Withanolide A production from Withania somnifera hairy root cultures with improved growth by altering the concentrations of macro elements and nitrogen source in the medium

Withania somnifera is an important medicinal plant that contains withanolides as bioactive compounds. We have investigated the effects of macroelements and nitrogen source in hairy roots of W. somnifera with the aim of optimizing the production of biomass and withanolide A content. The effects of the macroelements NH₄NO₃, KNO₃, CaCl₂, MgSO₄ and KH₂PO₄ at concentrations of 0, 0.5, 1.0, 1.5 and 2.0× strengths and of nitrogen source [NH₄ ⁺/NO₃ ^? (0.00/18.80, 7.19/18.80, 14.38/18.80, 21.57/18.80, 28.75/18.80, 14.38/0.00, 14.38/9.40, 14.38/18.80, 14.38/28.20 and 14.38/37.60 mM)] in Murashige and Skoog medium were evaluated for biomass and withanolide A production. The highest accumulation of biomass (139.42 g l^?1 FW and 13.11 g l^?1 DW) was recorded in the medium with 2.0× concentration of KH₂PO₄, and the highest production of withanolide A was recorded with 2.0× KNO₃ (15.27 mg g^?1 DW). The NH₄ ⁺/NO₃ ^? ratio also influenced root growth and withanolide A production, with both parameters being larger when the NO₃ ^? concentration was higher than that of NH₄ ⁺. Maximum biomass growth (148.17 g l^?1 FW and 14.79 g l^?1 DW) was achieved at NH₄ ⁺/NO₃ ^? ratio of 14.38/37.60 mM, while withanolide A production was greatest (14.68 mg g^?1 DW) when the NH₄ ⁺/NO₃ ^? ratio was 0.00/18.80 mM. The results are useful for the large scale cultivation of Withania hairy root culture for the production of withanolide A.     

Optimal C:N ratio for the production of red pigments by Monascus ruber

The carbon-to-nitrogen (C:N) ratio in the biomass of microfungi tends to be quite different (e.g. 10–15) compared with the C:N ratio in the red pigments (e.g. >20) of the fungus Monascus ruber. Therefore, determining an optimal C:N ratio in the culture medium for maximizing the production of the pigments is important. A culture medium composition is established for maximizing the production of the red pigment by the fungus M. ruber ICMP 15220 in submerged culture. The highest volumetric productivity of the red pigment was 0.023 AU L^?1 h^?1 in a batch culture (30 °C, initial pH of 6.5) with a defined medium of the following composition (g L^?1): glucose (10), monosodium glutamate (MSG) (10), MgSO₄·7H₂O (0.5), KH₂PO₄ (5), K₂HPO₄ (5), ZnSO₄·7H₂O (0.01), FeSO₄·7H₂O (0.01), CaCl₂ (0.1), MnSO₄·H₂O (0.03). This medium formulation had a C:N mole ratio of 9:1. Under these conditions, the specific growth rate of the fungus was 0.043 h^?1 and the peak biomass concentration was 6.7 g L^?1 in a 7-day culture. The biomass specific productivity of the red pigment was 1.06 AU g^?1 h^?1. The best nitrogen source proved to be MSG although four other inorganic nitrogen sources were evaluated.     

Decolorization of beet molasses vinasse by Bacillus megaterium ATCC 14581

Beet molasses vinasse is formed in large quantities as a waste product of the distillery industry, where the molasses derived from sugar beet is used as a raw material. This vinasse has a dark color, low pH, and chelating properties. Many microorganisms have the ability to decolorize and biodegrade beet molasses vinasse. In this work, different cultivation parameters (the type of the bacterial culture; static or agitated) and medium components ((NH₄)₂SO₄, KH₂PO₄, yeast extract, glucose, and peptone, and the vinasse concentration) were evaluated utilizing Plackett-Burman design to identify the important factors influencing the vinasse decolorization by Bacillus megaterium ATCC 14581. The significant variables were selected as follows: (NH₄)₂SO₄, KH₂PO₄, glucose, and the concentration of vinasse. These four factors should be chosen as being promising for further optimization studies. The maximum color removal was 38%.     

Biogeochemical regime shifts in coastal landscapes: the contrasting effects of saltwater incursion and agricultural pollution on greenhouse gas emissions from a freshwater wetland

Many coastal plain wetlands receive nutrient pollution from agricultural fields and are particularly vulnerable to saltwater incursion. Although wetlands are a major source of the greenhouse gases methane (CH₄) and nitrous oxide (N₂O), the consequences of salinization for greenhouse gas emissions from wetlands with high agricultural pollution loads is rarely considered. Here, we asked how saltwater exposure alters greenhouse gas emissions from a restored freshwater wetland that receives nutrient loading from upstream farms. During March to November 2012, we measured greenhouse gases along a ~2 km inundated portion of the wetland. Sampling locations spanned a wide chemical gradient from sites receiving seasonal fertilizer nitrogen and sulfate (SO₄ ^2?) loads to sites receiving seasonal increases in marine salts. Concentrations and fluxes of CH₄ were low (<100 µg L^?1 and <10 mg m^?2 h^?1) for all sites and sampling dates when SO₄ ^2? was high (>10 mg L^?1), regardless of whether the SO₄ ^2? source was agriculture or saltwater. Elevated CH₄ (as high as 1,500 µg L^?1 and 45 mg m^?2 h^?1) was only observed on dates when air temperatures were >27 °C and SO₄ ^2? was <10 mg L^?1. Despite elevated ammonium (NH₄ ⁺) for saltwater exposed sites, concentrations of N₂O remained low (<5 µg L^?1 and <10 µg m^?2 h^?1), except when fertilizer derived nitrate (NO₃ ^?) concentrations were high and N₂O increased as high as 156 µg L^?1. Our results suggest that although both saltwater and agriculture derived SO₄ ^2? may suppress CH₄, increases in N₂O associated with fertilizer derived NO₃ ^? may offset that reduction in wetlands exposed to both agricultural runoff and saltwater incursion.     

Improved production of <Emphasis Type="Italic">Oroxylum indicum</Emphasis> (L.) Kurz by altering the salts of the medium

The present study was conducted to test the effects of KNO₃, KH₂PO₄, and CaCl₂ on shoot multiplication, root proliferation, and accumulation of phytochemicals in in vitro cultures of Oroxylum indicum. The results indicate that modifying the MS salt formulation in relation to particular inorganic nutrients highly affected shoot multiplication, root proliferation, and accumulation of flavonoids in in vitro cultures. A concentration of 0.60 g L^?1 CaCl₂ resulted in the highest frequency of shoot regeneration (5.6 shoots per explant). A concentration of 0.40 g L^?1 CaCl₂ resulted in the highest frequency of root regeneration (7.8 roots per shoot). Modifications of the concentrations of inorganic salts were also found to be advantageous for production media for both multiple shoots and shoot-derived root in vitro cultures. Multiple shoots generated on shoot induction medium with a concentration of 0.60 g L^?1 CaCl₂ and roots generated on root induction medium with a concentration of 1.5 g L^?1 KNO₃ yielded about a five times higher flavonoid level than cultures generated on control medium respectively.     

An effective method for the detoxification of cyanide-rich wastewater by Bacillus sp. CN-22

The biodetoxification of cyanide-rich wastewater has become increasingly popular because of its cost-effectiveness and environmental friendliness. Therefore, we have developed an effective method, optimised by response surface methodology, for detoxifying cyanide-rich wastewater using Bacillus sp. CN-22, which was newly isolated from a cyanide-contaminated electroplating sludge and could tolerate a CN^? concentration of 700 mg L^?1. The concentration of CN^? in the treated wastewater decreased from 200 to 6.62 mg L^?1 after cultivation with 2.38 % inocula for 72 h on the medium, consisting of 0.05 % KH₂PO₄, 0.15 % K₂HPO₄, 1.0 mM MgCl₂, 1.0 mM FeCl₃, 0.1 % NH₄Cl, and 0.1 % glycerol. The CN^? degradability of 96.69 % is similar to the predicted value of 96.82 %. The optimal cultivation conditions were controlled as follows: initial pH, 10.3; temperature, 31 °C; and rotary speed, 193 rpm. The maintenance of higher pH in the overall treatment procedures may avoid the production of volatile HCN and the risk associated with cyanide detoxification. Additionally, the bacterial strain Bacillus sp. CN-22, with its potent cyanide-degrading activity at the initial CN^– concentration of 200 mg L^?1, may be employed to effectively treat cyanide-rich wastewater, especially electroplating effluent.     

Reduced late-season leaf potassium and phosphorus levels influence decreases in sugar contents of bagged apple fruit

Fruit bagging has been widely used in the fruit production industry; however, in apples, it is known to cause a significant decrease in fruit sugar contents. To address this issue and identify the changes in leaf mineral contents associated with fruit sugar levels in bagged apples, aqueous solutions of 10 g L^?1 CaCl₂, 5 g L^?1 KH₂PO₄, or 2 g L^?1 Na₂B₄O₇·10H₂O were foliar sprayed during four fruit developmental stages. The late-season leaf phosphorus (P) and potassium (K) contents after the rapid fruit growth period and the soluble sugar contents in ripening fruit were significantly lower in bagged fruit than in non-bagged fruit (11.1–15.09 %). The decreases in leaf P and K contents caused by bagging were almost completely compensated for by foliage applications of CaCl₂, KH₂PO₄, or Na₂B₄O₇ during the fruit set period. Therefore, the fruit soluble sugar contents were significantly higher in bagged ripening fruit with foliar spray than in bagged fruit without foliar sprays, reaching the levels of non-bagged apples. The decrease in the sugar contents of bagged apples was closely associated with the decrease in late-season leaf P and K levels caused by fruit bagging.     

Medium optimization for the feather-degradation by <Emphasis Type="Italic">Streptomyces fradiae Var</Emphasis><Emphasis Type="Italic">S-221</Emphasis> using the response surface methodology

In order to accelerate biodegradation of feather into more amino acids, the fermentation medium of feather-biodegrading Streptomyces fradiae Var S-221 was optimized in this paper. In the first optimization step, the effects of feather powder, beet molasses, (NH₄)₂SO₄ and KH₂PO₄ on amino acids formation were evaluated by using full factorial design. The results showed that feather powder and (NH₄)₂SO₄ had significant and positive effects on feather-biodegradation into amino acids. Then, the method of the steepest ascent was used to access the optimal region of the two significant factors. In the third step, the concentration of feather powder and (NH₄)₂SO₄ were further optimized with central composite design and response surface analysis. As a result, the composition of the optimal medium for S. fradiae Var S-221 fermentation were as follows (g/100 ml): feather powder, 19.504; beet molasses, 4.0; (NH₄)₂SO₄, 1.467; KH₂PO₄, 0.3; MgSO₄, 0.15; FeSO₄, 0.001; ZnSO₄, 0.0001; and MnSO₄, 0.0001. Using this optimal fermentation medium, the amino acids concentration was increased from 4.61 to 6.13 g/100 ml.     

Improved gellan gum production by a newly-isolated Sphingomonas azotifigens GL-1 in a cheese whey and molasses based medium

Gellan gum is a water-soluble exopolysaccharide, it has applications in the food, pharmaceutical and chemical industries. In this study, a gellan gum producing strain was isolated from rice root, and this strain was identified be the species of Sphingomonas azotifigens. The Plackett-Burman design was applied to investigate the main factors affecting gellan gum production by S. azotifigens GL-1 in a molasses and cheese whey based medium; the medium compositions were optimized by response surface methodology. The optimum cheese whey based medium consisted of cheese whey 68.34 g/L, Na₂HPO₄ 14.58 g/L and KH₂PO₄ 7.66 g/L, and the maximum gellan gum production that using this medium was 33.75 ± 1.55 g/L. 14.75 ± 0.65 g/L gellan gum was obtained with an optimized molasses medium, which consisted of molasses 50 g/L, Na₂HPO₄ 9.71 g/L and KH₂PO₄ 5.92 g/L. The molecular weight of gellan gum obtained from two medias were 1.06 × 10⁶ and 0.89 × 10⁶ Da, respectively. The cheese whey-derived gellan gum showed a higher rhamnose, lower glucuronic acid and higher glycerate content compared to the molasses-derived gellan gum. S. azotifigens GL-1 has a high gellan gum production capacity in a cheap medium suggesting it has great potential as an industrial gellan gum producer.     

Citric Acid Production from Hydrolysate of Pretreated Straw Cellulose by Yarrowia lipolytica SWJ-1b Using Batch and Fed-Batch Cultivation

In this study, crude cellulase produced by Trichoderma reesei Rut-30 was used to hydrolyze pretreated straw. After the compositions of the hydrolysate of pretreated straw were optimized, the study showed that natural components of pretreated straw without addition of any other components such as (NH₄)₂SO₄, KH₂PO₄, or Mg²⁺ were suitable for citric acid production by Yarrowia lipolytica SWJ-1b, and the optimal ventilatory capacity was 10.0 L/min/L medium. Batch and fed-batch production of citric acid from the hydrolysate of pretreated straw by Yarrowia lipolytica SWJ-1b has been investigated. In the batch cultivation, 25.4 g/L and 26.7 g/L citric acid were yields from glucose and hydrolysate of straw cellulose, respectively, while the cultivation time was 120 hr. In the three-cycle fed-batch cultivation, citric acid (CA) production was increased to 42.4 g/L and the cultivation time was extended to 240 hr. However, iso-citric acid (ICA) yield in fed-batch cultivation (4.0 g/L) was similar to that during the batch cultivation (3.9 g/L), and only 1.6 g/L of reducing sugar was left in the medium at the end of fed-batch cultivation, suggesting that most of the added carbon was used in the cultivation.     

Evaluation of different culture conditions ofClostridium bifermentans DPH-1 for cost effective PCE degradation

Clostridium bifermentans strain DPH-1 has already been found to dechlorinate perchloroethylene (PCE) tocis-dichloroethylene (cis-DCE)via trichloroethylene (TCE). In this study, our investigation on different culture conditions of this DPH-1 strain was extended to find a more efficient and cost effective growth medium composition for this DPH-1 strain in bioremediation practices. Temperature dependency of strain DPH-1 showed that the growth starting time and PCE degradation at 15°C was very slow compared to that of 30°C, but complete PCE degradation occurred in both cases. For the proper utilization of strain DPH-1 in more cost effective bioremediation practices, a simpler composition of an effective media was studied. One component of the culture medium, yeast extract, had been substituted by molasses, which served as a good source of electron donor. The DPH-1 strain in the medium containing molasses, in the presence of K₂HPO₄ and KH₂PO₄, showed identical bacterial multiplication (0.135 mg protein mL⁻¹h⁻¹) and PCE degradation rates (0.38 μM/h) to those of the yeast extract containing medium.     

Culture and exopolysaccharide production from sugarcane molasses by Gordonia polyisoprenivorans CCT 7137, isolated from contaminated groundwater in Brazil

Gordonia polyisoprenivorans CCT 7137 was isolated from groundwater contaminated with leachate in an old controlled landfill (São Paulo, Brazil), and cultured in GYM medium at different concentrations of sugarcane molasses (2%, 6%, and 10%). The strain growth was analyzed by monitoring the viable cell counts (c.f.u. mL^?1) and optical density and EPS production was evaluated at the end of the exponential phase and 24 h after it. The analysis of the viable cell counts showed that the medium that most favored bacterial growth was not the one that favored EPS production. The control medium (GYM) was the one that most favored the strain growth, at the maximum specific growth rate of 0.232 h^?1. Differences in bacterial growth when cultured at three different concentrations of molasses were not observed. Production of EPS, in all culture media used, began during the exponential phase and continued during the growth stationary phase. The highest total EPS production, after 24 h of stationary phase, was observed in 6% molasses medium (172.86 g L^?1) and 10% (139.47 g L^?1) and the specific total EPS production was higher in 10% molasses medium (39.03 × 10^?11 g c.f.u.^?1). After the exponential phase, in 2%, 6%, and 10% molasses media, a higher percentage of free exopolysaccharides (EPS) was observed, representing 88.4%, 62.4%, and 64.2% of the total, respectively. A different result was observed in pattern medium, which presented EPS made up of higher percentage of capsular EPS (66.4% of the total). This work is the first study on EPS production by G. polyisoprenivorans strain in GYM medium and in medium utilizing sugarcane molasses as the sole nutrient source and suggests its potential use for EPS production by G. polyisoprenivorans CCT 7137 aiming at application in biotechnological processes.     

Improving culture conditions for poly(3-hydroxybutyrate-co-3-hydroxyvalerate) production by Bacillus sp. ND153, a bacterium isolated from a mangrove forest in Vietnam

The production of polyhydroxyalkanoate (PHA) by Bacillus sp. ND153, a bacterium strain isolated from a mangrove forest in Vietnam, was studied. Bacillus sp. ND153 was grown on HM-1 medium with different carbon sources (e.g. glucose, sucrose, maltose, dextrin, and starch). Glucose was found to be the most suitable carbon source for PHA accumulation, whereas starch and dextrin favored cell growth over PHA accumulation. Optimization of the culture medium for PHA production was investigated by applying factorial design, and a maximum PHA content of 79 % (w/w) was obtained with low concentrations of NH₄Cl and MgSO₄ and a high concentration of KH₂PO₄ in the medium. Propionate was used as the precursor for the production of copolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), and the amount of 3-hydroxyvalerate (3HV) in the polymer showed an increasing linear trend with the increase in propionate concentration from 0.2 g l^?1 to 1.0 g l^?1. Thus, the production of PHBV by Bacillus sp. ND153, with 3HV fraction ranging from 1 mol% to 30 mol%, was noted to be high, and the characteristics of fast cell growth and accumulation of PHA exhibited by Bacillus sp. ND153 make it a promising choice for biopolyester production.     

Characterization of cell growth and starch production in the marine green microalga Tetraselmis subcordiformis under extracellular phosphorus-deprived and sequentially phosphorus-replete conditions

Microalgal starch is a potential feedstock for biofuel production. Nutrient stress is widely used to stimulate starch accumulation in microalgae. Cell growth and starch accumulation in the marine green microalga Tetraselmis subcordiformis were evaluated under extracellular phosphorus deprivation with initial cell densities (ICD) of 1.5, 3.0, 6.0, and 9.0?×?10⁶ cells mL^?1. The intracellular stored phosphorus supported cell growth when extracellular phosphorus was absent. The maximum starch content of 44.1 % was achieved in the lowest ICD culture, while the maximum biomass productivity of 0.71 g L^?1 day^?1, starch concentration of 1.6 g L^?1, and starch productivity of 0.30 g L^?1 day^?1 were all obtained in the culture with the ICD of 3.0?×?10⁶ cells mL^?1. Appropriate ICD could be used to regulate the intracellular phosphorus concentration and maintain adequate photosynthetic activity to achieve the highest starch productivity, along with biomass and starch concentration. The recovery of phosphorus-deprived T. subcordiformis in medium containing 0.5, 1.0, or 6.0 mM KH₂PO₄ was also tested. Cell growth and starch accumulation ability could be recovered completely. A phosphorus pool in T. subcordiformis was shown to manipulate its metabolic activity under different environmental phosphorus availability. Though lower starch productivity and starch content were achieved under phosphorus deprivation compared with nitrogen- or sulfur-deprived conditions, the higher biomass and starch concentration make T. subcordiformis a good candidate for biomass and starch production under extracellular phosphorus deprivation.     

Enhancing ethanol production from cellulosic sugars using <Emphasis Type="Italic">Scheffersomyces (Pichia) stipitis</Emphasis>

Studies were performed on the effect of CaCO₃ and CaCl₂ supplementation to fermentation medium for ethanol production from xylose, glucose, or their mixtures using Scheffersomyces (Pichia) stipitis. Both of these chemicals were found to improve maximum ethanol concentration and ethanol productivity. Use of xylose alone resulted in the production of 20.68 ± 0.44 g L^?1 ethanol with a productivity of 0.17 ± 0.00 g L^?1 h^?1, while xylose plus 3 g L^?1 CaCO₃ resulted in the production of 24.68 ± 0.75 g L^?1 ethanol with a productivity of 0.21 ± 0.01 g L^?1 h^?1. Use of xylose plus glucose in combination with 3 g L^?1 CaCO₃ resulted in the production of 47.37 ± 0.55 g L^?1 ethanol (aerobic culture), thus resulting in an ethanol productivity of 0.39 ± 0.00 g L^?1 h^?1. These values are 229 % of that achieved in xylose medium. Supplementation of xylose and glucose medium with 0.40 g L^?1 CaCl₂ resulted in the production of 44.84 ± 0.28 g L^?1 ethanol with a productivity of 0.37 ± 0.02 g L^?1 h^?1. Use of glucose plus 3 g L^?1 CaCO₃ resulted in the production of 57.39 ± 1.41 g L^?1 ethanol under micro-aerophilic conditions. These results indicate that supplementation of cellulosic sugars in the fermentation medium with CaCO₃ and CaCl₂ would improve economics of ethanol production from agricultural residues.     

Feasibility of Typha Latifolia for High Salinity Effluent Treatment in Constructed Wetlands for Integration in Resource Management Systems

High salinity wastewaters have limited treatment options due to the occurrence of salt inhibition in conventional biological treatments. Using recirculating marine aquaculture effluents as a case study, this work explored the use of Constructed Wetlands as a treatment option for nutrient and salt loads reduction. Three different substrates were tested for nutrient adsorption, of which expanded clay performed better. This substrate adsorbed 0.31 mg kg^?1 of NH₄ ⁺?N and 5.60 mg kg^?1 of PO₄ ^3??P and 6.9 mg kg^?1 dissolved salts after 7 days of contact. Microcosms with Typha latifolia planted in expanded clay and irrigated with aquaculture wastewater (salinity 2.4%, 7 days hydraulic retention time, for 4 weeks), were able to remove 94% NH₄ ⁺?N (inlet 0.25 ± 0.13 mg L^?1), 78% NO₂ ^??N (inlet 0.78 ± 0.62 mg L^?1), 46% NO₃ ^??N (inlet 18.83 ± 8.93 mg L^?1) whereas PO₄ ^3??P was not detected (inlet 1.41 ± 0.21 mg L^?1). Maximum salinity reductions of 52% were observed. Despite some growth inhibition, plants remained viable, with 94% survival rate. Daily treatment dynamics studies revealed rapid PO₄ ^3??P adsorption, unbalancing the N:P ratio and possibly affecting plant development. An integrated treatment approach, coupled with biomass valorization, is suggested to provide optimal resource management possibilities.     

Characterization and application of bioflocculant prepared by Rhodococcus erythropolis using sludge and livestock wastewater as cheap culture media

A new bioflocculant was produced by culturing Rhodococcus erythropolis in a cheap medium. When culture pH was 7.0, inoculum size was 2 % (v/v), Na₂HPO₄ concentration was 0.5 g L^?1, and the ratio of sludge/livestock wastewater was 7:1 (v/v), a maximum flocculating rate of 87.6 % could be achieved. Among 13 different kinds of pretreatments for sludge, the optimal one was the thermal-alkaline pretreatment. Different from a bioflocculant produced in a standard medium, this bioflocculant was effective over a wide pH range from 2 to 12 with flocculating rates higher than 98 %. Approximately, 1.6 g L^?1 of crude bioflocculant could be harvested using cold ethanol for extraction. This bioflocculant showed color removal rates up to 80 % when applied to direct and disperse dye solutions, but only 23.0 % for reactive dye solutions. Infrared spectrum showed that the bioflocculant contained functional groups such as –OH, –NH₂, and –CONH₂. Components in the bioflocculant consisted of 91.2 % of polysaccharides, 7.6 % of proteins, and 1.2 % of DNA. When the bioflocculant and copper sulfate (CuSO₄) were used together for decolorization in actual dye wastewater, the optimum decolorization conditions were specified by the response surface methodology as pH 11, bioflocculant dosage of 40 mg/L, and CuSO₄ 80 mg/L, under which a decolorization rate of 93.9 % could be reached.     

Medium factors on anaerobic production of rhamnolipids by <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> SG and a simplifying medium for in situ microbial enhanced oil recovery applications

Aerobic production of rhamnolipid by Pseudomonas aeruginosa was extensively studied. But effect of medium composition on anaerobic production of rhamnolipid by P. aeruginosa was unknown. A simplifying medium facilitating anaerobic production of rhamnolipid is urgently needed for in situ microbial enhanced oil recovery (MEOR). Medium factors affecting anaerobic production of rhamnolipid were investigated using P. aeruginosa SG (Genbank accession number KJ995745). Medium composition for anaerobic production of rhamnolipid by P. aeruginosa is different from that for aerobic production of rhamnolipid. Both hydrophobic substrate and organic nitrogen inhibited rhamnolipid production under anaerobic conditions. Glycerol and nitrate were the best carbon and nitrogen source. The commonly used N limitation under aerobic conditions was not conducive to rhamnolipid production under anaerobic conditions because the initial cell growth demanded enough nitrate for anaerobic respiration. But rhamnolipid was also fast accumulated under nitrogen starvation conditions. Sufficient phosphate was needed for anaerobic production of rhamnolipid. SO₄ ^2? and Mg²⁺ are required for anaerobic production of rhamnolipid. Results will contribute to isolation bacteria strains which can anaerobically produce rhamnolipid and medium optimization for anaerobic production of rhamnolipid. Based on medium optimization by response surface methodology and ions composition of reservoir formation water, a simplifying medium containing 70.3 g/l glycerol, 5.25 g/l NaNO₃, 5.49 g/l KH₂PO₄, 6.9 g/l K₂HPO₄·3H₂O and 0.40 g/l MgSO₄ was designed. Using the simplifying medium, 630 mg/l of rhamnolipid was produced by SG, and the anaerobic culture emulsified crude oil to EI₂₄ = 82.5 %. The simplifying medium was promising for in situ MEOR applications.     

Effect of nitrogen and phosphate on in vitro growth and metabolite profiles of <Emphasis Type="Italic">Stevia rebaudiana</Emphasis> Bertoni (Asteraceae)

The commercialization of Stevia rebaudiana Bertoni (Asteraceae) extracts as a natural sweetener is driving interest in the use of in vitro propagation systems as an alternative source of steviol glycosides. Out of this suite of chemicals, stevioside is the most abundant but rebaudioside A is the sweetest. We established an in vitro propagation method from germinated seedlings on a Murashige and Skoog (MS) (Physiol Plant 15:473–497, 1962) medium with aims to study the effects of nitrogen and phosphate on the growth and metabolite profiles of S. rebaudiana plants. Generally, NH₄NO₃ is supplied at a concentration of 20.61 mM in MS medium and together with 18.79 mM KNO₃, provide nitrogen to in vitro growing plants. In this study, we used a range of 0.3–72.1 mM NH₄NO₃ and 9.4–65.8 mM KNO₃ and generated six different media with altered nitrogen. Similarly, six different concentrations of KH₂PO₄, ranging from 0.6 to 4.4 mM were tested for the phosphate treatments and the control medium had 1.25 mM KH₂PO₄. By reducing the nitrogen and phosphate levels to half, respectively, this led to the tallest plants. Increasing concentrations of nitrogen in the medium significantly lowered the amount of rebaudioside A as plants on the control medium accumulated 270 mg g^?1 rebaudioside A compared to those that were on a medium with 3.5 times the nitrogen supply (30 mg g^?1 rebaudiose A). Steviol increased with increasing nitrogen available to the microplants. The highest levels of stevioside (740 mg g^?1) quantified was linked to microplants on a medium with half the phosphate concentration. To further assess changes to the metabolomic profiles of treated microplants, LC–MS/MS was used in combination with multivariate statistical analyses. Two distinct clusters were revealed after principal component analysis. Steviol hydrate, stevioside hydrate and rebaudioside A contributed significantly to the separation of phosphate-treated plants from those with variable nitrogen concentrations. Chlorogenic acid and its derivatives were linked to changing phosphate concentrations. The clustering suggests different molecular mechanisms at play that are affected by nitrogen and phosphate supply which serve to alter secondary metabolic flux, resulting in different chemical profiles.     

The production of bioflocculants by Bacillus licheniformis using molasses and its application in the sugarcane industry

A bioflocculant produced by B. licheniformis was investigated with regard to a low-cost culture medium and its industrial application. Molasses replaced sucrose as the sole carbon source in bioflocculant fermentation. The optimum low-cost culture medium was determined to be composed of 20 g/L molasses, 0.4 g/L urea, 0.4 g/L NaCl, 0.2 g/L KH₂PO₄, 1.6 g/L K₂HPO₄, and 0.2 g/L MgSO₄. The bioflocculant from B. licheniformis was then applied to treat sugarcane-neutralizing juice to remove colloids, suspended particles, and coloring matters in a sugar refinery factory. The optimal operation conditions were a bioflocculant dosage of 21 U/mL, pH 7.3 and a heating temperature of 100°C. The color and turbidity of the sugarcane juice reached IU 1267 and IU 206, respectively, after clarification with the bioflocculant; these values were almost the same as those acquired following treatment with polyacrylamide (PAM), the most widely applied flocculant in sugar industries. These results suggest the great potential for use of bioflocculants in the sugar refinery process.