Pathogenicity of Pochonia species on eggs of Meloidogyne javanica

Among fungi, species of the genus Pochonia Batista & O.M. Fonseca are considered as promising biological control agents with high potential to reduce root-knot nematode (RKN) and nematode populations. In this research we investigated Fars province of Iran for the presence of Pochonia spp., compared pathogenicity of different Pochonia species on eggs of RKN in vitro, and selected the best isolates for further studies. During 2004-2006, 128 soil samples of fields infested with cyst nematodes and 18 soil samples infested with RKN were collected from Fars province of Iran. In vitro pathogenicity tests were carried out on 36 isolates of Pochonia spp. obtained from CBS and IRAN culture collections. The seven best isolates of this experiment were selected for greenhouse test and their ability in controlling RKN was examined in natural soil. In greenhouse test fresh weight of plant’s tops and roots, gall index, nematode multiplication, second-stage juveniles’ population in soil, reproduction rate (P_f/P_i), proportion of infected eggs, control efficacy, root colonization and soil colony forming units were determined. In vitro pathogenicity of Pochonia on RKN eggs varied between 39% and 95% eggs infected. In greenhouse experiment, three isolates are promising for control of RKN and selected isolates are subjected to more extensive testing to determine their effectiveness in a range of conditions before being developed as commercial biological control agents.     

In vitro cultivation of Schistosoma japonicum-parasites and cells

Schistosomiasis is a serious parasitic zoonosis caused by blood-dwelling flukes of the genus Schistosoma. Understanding functions of genes and proteins of this parasite is important for uncovering this pathogen's complex biology, which will provide valuable information to design new strategies for schistosomiasis control. Effective applications of molecular tools reported to investigate schistosome gene function, such as inhibitor studies and transgenesis, rely on the developments of in vitro cultivation system of this parasite and cells. Besides the in vitro culture studies dealing with Schistosoma mansoni, there are also numerous excellent studies about the in vitro cultivation of Schistosoma japonicum, which were performed by Chinese researchers and published in Chinese journals. Nearly every stage of the life-cycle of S. japonicum, including miracidia, mother sporocysts, cercariae, schistosomula, and egg-laying adult worms, was employed for developing in vitro cultivation methods, being accompanied by the introduction of several media and supplements that helped to improve culture conditions. It was not only possible to generate mother sporocysts from miracidia in vitro, but also to obtain adult worms from cercariae through in vitro cultivation. The main obstacles to complete the life cycle of S. japonicum in the lab are the transition from mother sporocysts to cercariae, and the production of fertilized and completely developed eggs by adult worms generated in vitro. With regard to cells from S. japonicum, besides established isolation protocols and morphological observations, media optimizations were conducted by using different chemical reagents, biological supplements and physical treatment. Among these, mutagens like N-methyl-N-nitro-N-nitrosoguanidine and the addition of extracellular matrix were found to be able to induce mitogenic activities. Although enzyme activities or the level of silver-stained nucleolar region associated protein in cultured cells indicated still suboptimal conditions, the achievements made point to the possibility of reaching the aim of establishing cell lines for S. japonicum. Both the improvements of the in vitro culture of larval and adult worms of S. japonicum as well as the access of cells of this parasite provide excellent advances for research on this important parasite in the future.     

Multifunctional flavonoid dioxygenases: Flavonol and anthocyanin biosynthesis in Arabidopsis thaliana L.

Flavonols and conditionally also anthocyanins, aside from flavonols, are the predominant polyphenols accumulated in various tissues of the model plant Arabidopsis thaliana L. In vitro experiments suggested that the dioxygenases involved in their biosynthesis, flavonol synthase and anthocyanidin synthase, are “multifunctional” enzymes showing distinct side activities. The in vivo relevance of the additional activities attributed to these enzymes, however, has remained obscure. In this review we summarize the most recent results and present final proof of the complementing activities of these synthases for flavonol and anthocyanidin formation in the model plant A. thaliana. The impact of their modification on the biosynthetic pathway and the pattern of flavonoids in different plant tissues are discussed.     

内生真菌感染对宿主羊草抗病性的影响

以感染内生真菌的天然禾草羊草为实验材料,通过体外纯培养条件下的内生真菌、感染内生真菌的离体叶片和在体叶片对3种病原菌的抑菌实验,以探讨内生真菌对宿主植物羊草在抗病性方面的贡献。结果表明:体外纯培养条件下,分离自羊草的内生真菌Epichlobromicola对新月弯孢(Curvularia lunata)、根腐离蠕孢(Bipolaris sorokiniana)和枝孢霉(Cladosporium sp.)这3种病原菌都具有抑制作用,抑菌率分别达56.22%,46.93%和45.15%,且内生真菌培养滤液可以有效抑制这3种病原菌的孢子萌发,平均萌发率分别为30.4%,15.7%和16.4%;宿主植物叶片在离体条件下,内生真菌感染可以有效降低羊草叶片受C.lunata和C.sp.侵染后的病斑数或病斑长度,但对B.sorokiniana不起作用,甚至提高了叶片的病斑数及病斑长度,而离体叶片提取液对不同病原菌均有不同程度的抑制作用;在体条件下,内生真菌均可以通过降低叶片病斑数来增强羊草植株对这3种病原菌的抗性。由此看来,内生真菌E.bromicola对宿主植物羊草在抗病原菌侵染方面有一定的增益作用。     

Amelioration of intracerebroventricular streptozotocin induced cognitive impairment by Evolvulus alsinoides in rats: In vitro and in vivo evidence

Evolvulus alsinoides, also known as Shankpushapi, is a commonly used traditional medicine for enhancing memory. We evaluated the in vitro free radical scavenging and enzymes [acetylcholinesterase, butyrylcholinestrase, glycogen synthase kinase-3-β (GSK-3-β), rho kinase (ROCK II), prolyl endopeptidase (PEP), catechol-O-methyl transferase (COMT) and lipoxygenase (LOX)] inhibitory activities of aqueous and hydro-alcoholic extracts of E. alsinoides. Hydro-alcoholic extract of E. alsinoides demonstrated more free radical scavenging activity as compared to aqueous extract. Hydro-alcoholic extract also showed higher cholinesterase, GSK-3-β, ROCK II, PEP, COMT and LOX enzyme inhibitory activities as compared to aqueous extract. Phytochemical analysis revealed more flavanoids in hydro-alcoholic extract as compared to aqueous extract but no significant difference in phenolic content of the two extracts was observed. Based on in vitro data, hydro-alcoholic extract (100, 300 and 500 mg/kg, p.o.) was selected for in vivo study in intracerebroventricularly injected streptozotocin (STZ) induced cognitive impairment in male Wistar rats. Elevated plus maze, passive avoidance and Morris water maze were used for assessment of cognitive function on 14th, 21st and 28th day after STZ injection. Oxidative stress parameters (malondialdehyde, reduced glutathione, nitric oxide levels and superoxide dismutase activity), cholinergic dysfunction and rho kinase (ROCK II) expression were studied in cerebral cortex and hippocampus of rat brain at the end of the study. Hydro-alcoholic extract of E. alsinoides dose dependently prevented STZ induced cognitive impairment by reducing the oxidative stress, improving cholinergic function and preventing the increase in rho kinase expression. The results suggest an anti-Alzheimer potential of hydro-alcoholic extract of E. alsinoides.     

Toxocara canis: Potential activity of natural products against second-stage larvae in vitro and in vivo

The anthelmintic activity of extracts from Chenopodiumambrosioides, Pycnanthusangolensis and Nutridesintox® was in vitro and in vivo investigated, against Toxocaracanis larvae. The in vitro assays results showed that the aqueous extract of Nutridesintox® was the most effective, followed by C. ambrosioides extracts, hexane, dichloromethane and the infusion. P. angolensis extracts showed a lower anthelmintic activity compared to the other natural products. For the in vivo assays, Nutridesintox®, the hexane extract and the infusion of C. ambrosioides were administered orally to T. canis-infected mice, in single doses, during three consecutive days. The efficacy was evaluated on the 17th day post-infection, not only by counting T. canis larvae in the tissues but also by ELISA detection of IgM and IgG antibodies and histological analysis of liver and lungs. The different treatments did not reduce the larvae burden and had no influence on the antibodies dynamic. Interestingly, a reduction on the inflammatory infiltrates was observed in the liver and lung sections of the group treated with the hexane extract of C. ambrosioides. In conclusion, the hexane extract of C. ambrosioides is of further research interest, as it showed an anthelmintic activity in vitro and a reduction on the inflammatory reaction produced by the infection of T. canis larvae in vivo.     

Interactions of antimicrobial peptides with Leishmania and trypanosomes and their functional role in host parasitism

Antimicrobial peptides (AMPs) are multifunctional components of the innate systems of both insect and mammalian hosts of the pathogenic trypanosomatids Leishmania and Trypanosoma species. Structurally diverse AMPs from a wide range of organisms have in vitro activity against these parasites acting mainly to disrupt surface-membranes. In some cases AMPs also localize intracellularly to affect calcium levels, mitochondrial function and induce autophagy, necrosis and apoptosis. In this review we discuss the work done in the area of AMP interactions with trypanosomatid protozoa, propose potential targets of AMP activity at the cellular level and discuss how AMPs might influence parasite growth and differentiation in their hosts to determine the outcome of natural infection.     

In vitro antioxidant and antitumor activities of polysaccharides extracted from Asparagus officinalis

Ultrasonic circulating extraction technology was applied for the polysaccharide extraction from Asparagus officinalis. The crude polysaccharides were deproteinized by Sevag method and three main polysaccharide fractions, AOP-4, AOP-6 and AOP-8 were obtained by fractional precipitation with gradient concentrations of ethanol (40%, 60% and 80%). The in vitro antitumor and antioxidant activities of the polysaccharide fractions were evaluated by MTT assay and free radical-scavenging assay, respectively. Deproteinized AOPs showed higher antioxidant and antitumor activities than crude AOP. AOP-4 with molecular weight 5.75 × 10⁴ Da showed significant function of scavenging hydroxyl radical. Three AOP fractions had significant antitumor activity against HeLa and BEL-7404 cells in a dose dependent manner. Furthermore, the inhibit activity of AOP-4 against HeLa cells was higher than those of other AOPs and the inhibition rate reached 83.96% at the concentration of 10 mg/mL. These results indicated that the AOP might be useful for developing natural safe antitumor drugs or health food.     

Sesquiterpene lactones from Vernonia scorpioides and their in vitro cytotoxicity

Fresh leaves of Vernonia scorpioides are widely used in Brazil to treat a variety of skin disorders. Previous in vivo studies with extracts of this species had also demonstrated a high antitumor potential. This paper reports isolation of four sesquiterpene lactones (hirsutinolides and glaucolides), together with diacetylpiptocarphol, 8-acetyl-13-etoxypiptocarphol, luteolin, apigenin, and ethyl caffeate from fresh leaves and flowers of Vernonia scorpioides. The hypothesis that hirsutinolide 3 is formed during extraction was verified theoretically using Density Functional Theory. The effects of isolated compounds on in vitro tumor cells were investigated, as well as their genotoxicity by means of an in vitro comet assay. The results indicate that glaucolide 2 and hirsutinolide 4 are toxic to HeLa cells. These compounds were genotoxic in vitro, a property that appears to be related to the presence of their epoxy groups, which has been a more reliable indication of toxicity than substitution on C-13 or the presence of α,β-unsaturated keto-groups. These results need to be replicated in vivo in order to ascertain their toxicity.     

In silico and in vitro comparative activity of novel experimental derivatives against Leishmania major and Leishmania infantum promastigotes

This in silico and in vitro comparative study was designed to evaluate the effectiveness of some biurets (K1 to K8) and glucantime against Leishmania major and Leishmania infantum promastigotes. Overall, eight experimental ligands and glucantime were docked using AutoDock 4.3 program into the active sites of Leishmania major and Leishmania infantum pteridine reductase 1, which were modeled using homology modeling programs. The colorimetric MTT assay was used to find L. major and L. infantum promastigotes viability at different concentrations of biuret derivatives in a concentration and time-dependent manner and the obtained results were expressed as 50% and 90% of inhibitory concentration (IC₅₀ and IC₉₀). In silico method showed that out of eight experimental ligands, four compounds were more active on pteridine reductase 1. K3 was the most active against L. major promastigotes with an IC₅₀ of 6.8 μM and an IC₉₀ of 40.2 μM, whereas for L. infantum promastigotes was K8 with IC₅₀ of 7.8 μM. The phenylethyl derivative (K7) showed less toxicity (IC₅₀s > 60 μM) in both Leishmania strains. Glucantime displayed less growth inhibition in concentration of about 20 μM. In silico and especially docking results in a recent study were in accordance with the in vitro activity of these compounds in presented study and compound K3, K2 and K8 showed reasonable levels of selectivity for the Leishmania pteridine reductase 1.     

Association of a luminous Vibrio sp., taxonomically related to Vibrio harveyi, with Clytia linearis (Thornely, 1900) (Hydrozoa, Cnidaria)

A previously unknown association between a luminous Vibrio sp., taxonomically related to the species Vibrio harveyi and a common member of the shallow/mid water communities of the Mediterranean Sea, the hydrozoan Clytia linearis is described. All the specimens of C. linearis observed under blue light excitation showed both a natural luminescence appearing as a series of fine dots due to clytin, and a clear fluorescence on the external side of the perisarc around the colonies due to the presence of luminous bacteria. Luminous bacteria were isolated from the surface of C. linearis, their phenotypic characterization as isolates was performed by several morphological, biochemical, and cultural tests, completed with 16S rDNA sequence analysis. All the isolates were referred to a Vibrio sp. taxonomically related to V. harveyi. The association of the V. harveyi-related species with C. linearis, as already suggested for another hydroid, Aglaophenia octodonta, could be explained with the activity of these bacteria of feeding on the chitinous structures present in these hydroids. Moreover, the adhesion of the luminous bacterium (here referred to as Vibrio sp. CL1) on C. linearis may contribute to the survival of this Vibrio species in the marine environment providing a suitable growth habitat.     

Risk of Coxiella burnetii transmission via embryo transfer using in vitro early bovine embryos

Coxiella burnetii, an obligate intracellular bacterium of worldwide distribution, is responsible for Q fever. Domestic ruminants are the main source of infection for humans. The objectives of this study were to determine (1) whether C. burnetii would adhere to the intact zona pellucida (ZP-intact) of early in vitro–produced bovine embryos; (2) whether the bacteria would adhere to or infect the embryos (ZP-free) after in vitro infection; and (3) the efficacy of the International Embryo Transfer Society (IETS) washing protocol. One hundred and sixty, eight- to 16-cell bovine embryos produced in vitro, were randomly divided into 16 batches of 10 embryos. Twelve batches (eight ZP-intact and four ZP-free) were incubated in a medium containing C. burnetii CbB1 (Infectiologie Animale et Santé Publique, Institut National de Recherche Agronomique Tours, France). After 18 hours of incubation at 37 °C and 5% CO₂ in air, the embryos were washed in 10 successive baths of a PBS and 5% fetal calf serum solution in accordance with the IETS guidelines. In parallel, four batches (two ZP-intact and two ZP-free) were subjected to similar procedures but without exposure to C. burnetii to act as controls. Ten washing fluids from each batch were collected and centrifuged for 1 hour at 13,000× g. The embryos and wash pellets were tested using conventional polymerase chain reaction. C. burnetii DNA was found in all ZP-intact and ZP-Free embryos after 10 successive washes. It was also detected in the first four washing fluids for ZP-intact embryos and in the 10th wash fluid for two of the four batches of ZP-free embryos. In contrast, none of the embryos or their washing fluids in the control batches were DNA positive. These results demonstrate that C. burnetii adheres to and/or penetrates the early embryonic cells and the ZP of in vitro bovine embryos after in vitro infection, and that the standard washing protocol recommended by the IETS for bovine embryos, failed to remove it. The persistence of these bacteria after washing makes the embryo a potential means of transmission of the bacterium during embryo transfer from infected donor cows to healthy recipients and/or their offspring. Further studies are required to investigate whether enzymatic and/or antibiotic treatment of bovine embryos infected by C. burnetii would eliminate the bacteria from the ZP and to verify if similarly results are obtained with in vivo–derived embryos.     

Anti-thrombosis effect of diosgenin extract from Dioscorea zingiberensis C.H. Wright in vitro and in vivo

Thrombus formation in blood vessel plays an important role in the pathogenesis and progression of atherosclerosis and cardiovascular diseases. Extract of Dioscorea zingiberensis C.H. Wright (D. zingiberensis) is demonstrated to posses activities for curing cardiovascular diseases such as coronary heart disease and angina pectoris. However, there were few studies on anti-thrombosis activity of it. We investigated the anti-thrombosis effect of diosgenin from D. zingiberensis (Dio) in vitro and in vivo on inferior vena cava ligation thrombosis rat model and pulmonary thrombosis mice model. We evaluated the protective effect of Dio by measuring the platelet aggregation, activated partial thromboplastin time (APTT), thrombin time (TT), prothrombin time (PT) and the venous thrombosis in rats and the bleeding time, clotting time and protection rate in mice. Results showed that Dio inhibited platelet aggregation, thrombosis and prolonged APTT, PT and TT in rats in a dose-dependent manner. They also prolonged the bleeding time, clotting time and increased protection rate in mice in a dose-dependent manner. Taken together, these findings suggested that Dio which contained 95% diosgenin had anti-thrombosis activity. Dio executives the anti-thrombosis activity through improving the anticoagulation function, inhibiting platelet aggregation and thrombosis.     

Expression of messenger RNAs for insulin-like growth factors and their receptors in bovine fetuses at early gestation from embryos produced in vivo or in vitro

The objective of this study was to determine the effects of in vitro embryo production on physical development and levels of expression of mRNAs for insulin-like growth factor (IGF) ligands (IGF1, IGF2), their receptors (IGF1R, IGF2R), and IGF binding protein-2 (IGFBP2) in bovine fetuses during early gestation. In vivo embryos were recovered from superovulated Holstein cows. For production of embryos in vitro, Holstein oocytes were matured, fertilized, and subsequently cultured in M199 with 10% serum to 168 hpi. On Day 70 of gestation, fetuses (in vivo, n = 14; in vitro, n = 13) were recovered, serum samples collected, and physical measurements recorded. Semi-quantitative RT-PCR assays were used to determine the levels of expression of mRNAs for IGF1, IGF2, IGF1R, and IGF2R in fetal liver and skeletal muscle. Western blots were used to assess levels of IGFBP2 in fetal serum. Fetal body weight did not differ with treatment; however, production of embryos in vitro was associated with decreased crown-nose length and a tendency for increased paired kidney weight, which became significant when expressed on a per bodyweight basis. There was no effect of treatment on levels of IGFBP2 in fetal serum. Levels of IGF1 mRNA in fetal liver were decreased (P < 0.001) in the in vitro group. Levels of IGF2R mRNA in both liver and skeletal muscle were also decreased (P < 0.01) in fetuses from the in vitro group. In summary, fetuses at Day 70 of gestation from embryos produced in vitro had shortened crown-nose length and increased kidney weight on a per bodyweight basis, as well as decreased expression of mRNAs for IGF1 in liver and IGF2R in both liver and skeletal muscle, compared with fetuses from embryos produced in vivo. In conclusion, in vitro embryo culture was associated with subtle changes in fetal development as well as altered expression of both imprinted and non-imprinted genes.     

Lavandula angustifolia essential oil as a novel and promising natural candidate for tick (Rhipicephalus (Boophilus) annulatus) control

Lavandula angustifolia is a well known herbal medicine with a variety of useful properties, including its acaricidal effect. This experiment was carried out to study the bioacaricidal activity of L. angustifolia essential oil (EO) against engorged Rhipicephalus (Boophilus) annulatus (Acari; Ixodidae) females. For this purpose six serial concentrations (0.5, 1.0, 2.0, 4.0, 6.0 and 8.0% w/v) of L. angustifolia EO were used. There was considerable mortality in concentrations more than 4.0% although there were no differences between 6.0 and 8.0% in all measured criteria. The mortality rate 24 h after inoculation was 73.26 and 100% in groups treated with 4.0 and 8.0% EO, respectively. Lavender EO also reduced tick egg weight in a concentration-dependent manner. The amount of eggs produced varied from 0.12 g (at 0.5% EO) to 0.00 g (at 8.0% EO) but did not differ statistically from the control. L. angustifolia EO caused 100% failure in egg laying at 6.0 and 8.0% whereas this value in the control group was zero. A positive correlation between L. angustifolia EO concentration and tick control, assessed by relative mortality rate and egg-laying weight, was observed by the EO LC/EC₅₀, which, when calculated using the Probit test, was 2.76-fold higher than the control. Lavender is a promising acaricidal against R. (B.) annulatusin vitro.     

In vitro and in vivo screening of essential oils for the control of wet bubble disease of Agaricus bisporus

Proliferation of fungal pathogens, such as Mycogone perniciosa, can severely affect the yields of cultivated mushrooms, including that of the button mushroom, Agaricus bisporus. A reduction in the number of fungicidal products approved for commercial application is currently providing new challenges to the mushroom industry. Forty essential oils, seven pure terpenoids and one phenylpropanoid were screened in vitro to determine the abilities of these substances to inhibit the growth of M. perniciosa. The fungal growth medium of both A. bisporus and M. perniciosa was supplemented with each test substance at a concentration of 50 μL/L. Ten essential oils were further investigated at lower concentrations ranging from 5 to 40 μL/L. The main components of these oils were determined by GC–FID and GC–MS. Lemon verbena (Lippia citriodora), lemongrass (Cymbopogon citratus) and thyme (Thymus vulgaris) oils were found to substantially inhibit the growth of the pathogen, while demonstrating lower toxicity towards A. bisporus than any of the other oils tested. A preliminary in vivo trial using M. perniciosa-inoculated casings revealed that the preventative use of lemon verbena or thyme oils was able to control the development of the disease. A commercial trial using these oils, as well as two of their main components (nerol and thymol), at a concentration of 40 μL/L, revealed that none of these treatments were detrimental to the growth of the A. bisporus and an overall yield similar to that following application of a commercial fungicide (Chronos 450 SC) was obtained. These results suggest that essential oils or mixtures of selected pure components of essential oils may in future find application in button mushroom production, either as a substitute for synthetic fungicides or as an additional protective measure.     

Dibenzocyclooctadiene lignans from Kadsura philippinensis

Lignans with the dibenzocyclooctadiene skeleton, kadsuphilols I-L, and one C₁₉-homolignan, kadsuphilol M, were isolated by chromatographic fractionation of an ethyl acetate extract of the aerial parts of Kadsura philippinensis. Their structures were elucidated through extensive spectroscopic methods, including HRESIMS and 2D NMR experiments (HMQC, COSY and HMBC). The stereochemistry at the chiral centers and the biphenyl moist, were determined using NOESY, as well as analysis of CD spectra, respectively. The relative configuration of heteroclitin J was confirmed by single crystal X-ray crystallographic analysis. The in vitro radical-scavenging activities of these compounds by using DPPH were evaluated.     

In vitro anti-influenza virus activity of a cardiotonic glycoside from Adenium obesum (Forssk.)

Methanolic extracts of six Saudi plants were screened for their in vitro antiviral activity using influenza virus A/PR/8/34 (H1N1) and MDCK cells in an MTT assay. The results indicated that the extracts of Adeniumobesum and Tephorosianubica possessed antiviral activity (99.3 and 93.3% inhibition at the concentration of 10 μg/ml, respectively). Based on these results A. obesum was selected for further study by applying bioactivity-guided fractionation to isolate its antiviral principle. The antiviral principle was isolated from the chloroform fraction through solvent fractionation, combined open liquid chromatography and HPLC. The isolated active compound A was identified as oleandrigenin-β-d-glucosyl (1 → 4)-β-d-digitalose, on the basis of its spectral analysis (MS, 1D and 2D NMR). The isolated glycoside showed reduction of virus titre by 69.3% inhibition at concentration of 1 μg/ml (IC₅₀ = 0.86 μg/ml).     

Axenization and optimization of in vitro growth of clonal cultures of Tetratrichomonas gallinarum and Trichomonas gallinae

A rapid and simple procedure was established to obtain clonal axenic cultures of Tetratrichomonas gallinarum and Trichomonas gallinae and to optimize their in vitro growth conditions. Medium 199 was used for axenization of two genetically different clones of T. gallinarum and T. gallinae. Six different media were used to optimize the growth behaviour of axenically grown parasites: Medium 199, TYM, TYI-S-33, Hollander fluid (HF), Trichomonas vaginalis (TV) and modified TV media. The highest cell yields for both axenic clones of T. gallinarum were obtained in modified TV medium without antibiotics. The maximum numbers of trophozoites of T. gallinae were obtained in an optimized HF medium. This study demonstrated that axenic cultures for T. gallinarum and T. gallinae could be obtained avoiding the migration technique through a V-tube. Following axenization and optimization, both clones of T. gallinarum and T. gallinae could be propagated both aerobically and anaerobically.