Microbiological parameters as indicators of compost maturity

AIMS: The objectives of this study were to determine the changes of microbial properties of pig manure collected from pens with different management strategies and composted using different turning and moisture regimes; relate their association with humification parameters and compost temperature; and identify the most suitable microbial indicators of compost maturity. METHODS AND RESULTS: Six different microbial parameters, including total bacterial count, oxygen consumption rate, ATP content, dehydrogenase activity, and microbial biomass C and N, along with humification parameters [humic acid (HA), fulvic acid (FA) and HA : FA ratio] and compost temperature were monitored during composting. Significant positive correlations were found between temperature and microbial properties, including O2 consumption rate, ATP content, dehydrogenase activity, and microbial biomass N. The humification parameters also showed significant correlations with microbial properties of the manure compost. For instance, HA contents of pig manures was positively correlated with total aerobic heterotrophs, and microbial biomass N and C; and negatively correlated with O2 consumption rate, ATP content, and dehydrogenase activity. Among the six microbial parameters examined, dehydrogenase activity was the most important factor affecting compost temperature and humification parameters. Composting strategies employed in this study affected the speed of composting and time of maturation. If the moisture content is maintained weekly at 60% with a 4-day turning frequency, the pig manure will reach maturity in 56 days. CONCLUSIONS: The composting process went through predictable changes in temperature, microbial properties and chemical components despite differences in the initial pig manure and composting strategies used. Among the six microbial parameters used, dehydrogenase activity is the most suitable indicator of compost maturity. Compared with respiration rate, ATP content and microbial biomass procedures, dehydrogenase activity is the simplest, quickest, and cheapest method that can be used to monitor the stability and maturity of composts. SIGNIFICANCE AND IMPACT OF THE STUDY: The results presented here show that microbial parameters can be used in revealing differences between composts and compost maturity. The statistical relationship established between humification parameters and microbial parameters, particularly dehydrogenase activity, demonstrates that it is possible to monitor the composting process more easily and rapidly by avoiding longer and more expensive analytical procedures.     

COMPARISON OF MICROBIOLOGICAL METHODS FOR MONITORING CHICKEN CARCASS QUALITY

Many methods have been developed to determine microbial levels in food products and these include ATP bioluminescence, hydrophobic grid membrane filtration (HGMF), impediometry and turbidimetry. A comparison of these techniques for detecting microbial levels in chicken carcass rinses was conducted.
Only the ATP bioluminescence assay and the HGMF system showed a good correlation with plate counts (r = 0.82 and r = 0.83, respectively). The repeatability of these methods was acceptable. There was also a significant correlation between results obtained with two turbidimetric methods and HGMF as well as between HGMF and ATP bioluminescence data. However, only the ATP bioluminescence assay was able to provide results of microbial levels on a realtime basis (within 10 min). This would be beneficial for HACCP (Hazard Analysis of Critical Control Points) based programs.     

基于ATP生物发光法的微生物数量快速检测技术的研究进展

微生物数量的快速检测一直是工业生产与食品行业需要解决的问题,腺嘌呤核苷三磷酸(adenosine triphosphate,ATP)生物发光法具有操作简便、检测周期短等优点,可满足一般微生物检测的需求。然而,ATP生物发光法的准确性也受到不同因素的影响,如微生物的ATP检测限值较高、微生物自身及其他因素(如非微生物ATP、提取剂种类、荧光素酶活性等)均对微生物数量的检测产生影响。本文简述了不同微生物数量检测方法的优缺点,介绍了ATP生物发光法的发展历程及原理,综述了非微生物ATP与游离ATP、微生物量、ATP提取剂、荧光素酶等因素对ATP生物发光法灵敏度与稳定性的影响,归纳总结了ATP生物发光法及检测设备在食品、医疗、污水处理等领域的应用现状,并就ATP生物发光法体系的优化及ATP在线检测的应用等方面进行了展望,以期为ATP生物发光法的高效应用提供新的思路。     

Determination of minimal concentrations of biocorrosion inhibitors by a bioluminescence method in relation to bacteria, participating in biocorrosion

By using a bioluminescence ATP assay, we have determined the minimal concentrations of some biocorrosion inhibitors (Katon, Khazar, VFIKS-82, Nitro-1, Kaspii-2, and Kaspii-4) suppressing most common microbial corrosion agents: Desulfovibrio desulfuricans, Desulfovibrio vulgaris, Pseudomonas putida, Pseudomonas fluorescens, and Acidithiobacillus ferrooxidans. The cell titers determined by the bioluminescence method, including not only dividing cells but also their dormant living counterparts, are two- to sixfold greater than the values determined microbiologically. It is shown that the bioluminescence method can be applied to determination of cell titers in samples of oil-field waters in the presence of iron ions (up to 260 mM) and iron sulfide (to 186 mg/l) and in the absence or presence of biocidal corrosion inhibitors.     

Use of a newly developed rapid microbial ATP bioluminescence assay to detect microbial contamination on poultry carcasses

A newly developed rapid microbial ATP bioluminescence test (R-mATP) was shown to be an adequate means to assay the microbial load of poultry carcasses. This assay utilizes differential extraction and filtration to separate somatic from microbial ATP in a very rapid timeframe. The assay requires approximately 5 min to complete; approximately 3.5 min to sample and 90 s analytical time. Correlation coefficient (r) between aerobic colony counts and R-mATP test results (n=329) was 0.82. Post-test probabilities to correctly classify carcasses with different levels of microbial contamination were as high as 98% for samples of ≥3.5 log aerobic CFU per ml. Given the rapidity of this assay, the R-mATP holds potential for monitoring the microbial load of carcasses at poultry-processing critical control points. Other potential applications of this new version of the microbial ATP bioluminescence test are discussed.     

The influence of lignin content and temperature on the biodegradation of lignocellulose in composting conditions

The aim of this research was to study the influence of lignin content and composting temperature on the biodegradation of lignin-containing pulp and paper products in a controlled composting test (European standard prEN 14046). Lignin reduced the biodegradation of the samples, and there was a linear correlation between the lignin content and the biodegradation of pulp and paper products at 58°C. The influence of incubation temperature (35, 50 and 58°C) on biodegradation was studied using bleached kraft paper containing 0.2 wt% lignin and mechanical pulp (stone-ground wood) containing 24–27 wt% lignin. Mechanical pulp biodegraded better at lower temperatures, while kraft paper biodegraded well at all three temperatures. Microbial activity was evaluated by measuring CO₂ evolution and the change in ATP content, and fungal biomass by measuring the ergosterol content during the composting experiments. Kraft paper strongly increased microbial activity during the controlled composting test, but the activity returned to the background level at the end of the composting test. The proportion of sample carbon converted to microbial biomass carbon was considerably higher at lower incubation temperatures. Changes in microbial community structure during biodegradation of mechanical pulp and kraft paper at 50°C were studied by the PCR-based technique denaturing gradient gel electrophoresis. Changes in the microbial community were observed during the intensive degradation phase of kraft paper. Electronic Publication     

Proteomic Analysis of Sauvignon Blanc Grape Skin,Pulp and Seed and Relative Quantification of Pathogenesis-Related Proteins

Thaumatin-like proteins (TLPs) and chitinases are the main constituents of so-called protein hazes which can form in finished white wine and which is a great concern of winemakers. These soluble pathogenesis-related (PR) proteins are extracted from grape berries. However, their distribution in different grape tissues is not well documented. In this study, proteins were first separately extracted from the skin, pulp and seed of Sauvignon Blanc grapes, followed by trypsin digestion and analysis by liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS). Proteins identified included 75 proteins from Sauvignon Blanc grape skin, 63 from grape pulp and 35 from grape seed, mostly functionally classified as associated with metabolism and energy. Some were present exclusively in specific grape tissues; for example, proteins involved in photosynthesis were only detected in grape skin and proteins found in alcoholic fermentation were only detected in grape pulp. Moreover, proteins identified in grape seed were less diverse than those identified in grape skin and pulp. TLPs and chitinases were identified in both Sauvignon Blanc grape skin and pulp, but not in the seed. To relatively quantify the PR proteins, the protein extracts of grape tissues were seperated by HPLC first and then analysed by SDS-PAGE. The results showed that the protein fractions eluted at 9.3 min and 19.2 min under the chromatographic conditions of this study confirmed that these corresponded to TLPs and chitinases seperately. Thus, the relative quantification of TLPs and chitinases in protein extracts was carried out by comparing the area of corresponding peaks against the area of a thamautin standard. The results presented in this study clearly demonstrated the distribution of haze-forming PR proteins in grape berries, and the relative quantification of TLPs and chitinases could be applied in fast tracking of changes in PR proteins during grape growth and determination of PR proteins in berries at harvest.     

Determination of Minimal Concentrations of Biocorrosion Inhibitors by a Bioluminescence Method

By using a bioluminescence ATP assay, we have determined the minimal concentrations of some biocorrosion inhibitors (Kathon, Khazar, VFIKS-82, Nitro-1, Caspii-2, and Caspii-4) suppressing most common microbial corrosion inducers: Desulfovibrio desulfuricans, Desulfovibrio vulgaris, Pseudomonas putida, Pseudomonas fluorescens, and Acidithiobacillus ferrooxidans. The cell titers determined by the bioluminescence method, including not only fissiparous cells but also their dormant living counterparts, are two-to sixfold greater than the values determined microbiologically. It is shown that the bioluminescence method can be applied to determination of cell titers in samples of oil-field waters in the presence of iron ions (up to 260 mM) and iron sulfide (to 186 mg/l) and in the absence or presence of biocidal corrosion inhibitors.__________Translated from Prikladnaya Biokhimiya i Mikrobiologiya, Vol. 41, No. 4, 2005, pp. 429–434.Original Russian Text Copyright © 2005 by Efremenko, Azizov, Makhlis, Abbasov, Varfolomeev.     

生物发光及化学发光在生物医学领域中应用的进展

生物发光和化学发光在生物医学领域内的应用主要包括细胞学检测,分子生物学、卫生学检测,生物传感器、脂质过氧化检测和药物筛选等六个方面,其中细胞学检测主要是利用细胞内ＡＴＰ导致的虫荧光素酶发光进行活细胞计数,目前已实现快速、动态、单细胞分析;同时发现了一些新的与生物或化学发光有关的细胞学指标。分子生物学领域内的应用主要为报告基因和分子杂交,近年来又有人推出了生物发光实时ＤＮＡ测序技术。卫生学检测则主要     

The microbiological aspects of grape marc humification

Summary The microbiological aspects of a novel process of grape marc composting have been investigated. It has been possible to determine the succession of populations during the process which are required to obtain the required final product. The initial population comprises exclusively yeasts which, by autolysis and subsequent binding of the residual alcohol by esterifying reactions, enable rapid appearance of a mixed population of bacteria. The temperature increase continued by this bacterial flora favours growth of a thermophilic fungal flora, which is mainly responsible for the microbial decomposition process. The most important organism is Thermomyces lanuginosus Tsiklinski. Final humification is effected by a mixed population of Streptomycetes.It was possible to optimize the process by installing heat exchangers, thereby creating optimum conditions for the most important organism, T. lanuginosus.     

An efficient method for quantitative determination of cellular ATP synthetic activity

The authors have developed an efficient method to measure cellular activity of ATP synthesis. Although ATP is a major energy source of biological reactions, it has been difficult to measure cellular ATP synthetic activity quantitatively. In this report, bioluminescence from the luciferin-luciferase reaction was used for the quantitative measurement. Under the used condition, bioluminescence from standard ATP solution showed no attenuation within several minutes, and the intensity corresponded proportionally to ATP concentrations of the standards. To measure dynamic cellular ATP synthetic activity, combination of osmotic shock and detergent treatment was used to make Escherichia coli cells permeable. ATP was discharged from permeable cells and reacted with externally added luciferase. Because permeable cells used glucose to synthesize and accumulate ATP without further growth, intensity of bioluminescence was increasing during the cellular consumption of glucose. Cellular ATP biosynthetic activity was calculated form the slope of linearly increasing bioluminescence. This permeable cell assay could be applied to high-throughput measuring for dynamic cellular activity of glycolytic ATP synthesis.     

Assay of ATP in intact cells of the facultative phototroph Rhodobacter capsulatus expressing recombinant firefly luciferase

This study reports on the construction, calibration and use of recombinant cells of Rhodobacter capsulatus expressing the luciferase gene of the North American firefly Photinus pyralis to detect, by bioluminescence, variations of endogenous ATP levels under various physiological conditions. We show that the antibiotic polymyxin B allows luciferin to rapidly move into cell cytosol, but does not make external ATP freely accessible to intracellular luciferase. Notably, in toluene:ethanol-permeabilized cells, the apparent K(mATP) for luciferase (50 microM) is similar to that measured in soluble cell fractions. This finding limits the applicability of the firefly luciferase for monitoring intracellular maximal ATP concentration because dark/aerobic-grown recombinant cells of Rba. capsulatus contain approximately 1.3-2.6+/-0.5 mM ATP. Therefore, the effects of chemical and physical factors such as oxygen, light, carbonyl cyanide m-chlorophenyl hydrazone and antimycin A on ATP synthesis were examined in cells subjected to different starvation periods to reduce the endogenous ATP pool below the luciferase ATP saturation level (< or =0.2 mM). We conclude that the amount of endogenous ATP generated by light is maximal in the presence of oxygen, which is required to optimize the membrane redox poise.     

ATP concentrations in multicellular tumor spheroids assessed by single photon imaging and quantitative bioluminescence

To evaluate the interrelationship among the cellular energy status and the development of necrosis in tumor microregions, local ATP concentrations and the extent of necrosis were determined in multicellular tumor spheroids, i.e., in spherical tumor cell aggregates. The spheroids were grown in rotated suspension cultures using EMT6 cells that were derived from a murine mammary sarcoma. The distribution of viable and necrotic cell areas was assessed by histological investigations. The regional distribution of ATP concentrations was measured with a novel technique using quantitative bioluminescence and single photon imaging. This method makes it possible to determine ATP concentrations in absolute terms with a spatial resolution at the level of a single cell. The results show that ATP concentrations in the center of EMT6 spheroids decrease from values of 1.0 to 1.5 mM in small spheroids with 300 microns in diameter to values close to or at the background level in 750 microns spheroids. Necrosis was detectable in spheroids larger than 300 microns, and virtually no spheroid without necrosis was found at sizes larger than 600 microns. Since the emergence of central necrosis precedes the drop in ATP to undetectably low values, the data suggest that energy metabolism is not or not directly involved in the development of necrosis in tumor spheroids under the growth conditions investigated.     

利用ATP扩增反应与生物发光法结合检测微量微生物

摘要:【目的】腺苷酸激酶（adenylate kinase, ADK）和多聚磷酸盐激酶（polyphosphate kinase, PPK）偶联催化的ATP扩增反应结合生物发光检测法能够对微量微生物进行检测。但是PPK当中结合的内源性的ADP会产生背景干扰,影响测定。本文旨在融合表达ADK和PPK,并建立一种方便有效的内源性ADP的去除方法,降低背景,使之与传统生物发光法结合,实现高灵敏生物发光法检测微量ATP及微生物。【方法】PCR扩增得到PPK、ADK基因,插入表达载体pET28a (+)中构建重组表达质粒pET28a (+)-PPKADK,表达PPK-ADK融合蛋白。利用表面包裹聚胺醇（Polyurethane）的磁珠（magnetic beads）,通过化学反应将腺苷酸双磷酸酶（apyrase）固定于磁珠表面,制备固相腺苷酸双磷酸酶（Beads-apyrase）,用于除去与融合蛋白结合的内源性ADP,降低ATP扩增反应的背景,从而使之与生物发光反应相结合,测定微量外源ATP及细菌菌落数。【结果】表达的融合蛋白具有PPK和ADK的活性,利用Beads-apyrase可以方便而有效的去除内源性ADP,显著地降低反应背景,从而实现了利用ATP扩增反应与传统生物发光反应结合,测定了小于1 fmol的外源微量ATP,使生物发光法检测ATP及微生物的灵敏度提高至少100倍。【结论】利用Beads-apyrase能够方便、有效地降低PPK-ADK中的ADP背景,从而使PPK-ADK催化的ATP扩增反应能够与传统生物发光法相结合,极大地提高了生物发光法的灵敏度。     

Recycling of the asialoglycoprotein receptor in isolated rat hepatocytes. ATP depletion blocks receptor recycling but not a single round of endocytosis

Continuous endocytosis of 125I-asialo-orosomucoid (ASOR) mediated by the galactosyl receptor in rat hepatocytes is a cyclic process. 125I-ASOR-receptor complexes are internalized, processed, and the ligand is degraded while the receptor is returned to the cell surface for reutilization. Since a true cycle has a thermodynamic requirement for the input of external energy, we examined the effects of changes in intracellular ATP levels on the function of the receptor cycle. Hepatocytes were depleted of ATP to various extents prior to endocytosis by incubating cells at 15 degrees C in the presence of 2 mM NaF and 0-20 mM NaN3. A luciferase-luciferin bioluminescence assay was used to quantitate the amount of cellular ATP. ATP-depleted cells were allowed to bind 125I-ASOR at 0 degrees C, washed through discontinuous Percoll gradients, and only viable cells were isolated and incubated at 37 degrees C to initiate a synchronous single round of endocytosis. The extent of internalization of this surface-bound 125I-ASOR was unaffected by an ATP depletion to less than 1% of the control level. The rate of internalization of surface-bound ligand was unaffected until the ATP levels decreased to 30% or less; at greater than 98% ATP depletion the initial rate decreased by a maximum of 55% and the kinetics became biphasic. In contrast, continuous endocytosis in the presence of excess ASOR was inhibited by only a 25% decline in cellular ATP content and demonstrated a very sharp threshold response to changing ATP levels. Continuous endocytosis, which requires receptor recycling, was completely inhibited when the total cellular ATP level decreased by only 40%. We conclude that the internalization phase of endocytosis is not dependent on ATP but that the processing and/or externalization phases of the complete receptor cycle are either directly or indirectly dependent on ATP and very sensitive to changes in cellular ATP content.     

ATP as a biomarker of viable microorganisms in clean-room facilities

A new firefly luciferase bioluminescence assay method that differentiates free extracellular ATP (dead cells, etc.) from intracellular ATP (viable microbes) was used to determine the viable microbial cleanliness of various clean-room facilities. For comparison, samples were taken from both clean-rooms, where the air was filtered to remove particles >0.5 microm, and ordinary rooms with unfiltered air. The intracellular ATP was determined after enzymatically degrading the sample's free ATP. Also for comparison, cultivable microbial populations were counted on nutrient-rich trypticase soy agar (TSA) plates. Both the cultivable and ATP-based determinations indicate that the microbial burden was lower in clean-room facilities than in ordinary rooms. However, there was no direct correlation between the two sets of measurements because the two assays measured very different populations. A large fraction of the samples yielded no colony formers on TSA, but were positive for intracellular ATP. Subsequently, genomic DNA was isolated directly from selected samples and 16S rDNA fragments were cloned and sequenced, identifying nearest neighbors, many of which are known to be noncultivable in the media employed. It was concluded that viable microbial contamination can be reliably monitored by measurement of intracellular ATP, and that this method may be considered superior to cultivable colony counts due to its speed and its ability to report the presence of viable but noncultivable organisms. When the detection of nonviable microbes is of interest, the ATP assay can be supplemented with DNA analysis.     

Visualization and Measurement of ATP Levels in Living Cells Replicating Hepatitis C Virus Genome RNA

Adenosine 5′-triphosphate (ATP) is the primary energy currency of all living organisms and participates in a variety of cellular processes. Although ATP requirements during viral lifecycles have been examined in a number of studies, a method by which ATP production can be monitored in real-time, and by which ATP can be quantified in individual cells and subcellular compartments, is lacking, thereby hindering studies aimed at elucidating the precise mechanisms by which viral replication energized by ATP is controlled. In this study, we investigated the fluctuation and distribution of ATP in cells during RNA replication of the hepatitis C virus (HCV), a member of the Flaviviridae family. We demonstrated that cells involved in viral RNA replication actively consumed ATP, thereby reducing cytoplasmic ATP levels. Subsequently, a method to measure ATP levels at putative subcellular sites of HCV RNA replication in living cells was developed by introducing a recently-established Förster resonance energy transfer (FRET)-based ATP indicator, called ATeam, into the NS5A coding region of the HCV replicon. Using this method, we were able to observe the formation of ATP-enriched dot-like structures, which co-localize with non-structural viral proteins, within the cytoplasm of HCV-replicating cells but not in non-replicating cells. The obtained FRET signals allowed us to estimate ATP concentrations within HCV replicating cells as ∼5 mM at possible replicating sites and ∼1 mM at peripheral sites that did not appear to be involved in HCV replication. In contrast, cytoplasmic ATP levels in non-replicating Huh-7 cells were estimated as ∼2 mM. To our knowledge, this is the first study to demonstrate changes in ATP concentration within cells during replication of the HCV genome and increased ATP levels at distinct sites within replicating cells. ATeam may be a powerful tool for the study of energy metabolism during replication of the viral genome.     

Determination of sterilization effectiveness by measuring bacterial growth in a biological indicator through firefly luciferase determination of ATP

A bioluminescence procedure for measurement of microbial ATP allows a rapid determination of the effectiveness of autoclave sterilization. This determination is achieved faster than detection of acid production in a biological indicator via a pH indicator. Bacterial outgrowth from spores on test strips of the biological indicator was detected by measurement of ATP using the firefly luciferase reaction. A measureable increase in ATP was found after 5 hours of incubation of a biological indicator that had been treated under sterilizing conditions that produced 75% sterility of the biological indicator as measured by acid production. This is a marked improvement over the 24-48 hours of incubation currently required.     

Opposite effects of D-fructose on total versus cytosolic ATP/ADP ratio in pancreatic islet cells

D-fructose (10 mM) augments, in rat pancreatic islets, insulin release evoked by 10 mM D-glucose. Even in the absence of D-glucose, D-fructose (100 mM) displays a positive insulinotropic action. It was now examined whether the insulinotropic action of D-fructose could be attributed to an increase in the ATP content of islet cells. After 30-60 min incubation in the presence of D-glucose and/or D-fructose, the ATP and ADP content was measured by bioluminescence in either rat isolated pancreatic islets (total ATP and ADP) or the supernatant of dispersed rat pancreatic islet cells exposed for 30 s to digitonine (cytosolic ATP and ADP). D-fructose (10 and 100 mM) was found to cause a concentration-related decrease in the total ATP and ADP content and ATP/ADP ratio below the basal values found in islets deprived of exogenous nutrient. Moreover, in the presence of 10 mM D-glucose, which augmented both the total ATP content and ATP/ADP ratio above basal value, D-fructose (10 mM) also lowered these two parameters. The cytosolic ATP/ADP ratio, however, was increased in the presence of D-glucose and/or D-fructose. Under the present experimental conditions, a sigmoidal relationship was found between such a cytosolic ATP/ADP ratio and either (86)Rb net uptake by dispersed islet cells or insulin release from isolated islets. These data provide, to our knowledge, the first example of a dramatic dissociation between changes in total ATP content or ATP/ADP ratio and insulin release in pancreatic islets exposed to a nutrient secretagogue. Nevertheless, the cationic and insulinotropic actions of d-glucose and/or d-fructose were tightly related to the cytosolic ATP/ADP ratio.     

Characterization of the binding of the fluorescent ATP analog TNP-ATP to insulysin

It has recently been reported that insulin-degrading enzyme (IDE) contains an allosteric site which binds polyanions such as ATP and PPPi. This site is distinct from the catalytic site where homotrophic allosteric effects are produced. In this study, we have characterized the binding of ATP to this anion binding site using the fluorescent ATP analog 2',3'-O-(2,4,6-trinitrophenyl)-adenosine triphosphate (TNP-ATP), which exhibits a higher affinity to the enzyme than ATP itself. TNP-ATP binding to IDE was accompanied by a more than 4-fold increase in fluorescence. The dissociation constant (K(D)) of TNP-ATP was determined as 1.15 microM, while the activation constant (K(A)) was determined to be 1.6 microM. Competition experiments were used to show that ATP (Ki = 1.3 mM) and PPPi (Ki = 0.9mM) bind with a higher affinity than ADP (2.2 mM) and AMP (4.0 mM). Adenosine did not bind to the anion binding site.