Key determinants affecting sheep wool biodegradation directed by a keratinase-producing <Emphasis Type="Italic">Bacillus subtilis</Emphasis> recombinant strain

OVAT (one variable at a time) approach was applied in this study to screen the most important physicochemical key determinants involved in the process of sheep wool biodegradation. The process was directed by a keratinase-producing Bacillus subtilis DB 100 (p5.2) recombinant strain. Data indicate that, sheep wool could be degraded efficiently in cultures incubated at 30°C, with initial pH of 7 with agitation at 150 rpm. Two times autoclaved alkali treated and undefatted chopped sheep wool is more accessible to biodegradation. B. subtilis recombinant cells could utilize sheep wool as a sole source of carbon and nitrogen. Sheep wool-based modified basal medium II, lacking NH₄Cl and yeast extract, could greatly support the growth of these bacterial cells. Sheep wool biodegradation was conducted efficiently in the absence of kanamycin consequently; high stability of the recombinant plasmid (p5.2) represents a great challenge upon scaling up this process. Three key determinants (sheep wool concentration, incubation time and inoculum size) imposing considerable constraints on the process are highlighted. Sheep wool-based tap water medium and sheep wool-based distilled water medium were formulated in this study. High levels of released end products, produced from sheep wool biodegradation are achieved upon using these two sheep wool-based water media. Data indicate that, sheep wool hydrolysate is rich in some amino acids, such as tyrosine, phenylalanine, lysine, proline, isoleucine, leucine, valine, aspartic acid and glutamic acid. Moreover, the resulting sheep wool hydrolysate contains soluble proteins of high and intermediate molecular weights. The present study demonstrates a feasible, cheap, reproducible, efficient and rapid biotechnological approach towards utilization of raw sheep wool waste through a recombinant bacterium.     

Biodegradation of chicken feathers waste directed by Bacillus subtilis recombinant cells: scaling up in a laboratory scale fermentor

Biodegradation of chicken feathers waste directed by Bacillus subtilis DB 100 (p5.2) cells was successfully carried out in 14 L Bio Flo 110 laboratory scale fermentor. Seven liters of feathers-based modified basal medium II, feathers-based tap water and feathers-based distilled water separately in the fermentor were inoculated with activated bacterial cells. The fermentation processes were conducted at 37 °C, 700 rpm agitation speed and 0.7 vvm air flow rate in the absence of kanamycin. Highest net levels of released feathers hydrolysis end products [soluble proteins and NH₂-free amino groups] and keratinolytic alkaline protease activity in the fermentor were greatly comparable to those of shake flasks. Interestingly, the plasmid (p5.2) inside the recombinant B. subtilis cells growing in the fermentor displayed 100% stability till the fifth day of incubation and this presents a great challenge. Data certainly would encourage the transfer to larger scale fermentors to carry out feathers biodegradation process.     

Statistical optimization of cellulases production by <Emphasis Type="Italic">Penicillium</Emphasis><Emphasis Type="Italic">chrysogenum</Emphasis> QML-2 under solid-state fermentation and primary application to chitosan hydrolysis

Solid-state fermentation conditions for cellulases production by a newly isolated Penicillium chrysogenum QML-2 were investigated using statistical methods. At first, significant variables for cellulases production including (NH₄)₂SO₄, initial pH and inoculum size were screened by using Plackett-Burman Design. Then the optimal regions of the significant variables were investigated by using the method of steepest ascent. Finally, central composite design and response surface analysis were adopted to determine the optimal values of the significant variables and investigate the combined effects of each variable’s pair on cellulases production. The results showed that the optimal ranges of (NH₄)₂SO₄ concentration, initial pH and inoculum size for three types of cellulases activities were 1.97–2.15 g, pH 4.32–4.41 and 13.3–13.7% (v/w), respectively. Using the mixture of corn stover powder and wheat bran (CSP/WB, 1/1) as carbon source, the optimization resulted in 370.15, 101.76 and 321.56 U/g for maximal endoglucanase activity, filter paper activity and β-glucosidase activity, respectively. Compared with maximum values of cellulases activities (endoglucanase activity 85.21 U/g, filter paper activity 16.62 U/g and β-glucosidase activity 67.68 U/g) obtained under unoptimized conditions, the optimization resulted in 3.34, 5.12 and 3.75 folds improvement for endoglucanase activity, filter paper activity and β-glucosidase activity, respectively. For chitosan hydrolysis, the crude cellulases had the optimal temperature of 55°C, pH of 4.4 and exhibited Michaelis constant (K _m) value of 8.34 mg/ml and maximum velocity (V _max) of 2.21 μmol glucosamine/min by 1 ml of the crude cellulases.     

Characterization of a multifunctional feather-degrading <Emphasis Type="Italic">Bacillus subtilis</Emphasis> isolated from forest soil

In this study, we isolated and characterized a novel feather-degrading bacterium that shows keratinolytic, antifungal and plant growth-promoting activities. A bacterium S8 was isolated from forest soil and confirmed to belong to Bacillus subtilis by BIOLOG system and 16S rRNA gene analysis. The improved culture conditions for the production of keratinolytic protease were 0.1% (w/v) sorbitol, 0.3% (w/v) KNO₃, 0.1% (w/v) K₂HPO₄, 0.06% (w/v) KH₂PO₄ and 0.04% (w/v) MgCl₂·6H₂O (pH 8.0 and 30°C), respectively. In the improved medium containing 0.1% (w/v) feather, keratinolytic protease production was around 53.3 ± 0.3 U/ml at 4 day; this value was 10-fold higher than the yield in the basal feather medium (5.3 ± 0.1 U/ml). After cultivation for 5 days in the improved medium, intact feather was completely degraded. Feather degradation resulted in free –SH group, soluble protein and amino acids production. The concentration of free –SH group in the culture medium was 15.5 ± 0.2 μM at 4 days. Nineteen amino acids including all essential amino acids were produced in the culture medium; the concentration of total amino acid produced was 3360.4 μM. Proline (2809.9 μM), histidine (371.3 μM) and phenylalanine (172.0 μM) were the major amino acids released in the culture medium. B. subtilis S8 showed the properties related to plant growth promotion: hydrolytic enzymes, ammonification, indoleacetic acid (IAA), phosphate solubilization, and broad-spectrum antimicrobial activity. Interestingly, the strain S8 grown in the improved medium produced IAA and antifungal activity, indicating simultaneous production of keratinolytic and antifungal activities and IAA by B. subtilis S8. These results suggest that B. subtilis S8 could be not only used to improve the nutritional value of feather wastes but also is useful in situ biodegradation of feather wastes. Furthermore, it could also be a potential biofertilizer or biocontrol agent applicable to crop plant soil.     

The Influence of Nitrogen Nutrition on the Carbohydrate and Nitrogen Status of Emergent Macrophyte Acorus calamus L.

Carbon and nitrogen balance in Acorus calamus, a wetland species colonising littoral zones with a high trophic status, was studied under experimental conditions using water or sand culture with a defined composition of the nutrient solution. Influence of graded level of N (1.86, 7.5 and 18.6 mM) and/or forms of N (NH₄⁺ versus NO ₃^–) on the content of non-structural carbohydrates, free amino acids, total C, and total N was studied in Acorus rhizomes and roots to find possible connection with a reduced growth of Acorus plants under high N and NH₄⁺–N nutrition described in our previous study [Vojtíšková et al., 2004. Hydrobiologia 518: 9–22]. High N availability and pure NH₄⁺–N nutrition affected the C/N balance of rhizome and root systems of Acorus in a similar way. NH₄⁺–N was the only form of N elevated under the high N treatment. The major proportion of the total non-structural carbohydrates (TNC) was starch (91–93% and 51–64% in rhizomes and roots, respectively). The content of starch was significantly and and negatively affected by high N availability (P = 0.001), as well as by NH₄⁺–N nutrition (P=0.001). Amounts of simple soluble carbohydrates (sucrose, glucose, and fructose) were negligible in comparison to starch in rhizomes and branched roots (up to 5% of TNC), while roots without developed lateral roots (unbranched) contained up to 33% of TNC in the form of simple soluble sugars. Moreover, high hexoses/sucrose ratio, low starch/soluble sugars ratio, high content of N, and low C/N ratio support the notion that unbranched roots are metabolically active young roots with tissue differentiation in progress. A high content of free amino acids, typically with dominance of N-rich amino acids (Arg-46%, Gln-8%, Asn-7%), was found simultaneously with a low carbohydrate content under high N supply, which indicates that NH₄⁺ received is effectively incorporated into the organic form by this species. Since the decrease in carbohydrate content was not accompanied by luxurious growth, other possible carbon consuming processes were discussed in relation to NH₄⁺ nutrition. More dramatic changes in total N than C were found under high N availability resulting a shift in C/N ratio in favour of N. Although the shift towards N metabolism was obvious, no serious carbohydrate depletion occurred, which could explain the reduced growth of Acorus plants under high N and sole NH₄⁺–N nutrition described previously.     

Inhibition of ribonuclease and protease activities in germinating rice seeds exposed to nickel

When the seeds of two rice cvs. Malviya-36 and Pant-12 were germinated up to 120 h in the presence of 200 and 400 μM NiSO₄, a significant reduction in the germination of seeds occurred. Seeds germinating in the presence of 400 μM NiSO₄ showed about 12–20% decline in germination percent, about 20–53% decline in lengths and about 8–34% decline in fresh weights of roots and shoots at 120 h of germination. Ni²⁺ exposure of germinating seeds resulted in apparent increased levels of RNA, soluble proteins, and free amino acids in endosperms as well as embryo axes. A 400 μM Ni²⁺ treatment led to about 58–101% increase in the level of soluble proteins and about 39–107% increase in the level of free amino acids in embryo axes at 96 h of germination. Activities of ribonuclease and protease declined significantly with increasing levels of Ni²⁺ treatment. Isoenzyme profile of RNase as revealed by activity staining indicated decline in the intensities of 3–4 preexisting enzyme isoforms in embryo axes of both the rice cultivars and disappearance of one of the two isoforms in endosperms of cv. Pant-12 due to 400 μM Ni²⁺ treatment. Results suggest that the presence of high level of Ni²⁺ in the medium of germinating rice seeds serves as a stress factor resulting in decreased hydrolysis as well as delayed mobilization of endospermic RNA and protein reserves and causing imbalance in the level of biomolecules like RNA, proteins, and amino acids in growing embryo axes. These events would ultimately contribute to decreased germination of rice seeds in high Ni²⁺ containing environment.     

Production and optimization of polygalacturonase from mango (<Emphasis Type="Italic">Mangifera indica</Emphasis> L.) peel using <Emphasis Type="Italic">Fusarium moniliforme</Emphasis> in solid state fermentation

Mango peel is one of the major wastes from fruit processing industries, which poses considerable disposal problems and ultimately leads to environmental pollution. The objective of the current research was to determine the significant parameters on the production of polygalacturonase from mango peel which is a major industrial waste. Solid state culture conditions for polygalacturonase production by Fusarium moniliforme from dried mango peel powder were optimized by Taguchi’s L-18 orthogonal array experimental design methodology. Eight fungal metabolic influencing variables, viz. temperature, mango peel, inoculum, peptone, ammonium nitrate (NH₄NO₃₎, magnesium sulphate (MgSO₄), zinc sulphate (ZnSO₄) and potassium dihydrogen phosphate (KH₂PO₄) were selected to optimize polygalacturonase production. The optimized parameters composed of temperature (30°C), mango peel (6.5%, g, w/v), inoculum (8%, ml, v/v), peptone (1%, g, w/v), NH₄NO₃ (0.60%, g, w/v), MgSO₄ (0.05%, g, w/v), ZnSO₄ (0.06%, g, w/v) and KH₂PO₄ (0.4%, g, w/v). Based on the influence of interaction of fermentation components of fermentation, these could be classified as the least significant and the most significant at individual and interaction levels. The temperature, inoculum level, mango peel substrate and KH₂PO₄ showed maximum production impact at optimized conditions. From the optimized conditions the polygalacturonase activity was maximized to 43.2 U g⁻¹.     

Efficient COD removal and nitrification in an upflow microaerobic sludge blanket reactor for domestic wastewater

The treatment performance of an upflow microaerobic sludge blanket reactor (UMSB) for synthetic domestic wastewater was investigated at two dissolved oxygen (DO) levels, 0.3–0.5 and 0.7–0.9 mg l⁻¹, focusing on nitrification performance. The higher DO level induced complete nitrification of ammonia nitrogen (NH₃–N), achieving chemical oxygen demand and NH₃–N removals of 97 and 92%, respectively. There were consistently significantly higher nitrate nitrogen (NO₃–N) and nitrite nitrogen (NO₂–N) levels in the effluent, with ~66% of newly-produced oxidised nitrogen as NO₂–N. Despite the high nitrification efficiency, only about 23% of the removed NH₃–N amount from the influent was ultimately transformed into oxidised nitrogen due to the simultaneous nitrification-denitrification. Sludge blanket development and granulation occurred simultaneously in the UMSB.     

Alkaline lipase from a novel strain Burkholderia multivorans: Statistical medium optimization and production in a bioreactor

An alkaline lipase from Burkholderia multivorans was produced within 15 h of growth in a 14 L bioreactor. An overall 12-fold enhanced production (58 U mL⁻¹ and 36 U mg⁻¹ protein) was achieved after medium optimization following the “one-variable-at-a-time” and the statistical approaches. The optimal composition of the lipase production medium was determined to be (% w/v or v/v): KH₂PO₄ 0.1; K₂HPO₄ 0.3; NH₄Cl 0.5; MgSO₄·7H₂O 0.01; yeast extract 0.36; glucose 0.1; olive oil 3.0; CaCl₂ 0.4 mM; pH 7.0; inoculum density 3% (v/v) and incubation time 36 h in shake flasks. Lipase production was maximally influenced by olive oil/oleic acid as the inducer and yeast extract as the additive nitrogen. Plackett–Burman screening suggested catabolite repression by glucose. Amongst the divalent cations, Ca²⁺ was a positive signal while Mg²⁺ was a negative signal for lipase production. RSM predicted that incubation time, inoculum density and oil were required at their higher levels (36 h, 3% (v/v) and 3% (v/v), respectively) while glucose and yeast extract were required at their minimal levels for maximum lipase production in shake flasks. The production conditions were validated in a 14 L bioreactor where the incubation time was reduced to 15 h.     

Nutrient and hormone levels in cotton ovules during embryony

In vitro zygotic and somatic embryogenesis protocols rely on nutrient and hormone levels from media to satisfy the physiological and developmental requirements of embryony. To better understand these requirements for cotton, we quantified levels of major and minor elements, carbohydrates, NH₄ ⁺, free amino acids and six hormones in whole cotton ovules (with fibers removed), nucelli (ovules with integuments removed), or ovule fluid (extracted from the endosperm region). Samples were collected from field-grown cotton at 1–18 days-past-anthesis (DPA) during each of three growing seasons. Replication across 2 years was obtained for carbohydrates, NH₄ ⁺, free amino acids and hormones from nucellus samples. The year effect was large primarily for hormones only. The most abundant minerals across tissue types and years were K, P, Mg and S. Potassium was the most abundant at 260, 600 and 1,660 mmol kg⁻¹ dry mass (DM) in nucelli, whole ovules and ovule fluid, respectively. Magnesium, Ca, Zn and Mn levels were 2–8-fold higher in ovule fluid compared to whole ovules or nucelli. In the free amino acid plus NH₄ ⁺ category, NH₄ ⁺, alanine, serine, glycine, asparagine (plus aspartic acid), glutamine (plus glutamic acid), leucine, threonine and arginine predominated in nucelli and ovule fluid, and levels tended to be higher in the older samples across years and tissue types. Fructose and glucose levels also increased with age with very high levels being found in late DPA ovule fluid. Arabinose, inositol and melibiose were also prominent sugars. Indole-3-acetic acid levels were similar between nucelli and ovule fluid and ranged from 10 to 80 μmol kg⁻¹ DM. An abscisic acid spike, from 15 to 400 μmol kg⁻¹ DM, occurred in nucelli and whole ovules from 2 to 8 DPA. Thereafter, abscisic acid levels remained between 5 and 10 μmol kg⁻¹ DM. Zeatin and zeatin riboside were the most abundant cytokinins, and levels of these hormones fluctuated between 1 and 4 μmol kg⁻¹ DM in both nucelli and ovule fluid.     

Zhihengliuella salsuginis sp. nov., a moderately halophilic actinobacterium from a subterranean brine

A novel moderately halophilic, alkaliphilic, non-motile, non-sporulating, catalase-positive, oxidase-negative, aerobic, coccus-shaped, Gram-positive bacterium, designated strain JSM 071043^T, was isolated from a subterranean brine sample collected from a salt mine in Hunan Province, China. Growth occurred with 0.5–20% (w/v) NaCl (optimum 5–10%) at pH 6.5–10.5 (optimum pH 8.5) and at 10–40°C (optimum 25–30°C). Good growth also occurred in the presence of 0.5–20% (w/v) KCl (optimum 5–8%) or 0.5–25% (w/v) MgCl₂·6H₂O (optimum 5–10%). The peptidoglycan type was A4α (l-Lys–l-Ala–l-Glu) and major cell-wall sugars were tyvelose and mannose. The major cellular fatty acids were anteiso-C_15:0, iso-C_16:0 and anteiso-C_17:0. Strain JSM 071043^T contained MK-9 and MK-8 as the predominant menaquinones and diphosphatidylglycerol, phosphatidylglycerol and phosphatidylinositol as the major polar lipids. The DNA G + C content was 67.8 mol%. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain JSM 071043^T was a member of the suborder Micrococcineae, and was most closely related to Zhihengliuella halotolerans YIM 70185^T (sequence similarity 98.9%) and Zhihengliuella alba YIM 90734^T (98.2%), and the three strains formed a distinct branch in the phylogenetic tree. The combination of phylogenetic analysis, DNA–DNA relatedness values, phenotypic characteristics and chemotaxonomic data supports the proposal that strain JSM 071043^T represents a novel species of the genus Zhihengliuella, for which the name Z. salsuginis sp. nov. is proposed. The type strain is JSM 071043^T (= DSM 21149^T = KCTC 19466^T).     

Thermostable invertases from Paecylomyces variotii produced under submerged and solid-state fermentation using agroindustrial residues

The filamentous fungus Paecylomices variotii was able to produce high levels of cell extract and extracellular invertases when grown under submerged fermentation (SbmF) and solid-state fermentation, using agroindustrial products or residues as substrates, mainly soy bran and wheat bran, at 40°C for 72 h and 96 h, respectively. Addition of glucose or fructose (≥1%; w/v) in SbmF inhibited enzyme production, while the addition of 1% (w/v) peptone as organic nitrogen source enhanced the production by 3.7-fold. However, 1% (w/v) (NH₄)₂HPO₄ inhibited enzyme production around 80%. The extracellular form was purified until electrophoretic homogeneity (10.5-fold with 33% recovery) by DEAE-Fractogel and Sephacryl S-200 chromatography. The enzyme is a monomer with molecular mass of 102 kDa estimated by SDS–PAGE with carbohydrate content of 53.6%. Optima of temperature and pH for both, extracellular and cell extract invertases, were 60°C and 4.0–4.5, respectively. Both invertases were stable for 1 h at 60°C with half-lives of 10 min at 70°C. Mg²⁺, Ba²⁺ and Mn²⁺ activated both extracellular and cell extract invertases from P. variotii. The kinetic parameters K_m and V_max for the purified extracellular enzyme corresponded to 2.5 mM and 481 U/mg prot⁻¹, respectively.     

Nitrogen retention by peatland buffer areas at six forested catchments in southern and central Finland

We studied the nitrogen retention capacity of six peatland buffer areas constructed in forested catchments in southern and central Finland. The buffers (0.1–4.9% of the total catchment area) were either undrained mires or drained peatlands rewetted 4–7 years before the present study. The N retention capacity was studied by adding ammonium nitrate (NH₄NO₃–N) solution into the inflow waters of the buffers once (one area) or twice (five areas) during a period of 4–6 years. Except for the first N addition in one area, the three largest buffer areas (relative size > 1%) retained the added inorganic N almost completely; their retention efficiencies during the year of addition were >93% for both NO₃–N and NH₄–N. Two of the three small buffers (relative size < 0.25%) were also able to reduce inorganic N from the through-flow waters effectively; their retention capacities for inorganic nitrogen varied between 58 and 89%. However, one small buffer area had a retention capacity of only <20%. The factors contributing to efficient N retention were hydrological load during N addition, relative size of the buffer area, and its length, i.e. the distance between the inflow and outflow points. If there was any release of the added N, it mostly occurred within a relatively short-time period (<100 days) after the treatment. The buffer areas appeared to be efficient and long-term sinks for inorganic nitrogen because the release of N during the 2–4 years after N addition was minor.     

The differing responses of two <Emphasis Type="Italic">Arabidopsis</Emphasis> ecotypes to ammonium are modulated by the photoperiod regime

Responses to excessive ammonium (NH₄ ⁺) were compared between two Arabidopsis ecotypes (Col-0, JA22) with respect to different photoperiods in hydroponics. In this study, we showed that external extra NH₄ ⁺ led to severe growth suppression, accumulations of free NH₄ ⁺ and amino acids and increased the activities of glutamine synthetase (GS) and glutamate dehydrogenase (GDH) in shoots of the two Arabidopsis ecotypes. However, the levels of free NH₄ ⁺ and total amino acids increased, whereas the activities of GS, NADH-dependent glutamate synthase and GDH decreased under the continuous light when compared with the light (16 h)–dark (8 h) cycle photoperiod. Statistical analyses suggested that strong correlations exist among the growth reduction, accumulations of free NH₄ ⁺, total amino acids and levels of GS activity in shoots under the high NH₄ ⁺ stress regardless of the photoperiod regimes. Interestingly, under the continuous light, Col-0 showed more resistant to such growth reduction and maintained about onefold higher capability of converting excess free NH₄ ⁺ into amino acids, with onefold higher GS activity induced by the external NH₄ ⁺ when compared with JA22. In contrast, these differences were abolished between Col-0 and JA22 under the light–dark cycle condition. Taken together, our results conclude that the sensitivity to NH₄ ⁺ of Col-0 and JA22 is changed between the continuous light and the light–dark cycle photoperiod, which is correlative to the alteration of the GS activity in shoots.     

Nitrifying granules cultivation in a sequencing batch reactor at a low organics-to-total nitrogen ratio in wastewater

It is possible to cultivate aerobic granular sludge at a low organic loading rate and organics-to-total nitrogen (COD/N) ratio in wastewater in the reactor with typical geometry (height/diameter = 2.1, superficial air velocity = 6 mm/s). The noted nitrification efficiency was very high (99%). At the highest applied ammonia load (0.3 ± 0.002 mg NH₄⁺–N g total suspended solids (TSS)⁻¹ day⁻¹, COD/N = 1), the dominating oxidized form of nitrogen was nitrite. Despite a constant aeration in the reactor, denitrification occurred in the structure of granules. Applied molecular techniques allowed the changes in the ammonia-oxidizing bacteria (AOB) community in granular sludge to be tracked. The major factor influencing AOB number and species composition was ammonia load. At the ammonia load of 0.3 ± 0.002 mg NH₄⁺–N g TSS⁻¹ day⁻¹, a highly diverse AOB community covering bacteria belonging to both the Nitrosospira and Nitrosomonas genera accounted for ca. 40% of the total bacteria in the biomass.     

Effect of different operational conditions on the decolorization of molasses spent wash using once developed soil inoculum

A stirred vessel coupled with membrane unit containing cellulose acetate (0.45 μm) membrane was used to study the decolorization of anaerobically digested molasses spent wash (MSW). The soil collected from the MSW disposal site was used as inoculum to study the decolorization without addition of any additives. The same inoculum was used over a period of 163 days at room temperature to study the decolorization of 12.5–50% (v/v) MSW using different operational conditions. The reactor was entered in to the inhibition mode after the feeding of 50% MSW, which was restored 100% without changing any operational condition. The maximum decolorization obtained for 12.5% (v/v) MSW was 77.22 ± 0.13%. The decolorization achieved for 25, 37.5, and 50% (v/v) MSW was 70.41 ± 0.12, 56.47 ± 0.17, and 48.78 ± 0.09%, respectively. Increase in the utilization of protein and reducing sugar was observed up to 25% MSW whereas, higher concentration showed decrease in the utilization. Results indicate 63% removal of chemical oxygen demand for 12.5% (v/v) MSW. Membrane flux which was significantly reduced after the feeding of 50% MSW was regenerated without changing the washing procedure, however, 35% decrease in sample flux was observed over the continuous use of membrane for the period of 198 days.     

Automated, high-throughput platform for protein solubility screening using a split-GFP system

Overproduction of soluble and stable proteins for functional and structural studies is a major bottleneck for structural genomics programs and traditional biochemistry laboratories. Many high-payoff proteins that are important in various biological processes are “difficult to handle” as protein reagents in their native form. We have recently made several advances in enabling biochemical technologies for improving protein stability (), allowing stratagems for efficient protein domain trapping, solubility-improving mutations, and finding protein folding partners. In particular split-GFP protein tags are a very powerful tool for detection of stable protein domains. Soluble, stable proteins tagged with the 15 amino acid GFP fragment (amino acids 216–228) can be detected in vivo and in vitro using the engineered GFP 1–10 “detector” fragment (amino acids 1–215). If the small tag is accessible, the detector fragment spontaneously binds resulting in fluorescence. Here, we describe our current and on-going efforts to move this process from the bench (manual sample manipulation) to an automated, high-throughput, liquid-handling platform. We discuss optimization and validation of bacterial culture growth, lysis protocols, protein extraction, and assays of soluble and insoluble protein in multiple 96 well plate format. The optimized liquid-handling protocol can be used for rapid determination of the optimal, compact domains from single ORFS, collections of ORFS, or cDNA libraries.     

How aphids decide what is good for them: experiments to test aphid feeding behaviour on Tanacetum vulgare (L.) using different nitrogen regimes

Leaf-chewing herbivores select food with a protein/carbohydrate ratio of 0.8–1.5, whereas phloem sap, which aphids feed on, has a ratio of ~0.1. Enhanced N fertilization increases the amino acid concentration in phloem sap and elevates the N/C ratio. The study examines: (1) whether aphids select between plants of different N nutrition, (2) whether feeding time correlates with the amino acid composition of phloem sap, and (3) at which stage of probing aphids identify the quality of the plant. Uroleucon tanaceti (Mordvilko) and Macrosiphoniella tanacetaria (Kaltenbach), specialist aphids feeding on tansy (Tanacetum vulgare L.), were reared on this host plant grown essentially hydroponically (in Vermiculite) in the greenhouse on 1, 3, 6, or 12 mM NH₄NO₃. One and 3 mM NH₄NO₃ corresponds to the situation found in natural tansy stands. Aphid stylet penetration was monitored by electrical penetration graphs whilst phloem sap was sampled by stylectomy. Both aphid species settled 2–3 times more frequently on plants fertilized with 6 or 12 mM NH₄NO₃. The phloem sap of these plants contained up to threefold higher amino acid concentrations, without a change in the proportion of essential amino acids. No time differences were observed before stylet penetration of plant tissue. After the first symplast contact, most aphids penetrated further, except M. tanacetaria on low-N plants, where 50% withdrew the stylet after the first probing. The duration of phloem feeding was 2–3 times longer in N-rich plants and the time spent in individual sieve tubes was up to tenfold longer. Aphids identified the nutritional quality of the host plant mainly by the amino acid concentration of phloem sap, not by leaf surface cues nor the proportion of essential amino acids. However, U. tanaceti infestation increased the percentage of methionine plus tryptophan in phloem tenfold, thus manipulating the plants nutritional quality, and causing premature leaf senescence.     

<Emphasis Type="Italic">Yaniella soli</Emphasis> sp. nov., a new actinobacterium isolated from non-saline forest soil in China

A novel Gram-stain-positive, slightly halophilic, facultatively alkaliphilic, non-motile, non-sporulating, catalase-positive, oxidase-negative, aerobic bacterium, designated strain JSM 070026^T, was isolated from non-saline forest soil in China. Growth occurred with 0–20% (w/v) NaCl (optimum, 2–4%) and at pH 6.0–10.5 (optimum, pH 8.0) and 5–40°C (optimum, 30°C). Good growth also occurred in the presence of 0–28% (w/v) KCl (optimum, 2–5%) or 0–25% (w/v) MgCl₂·6H₂O (optimum, 1–4%). The peptidoglycan type was A4α (l-Lys–Gly–l-Glu). Cell-wall sugars contained mannose and xylose. The major cellular fatty acids were anteiso-C15:0 and iso-C15:0. Strain JSM 070026^T contained menaquinone 8 as the major respiratory quinone and diphosphatidylglycerol, phosphatidylglycerol and phosphatidylinositol as the major polar lipids. The DNA G + C content of strain JSM 070026^T was 56.7 mol%. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain JSM 070026^T was a member of the suborder Micrococcineae and most closely related to Yaniella flava YIM 70178^T (sequence similarity 99.4%) and Yaniella halotolerans YIM 70085^T (97.9%). The three strains formed a distinct branch in the phylogenetic tree. The combination of phylogenetic analysis, DNA–DNA relatedness values, phenotypic characteristics and chemotaxonomic data supports the proposal that strain JSM 070026^T represents a novel species of the genus Yaniella, for which the name Yaniella soli sp. nov. is proposed. The type strain is JSM 070026^T (=DSM 22211^T = KCTC 13527^T).     

Isolation and characterization of alkalotolerant bacteria and optimization of process parameters for decolorization and detoxification of pulp and paper mill effluent by Taguchi approach

Four different bacterial strains were isolated from pulp and paper mill sludge in which one alkalotolerant isolate (LP1) having higher capability to remove color and lignin, was identified as Bacillus sp. by 16S RNA sequencing. Optimization of process parameters for decolorization was initially performed to select growth factors which were further substantiated by Taguchi approach in which seven factors, % carbon, % black liquor, duration, pH, temperature, stirring and inoculum size, at two levels, applying L-8 orthogonal array were taken. Maximum color was removed at pH 8, temperature 35°C, stirring 200 rpm, sucrose (2.5%), 48 h, 5% (w/v) inoculum size and 10% black liquor. After optimization 2-fold increase in color and lignin removal from 25–69% and 28–53%, respectively, indicated significance of Taguchi approach in decolorization and delignification of lignin in pulp and paper mill effluent. Enzymes involved in the process of decolorization of effluent were found to be xylanase (54 U/ml) and manganese peroxidase (28 U/ml). Treated effluent was also evaluated for toxicity by Comet assay using Saccharomyces cerevisiae MTCC 36 as model organism, which indicated 58% reduction after treatment by bacterium.