated shoots started turning brown and necrotic after 10?C15?days in culture. To overcome the browning problem, the explants along with the developing shoot buds were transferred to modified MS medium containing 0.50?mg/l BAP, 0.20?mg/l IAA, 15?mg/l AdS, 0.1% PVP, 100?mg/l casein hydrolysate, 50?mg/l L-glutamine, 250?mg/l (NH₄)₂SO₄ and 0.5% agar. Shoot multiplication and elongation took place on the same medium. Indole-3-acetic acid at 0.10?mg/l was most effective for root regeneration. Using the current protocol, it took 2?months to regenerate plantlets. The in vitro regenerated plantlets were successfully acclimatized and established in greenhouse conditions. This regeneration system using petiole explants provides a foundation for Agrobacterium-mediated genetic transformation of P. deltoides clone G48 for incorporation of various silviculturally important traits.     

Lactic Acid Production from Pretreated Hydrolysates of Corn Stover by a Newly Developed Bacillus coagulans Strain

An inhibitor-tolerance strain, Bacillus coagulans GKN316, was developed through atmospheric and room temperature plasma (ARTP) mutation and evolution experiment in condensed dilute-acid hydrolysate (CDH) of corn stover. The fermentabilities of other hydrolysates with B. coagulans GKN316 and the parental strain B. coagulans NL01 were assessed. When using condensed acid-catalyzed steam-exploded hydrolysate (CASEH), condensed acid-catalyzed liquid hot water hydrolysate (CALH) and condensed acid-catalyzed sulfite hydrolysate (CASH) as substrates, the concentration of lactic acid reached 45.39, 16.83, and 18.71 g/L by B. coagulans GKN316, respectively. But for B. coagulans NL01, only CASEH could be directly fermented to produce 15.47 g/L lactic acid. The individual inhibitory effect of furfural, 5-hydroxymethylfurfural (HMF), vanillin, syringaldehyde and p-hydroxybenzaldehyde (pHBal) on xylose utilization by B. coagulans GKN316 was also studied. The strain B. coagulans GKN316 could effectively convert these toxic inhibitors to the less toxic corresponding alcohols in situ. These results suggested that B. coagulans GKN316 was well suited to production of lactic acid from undetoxified lignocellulosic hydrolysates.     

Azadirachtin production by hairy root cultivation of Azadirachta indica in a modified stirred tank reactor

Present investigation involves hairy root cultivation of Azadirachta indica in a modified stirred tank reactor under optimized culture conditions for maximum volumetric productivity of azadirachtin. The selected hairy root line (Az-35) was induced via Agrobacterium rhizogenes LBA 920-mediated transformation of A. indica leaf explants (Coimbatore variety, India). Liquid culture of the hairy roots was developed in a modified Murashige and Skoog medium (MM2). To further enhance the productivity of azadirachtin, selected growth regulators (1.0?mg/l IAA and 0.025?mg/l GA₃), permeabilizing agent (0.5?% v/v DNBP), a biotic elicitor (1?% v/v Curvularia (culture filtrate)) and an indirectly linked biosynthetic precursor (50?mg/l cholesterol) were added in the growth medium on 15th day of the hairy root cultivation period in shake flask. Highest azadirachtin production (113?mg/l) was obtained on 25th day of the growth cycle with a biomass of 21?g/l DW. Further, batch cultivation of hairy roots was carried out in a novel liquid-phase bioreactor configuration (modified stirred tank reactor with polyurethane foam as root support) to investigate the possible scale-up of the established A. indica hairy root culture. A biomass production of 15.2?g/l with azadirachtin accumulation in the hairy roots of 6.4?mg/g (97.28?mg/l) could be achieved after 25?days of the batch cultivation period, which was ~27 and ~14?% less biomass and azadirachtin concentration obtained respectively, in shake flasks. An overall volumetric productivity of 3.89?mg/(l?day) of azadirachtin was obtained in the bioreactor.     

Thermodynamic equilibrium in reversibly interacting ribosomal systems of E. coli and Bac. coagulans

The results which strongly confirm the existence of dynamic equilibrium 50S-30S ? 50S + 30S in ribosomal systems of E. Coli and Bac. coagulans are presented. A method has been devised which permitted to determine quantitatively the detailed composition of the reaction mixture of ribosomal subparticles and evaluate the equilibrium constant.The analysis of equilibrium ribosomal systems of E. coli and Bac. coagulans at different temperatures allowed evaluation of appropriate thermodynamic parameters. In particular, the free energy of dissociation was shown to be higher in the case of thennophilic Bac. coagulans rihosomes.     

Purification and characterization of antifungal compounds from Bacillus coagulans TQ33 isolated from skimmed milk powder

Bacillus coagulans TQ33, isolated from skimmed milk powder, displays strong antifungal activity against plant pathogenic fungi. The antifungal compound of the B. coagulans TQ33 culture was extracted by thin-layer chromatography and column chromatography, and its structure was elucidated based on HPLC, LC-MS, and NMR analysie. The antifungal compound was identified as phenyllactic acid (PLA), and it was found to have a minimum inhibitory concentration on Phytophthora drechsleri Tucker of 18 mg/mL. Bio-control activity tests indicated that PLA has a wide spectrum of antagonistic effects against Fusarium oxysporum, Botrytis cinerea, Glomerella cingulata, Penicillium citrinum, Penicillium digitatum, particularly against F. oxysporum. PLA is the most notable antimicrobial compound with broad and effective antimicrobial activity against both bacteria and fungi that has been isolated and identified to date. These results indicate that B. coagulans TQ33 has the potential for application in biological pesticides.     

Studies on dl-Alanine Formation by Thermophilic Bacteria

During the course of the screening of thermophilic microorganisms, several strains were found to accumulate amino-acids. Of those strains that were isolated from feces source with Bennett medium, one strain was found to produce a large amount of an amino acid. This amino acid was isolated in crystalline form and identified as dl-alanine by IR absorption spectrum, specific rotatory power and elementary analysis. The taxonomic studies were carried out and this strain was identified as Bacillus coagulans. The strain B. coagulans B₉-17 produced dl-alanine as much as 1000 mg/l after 24 hours at 50°C in shake culture.

The yield of dl-alanine was increased up to 16.5 g/l with some improvements.     

The role of salvinia rotundifolia in scavenging aquatic Pb (II) pollution: a case study

Bio-surveillance of environmental pollution is increasingly gaining ground, as it is felt that if and when standardized, it might prove to be a long term cost-effective alternative technology. In aquatic media, for monitoring heavy metal pollutants, which are mostly xenobiotic, a number of hydrophytes have been tried by earlier researchers. These hydrophytes have shown to have varying degree of accumulation capacities, which could be preferably utilized in scavenging the toxicants such as Pb(II) among others. The screening criteria of such a suitable scavenger also take into account the ease of harvesting and handling the biomass on a large scale. Here comes the use of a macrophyte, and its potentiality of forming metallo-protein complexes (phytochelatin) to hold back the diffused metal, Pb(II), ions. In the present investigation, the experimental test system was the common tropical aquatic weed, Salvinia rotundifolia, Willd. It has shown a high promise for Pb(II) removal from synthetic as well as industrial (battery producing unit) wastewater. Within a span of 4 days, 50?gm (wet weight) of the plant was capable of removing about 85–95% of Pb(II) from 1.50 litres of both kinds of wastewaters containing 0.65–1.8?ppm of the metal at an optimum pH of 5.5. The uptake potentials were examined under various combinations of pH, plant weight, and metal concentration. It is suggested that the high uptake and recovery of Pb(II) by Salvinia Rotundifolia can be efficiently used as a possible future biotechnological solution for industrial wastewater treatment in a tropical and developing country like India. In the present study, a reduction of biomass weight from 80 to 72.3 (in mg dry wt/g of fresh wt.) was noted, while an increase of tissue conductivity from 161 to 181.5 in micro-mhos/cm was observed. This reflects the potential injurious effect of heavy metals to the cell membrane of the biomass. These changes, when standardized, are likely to serve as a suitable `biomonitoring' device for aquatic metal pollution. In the present case, effluent quality of a battery manufacturing unit, India was evaluated. Even though there is an existing effluent treatment plant (ETP) of the industry, the effluent from the balancing tank (discharge point) was found to contain high concentrations of lead (0.657–1.021?mg/l), BOD (470?mg/l) and COD (680?mg/l) as against State Pollution Control Board's standards of 0.1?mg/l, 30?mg/l, and 250?mg/l respectively. In the present investigation, an attempt has also been made to treat wastewater from the balancing tank of the existing effluent treatment plant (ETP) of the above-stated industrial unit to bring down the concentrations of Pb(II), BOD (5 days), and COD within the permissible limits prescribed by the regulatory board. The treatment was done in a laboratory-scale oxidation pond, cultured with Salvinia rotundifolia, of 85 litres capacity using a hydraulic retention period of about 10 days.     

Influence of isolation technique of half-anthers and of initiation culture medium on callus induction and regeneration in Anthurium andreanum

Optimization of the isolation technique and initiation culture medium are two critical aspects that can determine the success of anthurium half-anther culture. Both aspects in half-anther culture of Anthurium andreanum Linden ex André cv. ??Tropical?? were studied and successfully improved. Untreated half-anthers, when cultured abaxial side down on medium, was the most suitable means of inducing callus. Callus formation was further improved by culturing half-anthers adaxial side down on Winarto-Teixeira (WT) medium (Winarto et al. in Plant Growth Regul 65:513?C529, 2011b) supplemented with 0.01?mg/l ??-naphthaleneacetic acid (NAA), 1.0?mg/l 6-benzyladenine (BA) and 0.5?mg/l thidiazuron (TDZ). Gelrite enhanced callus formation (compared to agar) when the concentration was reduced from 2.0 to 1.5?g/l on WT medium. Application of 0.5?mg/l 2,4-dichlorophenoxy acetic acid (2,4-D) in WT medium increased callus formation. Most callus formed when half-anthers were cultured adaxial-side down on WT medium supplemented with 0.5?mg/l 2,4-D in combination with 0.01?mg/l NAA, 1.0?mg/l BA and 0.5?mg/l TDZ using 1.5?g/l Gelrite. This ideal medium induced the growth of half-anthers, with as much as 38?% of half-anthers producing callus, or, on average, 3.4 half-anthers/treatment. Callus derived from this optimized protocol regenerated easily and could be multiplied on New Winarto-Teixeira medium (NWT) (Winarto et al. in Plant Growth Regul 65:513?C529, 2011b) containing 0.25?mg/l 2,4-D, 0.02?mg/l NAA, 0.75?mg/l BA, 1.5?mg/l TDZ and 2.0?g/l Gelrite. Shoots rooted well on hormone-free NWT medium with 2.0?g/l Gelrite. The plantlets could be easily acclimatized in a substrate of raw rice husk, burned-rice husk and organic manure (1:1:1, v/v/v) with a high survival (100?%) ex vitro in a greenhouse. The results of this study would benefit half-anther culture of other Anthurium cultivars, particularly at the initial stage of callus induction.     

Microalgae-based agro-industrial wastewater treatment: a preliminary screening of biodegradability

The potential of algal–bacterial symbiosis for the removal of carbon, nitrogen and phosphorus from five agro-industrial wastewaters was investigated in enclosed batch biodegradation tests using a mixed microalgae consortium and activated sludge as model microorganisms. The target wastewaters were obtained from potato processing (PW), fish processing (FW), animal feed production (MW), coffee manufacturing (CW) and yeast production (YW). The initial C/N/P ratio of the agro-industrial wastewater was correlated with its biodegradability. Thus, the highest removals of total organic carbon (TOC) and nitrogen were recorded in two fold diluted FW (64?±?2 % and 85?±?1 %, respectively), while the maximum P-PO₄ ^3? removal achieved was 89?±?1 % in undiluted PW. The biodegradable TOC was in most cases the limiting component in the treatment of the wastewaters evaluated. This study confirmed the potential of coupling carbon and nutrient recovery from agro-industrial effluents with the production of a valuable algal–bacterial biomass, despite their poor biodegradability.     

Nutrient removal from high strength nitrate containing industrial wastewater using <Emphasis Type="Italic">Chlorella</Emphasis> sp. strain ACUF_802

the research aim of this study was to characterize an isolated native strain of Chlorella sp. ACUF_802, well adapted to a high nitrate concentration environment and to investigate its potential to nitrate and phosphate removal from industrial wastewaters with the minimal addition of chemical reagents and energy. The isolated strain was identified and evaluated for its capability to support biomass growth and nutrient removal from synthetic wastewater in batch tests using different concentrations of carbon and nitrogen, different carbon sources and N:P ratios. The strain was isolated via the plating method from the settler of a pilot scale moving bed biofilm reactor performing a nitrification process. The strain was identified using molecular analysis with rDNA primers. Using sodium bicarbonate as carbon source, the batch productivity (71.43 mg L^?1 day^?1) of the strain Chlorella sp. ACUF_802 was calculated with a logistic model and compared to the values reported in the literature. Assays on the effect of the N:P ratio indicated that the productivity was increased 36% when the N:P ratio was close to 1 (111.96 mg L^?1 day^?1), but for a complete phosphorus removal a 5:1 N:P ratio with nitrate concentrations ≤125 mg?L^?1 is recommended. The isolated microalgae strain Chlorella sp. ACUF_802 showed versatility to grow in the synthetic industrial wastewaters tested and can be considered as an appropriate organism for nitrogen removal from industrial wastewaters in the presence of an organic or inorganic carbon source.     

Molecular Characterization and In Vitro Analyses of a Sporogenous Bacterium with Potential Probiotic Properties

The Gram-positive thin rods of a Bacillus species were identified and designated as Bacillus coagulans RK - 02 through the standard microbiological and biochemical characterization procedures, followed by data analysis and comparison with the characteristics given in Bergey’s manual of systematic bacteriology. The culture was further characterized and confirmed as Bacillus coagulans by 16S rDNA sequence analysis wherein about 755 nucleotides of 16S rDNA was amplified and cloned in pGEM-T vector and subsequently sequenced. Sequence was blasted against the nr database of NCBI. Multiple alignments were done with some selected and related sequences using Clustal W. Phylogenetic trees were drawn with the same software after the distances were determined by neighbor-joining algorithm. The in vitro studies on the probiotic properties demonstrated that our isolate could prove to be a potential probiotic with spore-forming and lactic acid-producing abilities coupled with acid and bile tolerance properties and antimicrobial action. In addition to these characteristics, the bacterium also produced enzymes such as amylase, cellulase, lipase, protease, lactase and catalase, which can help in improving digestion and overall health, alleviate lactose intolerance and remove oxidative stresses, required for the well-being of the consumers. In our previously reported studies, an exopolysaccharide (EPS), a probioactive molecule produced by the same bacterium, showed very significant antioxidant, antihyperglycemic and emulsification activities. Thus, Bacillus coagulans RK - 02 is a well-characterized promising probiotic for its potential commercial applications to pharmaceutical, nutraceutical and functional food formulations with care-free storage.     

Formaldehyde and methanol biodegradation with the methylotrophic yeast Hansenula polymorpha in a model wastewater system

In search of the optimal way to reduce the hazards of environmental contamination by formaldehyde (FD) and methanol the use of unconventional yeasts is proposed as exemplified by the methylotrophic yeast Hansenula polymorpha. In a very simplified environment of a model wastewater solution, H. polymorpha cells were able to grow on, and metabolize formaldehyde and methanol, applied as sole carbon sources, at concentrations typical for wastewaters of the chemical industry. Several experimental conditions were tested for cell growth and biodegradation kinetics. It was found that the yeast culture inoculated at low cell density was able to grow on initial FD levels up to 400mg/l and the biomass yield was dependent on both, the amount of total carbon added and the physiological state of the cells. When high density of pre-adapted cell culture was used, the methylotrophs were fully viable and able to degrade formaldehyde present at initial concentrations up to 700 mg/l. The maximum limiting FD consumption rate was determined as approx. 400 mg/1 per hour. Methanol, at concentrations up to 2%, was easily utilized and did not have a negative effect on cell growth and respiration. It is suggested that in real wastewaters the eukaryotic microorganisms--in contrast to bacteria--might reveal greater adaptation potential to toxic levels of formaldehyde as well as to other wastewater constituents.     

Removal of nitrogen from industrial wastewaters by three-step nitrification and denitrification.

The removal of nitrogen from industrial wastewaters carrying about 1,000 mg NH4-N and urea-N/l was investigated on a laboratory scale. The use of a three-step nitryfying activated sludge with adjustment of pH from step to step resulted in 99% oxidation of both forms of nitrogen to nitrites. The efficiency of nitrification was 18 mg N/l/h. Total time of wastewater aeration depended on nitrogen concentration and was 33-54 hours. Complete dentrification of NO2-N was obtained in packed-bed reactor with the use of acetic acid as a carbon source. Efficiency of denitrification was 361 mg N/l/h.     

Aerobic degradation of pyridine by a new bacterial strain, <Emphasis Type="Italic">Shinella zoogloeoides</Emphasis> BC026

A new bacterial strain, Shinella zoogloeoides BC026, which utilizes pyridine as its sole carbon, nitrogen and energy source, was isolated from the activated sludge of a coking wastewater treatment plant. The BC026 strain completely degraded up to 1,806 mg/l of pyridine in 45.5 h. The optimum degradation conditions were pH 8.0 and temperature 30–35°C. According to product monitoring and genetic analysis, the pyridine ring was cleaved between C₂ and N, resulting in 58% of pyridine-N being directly converted into ammonium. Providing glucose as the extra carbon source, the degradation of pyridine was not affected, while the growth of the strain was promoted, and 41% of pyridine-N was converted into ammonium with a C/N ratio of 35. The ammonium was utilized rapidly by the strain, and a portion of it was transformed into nitrate, then to nitrite, and finally to dinitrogen if enough extra carbon was provided. Considering these characteristics, this strain may accomplish heterotrophic nitrification and aerobic denitrification simultaneously.     

Isolation and characterization of histamine-producing bacteria from fermented fish products

Histamine is mainly produced by microorganisms that are found in fermented foods, and is frequently involved in food poisoning. Two histamine-producing bacteria were isolated from fermented fish products, anchovy sauce, and sand lance sauce by using a histidine decarboxylating medium. The species were identified as Bacillus licheniformis A7 and B. coagulans SL5. Multiplex PCR analysis showed the presence of the conserved histidine decarboxylase (hdc) gene in the chromosome of these bacteria. B. licheniformis A7 and B. coagulans SL5 produced the maximum amount of histamine (22.3±3.5 and 15.1±1.5 mg/L, respectively). As such, they were determined to be potential histamine-producing bacteria among the tested cultures.     

Nitrosomonas sp. Based biosensor for ammonium nitrogen measurement in wastewater

A bacterial culture of Nitrosomonas sp. was isolated from a nitrifying biofilm to construct a biosensor for ammonium nitrogen (NH ₄ ⁺ ?N) measurements in high ammonia wastewaters. The pure culture of microorganisms was immobilized into agarose gel matrix to attain a stable biosensor with a long service life. Biosensors were calibrated using (NH₄)₂SO₄ solution and a steady-state method. Subsequently, several experiments with synthetic and industrial wastewaters were conducted. A linear range up to 20 mg/L of NH ₄ ⁺ ?N, and sensitivities between 0.030 and 0.036 were gained with biosensors. During 14 days of stable service life of the Nitrosomonas sp. biosensors, variation of the signal was less than 7%. Response times of biosensors were 15 ～ 25 min, while recovery times were up to 25 min. Measurements with high ammonia content synthetic and industrial wastewaters were conducted, and 8.3 and 5.6% over estimation of NH ₄ ⁺ ?N was gained, respectively, compared with results of Nessler method. In spite of the small overestimation, the biosensor based on a pure culture of Nitrosomonas sp. and calibrated with (NH₄)₂SO₄ is suitable for the analysis of NH ₄ ⁺ ?N in high ammonia content wastewaters.