Urease

At the same time that the number of microorganisms was determined, i.e. on days 30 and 60 of the experiment in soil samples, from each repetition in three subsequent replications, the activity of dehydrogenases, catalase, urease, acid phosphatase, alkaline phosphatase, β-glucosidase and arylsulphatase was determined. The substrates used for the determination of the enzyme activity, as well as the units in which the activity of particular enzymes was expressed, are presented in Table 4. The activity of all enzymes, with the exception of catalase, was determined using a Perkin-Elmer Lambda 25 spectrophotometer (MA, USA). The activity of dehydrogenases was determined at a wavelength (λ) of 485 nm; the activity of urease, acid phosphatase and alkaline phosphatase at 410 nm; the activity of β-glucosidase at 400 nm; and the activity of arylsulphatase at 420 nm. The activity of catalase was determined based on the reaction of hydrogen peroxide decomposition using potassium permanganate.Table 4

Methods of determination of soil enzyme activity

Enzyme	Substrate	Product/unit	References
Deh—dehydrogenases (EC 1.1)	2,3,5-Triphenyl tetrazolium chloride (TTC)	Triphenyl fomazan (TFF), μmol kg−1 DM of soil h−1	Öhlinger (1996 )
Cat—catalase (EC 1.11.1.6)	H2O2—aqueous solution	O2, mol kg−1 DM of soil h−1	Alef and Nannipieri (1998 )
Ure—urease (EC 3.5.1.5)	Urea—aqueous solution	N-NH4, mmol kg−1 DM of soil h−1	Alef and Nannipieri (1998 )
Glu—β-glucosidase (EC 3.2.1.21)	4-Nitrophenyl-β-D-glucopyranoside (PNG)	4-Nitrophenol (PN), mmol kg−1 DM of soil h−1	Alef and Nannipieri (1998 )
Pac—acid phosphatase (EC 3.1.3.2)	Disodium 4-nitrophenyl phosphate hexahydrate (PNP)	4-Nitrophenol (PN), mmol kg−1 DM of soil h−1	Alef and Nannipieri (1998 )
Pal—alkaline phosphatase (EC 3.1.3.1)	Disodium 4-nitrophenyl phosphate hexahydrate (PNP)	4-Nitrophenol (PN), mmol kg−1 DM of soil h−1	Alef and Nannipieri (1998 )
Aryl—aryosulphatase (EC 3.1.6.1)	Potassium-4-nitrophenylsulfate (PNS)	4-Nitrophenol (PN), mmol kg−1 DM of soil h−1	Alef and Nannipieri (1998 )

Measurement of the serum anti-H. pylori antibody titer is a noninvasive, inexpensive, and readily available method for detection of H. pylori infection. Histology, culture, polymerase chain reaction (PCR), and the rapid urease test all require biopsy and/or collection of specimens by endoscopy, an invasive technique that is not suitable for mass screening [9 (link), 10 (link)]. The urea breath test and stool antigen test are regarded as noninvasive tests, but the results of both methods are significantly affected by proton pump inhibitor therapy [11 (link)–13 (link)]. However, validated serology tests can be used even in patients being treated with proton pump inhibitors.
H. pylori strains possessing the cytotoxin-associated gene A (CagA) protein, a well-known virulence factor, cause more extensive inflammation and severe atrophy in gastric mucosa than nonproducers [14 (link), 15 (link)]. However, there is still controversy regarding the significance of CagA serology, especially in East Asia, where most strains of H. pylori are CagA producers [16 (link)–19 (link)]. Therefore, gastric cancer screening is usually performed using the H. pylori antibody titer alone, except in limited areas [20 (link)].
Burucoa et al. [21 (link)] investigated the accuracy of 29 different serological tests and reported positive and negative predictive values of 70% and 100%, respectively. In general, better performance in serological screening depends on the use of the appropriate antigens and adjustment of cut-off values [22 (link)]. These considerations are among the disadvantages of using serum H. pylori antibody as a screening test for gastric cancer. Another disadvantage of using H. pylori antibody is that serology alone presents a challenge in distinguishing past and current infections [23 (link)]. The use of serology to identify posteradicated cases is considered later in this review.

Morphological, biochemical, culture and physiological characterization of the actinobacterial isolates of Minnie Bay were performed as recommended by the International Streptomyces Project (ISP) which were described by Shirling and Gottileb [18 ]. Microscopic study was performed with cover slip culture and cellophane method [19 ]. Formation of aerial, substrate mycelium and spore arrangements on mycelium were monitored under a phase contrast microscope (Nikon ECLIPSE E600, USA) at 100× magnification. Culture characteristics such as growth, coloration of aerial and substrate mycelia, formation of soluble pigment were investigated in eight different media including SCA, nutrient agar, yeast malt agar (ISP-2), oat meal agar (ISP-3), inorganic salt agar (ISP-4), glycerol-asparagine agar (ISP-5), peptone yeast extract agar (ISP-6) and tyrosine agar (ISP-7) with the procedures as recommended by ISP. Biochemical characterization, namely, Gram’s reaction, MR-VP, H₂S production, nitrate reduction, oxidase, catalase, urease, starch, casein and gelatin hydrolysis, blood hemolysis, TSI, citrate utilization, esculin and hippurate hydrolysis was also performed as suggested by ISP. Physiological characterization such as, effect of pH (5–11), growth range in NaCl (5-30%) and survival at 50°C was also evaluated. Capability of the isolates to utilize various carbon sources was performed in ISP-2 agar medium with phenol red as indicator [20 ]. Carbon sources viz., fructose, lactose, starch, dextrose, rhamnose, mannitol, maltose, adonitol, arabinose and raffinose were used in this study. Identification of the isolates was made with reference to Bergey’s manual of Systematic Bacteriology [21 ] and Waksman [22 ].

The following chemicals were obtained from Sigma‒Aldrich (USA): Na⁺-selective grade ionic carrier X, Na⁺ tetra [3,5-bis(trifluoromethyl)phenyl]borate (Na–TFPB), valinomycin (ionic carrier K⁺), Na⁺ tetraphenylborate (NaTPB), ETH 129 (Ca²⁺ carrier), 1-nitro-2-(n-octyloxy)benzene (NPOE), bis(2-ethylhexyl)sebacate (DOS), 3,4-ethylenedioxythiophene (EDOT), poly(4-styrenesulfonate) (NaPSS), HCl, H₂SO₄, urease (≥2 units/mg solid, from Candida spp.), bovine serum albumin (BSA), glutaraldehyde solution (20–25%), polyvinyl butyral resin BUTVAR B-98 (PVB), sodium chloride (NaCl), potassium chloride (KCl), calcium chloride (CaCl₂), magnesium chloride (MgCl₂), PBS (pH = 7.2), and methanol.
Au sulfite solution and Ag/AgCl ink were obtained from Yuncaitaotao Company. All the chemicals were used as received. All solutions were prepared using deionized water produced by Millipore Water Purification Systems, unless otherwise noted.

Nitrous oxide flux was analyzed using the closed chamber method (Herr et al., 2020 (link)). In this method, dark PVC boxes were installed, and the samples were drawn every 24 h in the morning using syringes, evacuated into plastic vials, and analyzed chromatographically. Denitrification losses were estimated by the denitrification enzyme assay method described by Smith & Tiedje (1979) (link).
Soil urease activity was analyzed at the 50% flowering stage, calorimetrically, by Bremner & Douglas (1971) (link) method. The normalized difference vegetation index (NDVI) was measured using a green seeker (handheld crop sensor by Trimble, Westminster, CO, USA) at the 50% flowering stage. Infrared gas analyzer (LI-COR Model LI-6400X7 portable photosynthetic system) (IRGA) was used to measure the photosynthetic rate and stomatal conductance. Soil microbial biomass carbon (MBC) and soil microbial biomass nitrogen (MBN) were determined by the chloroform fumigation–extraction method described by Vance, Brookes & Jenkinson (1987) (link) and Brookes et al. (1985) (link), respectively. The N content in grains and straws was also measured using the Kjeldahl method (Kjeldahl, 1883 (link)). After harvesting the crop, yield attributes were calculated from each plot.