Modelling the growth of Weissella cibaria as a function of fermentation conditions

Aims:  To investigate the effect of pH, water activity ( a _w) and temperature on the growth of Weissella cibaria DBPZ1006, a lactic acid bacterium isolated from sourdoughs.
Methods and Results:  The kinetics of growth of W. cibaria DBPZ1006 was investigated during batch fermentations as a function of pH (4·0–8·0), a _w (0·935–0·994) and temperature (10–45°C) in a rich medium. The growth curve parameters (lag time, growth rate and asymptote) were estimated using the dynamic model of Baranyi and Roberts (1994. A dynamic approach to predicting bacterial growth in food. Int J Food Microbiol 23, 277–294). The effect of pH, a _w and temperature on maximum specific growth rate (μ_max) were estimated by fitting a cardinal model. μ_max under optimal conditions (pH = 6·6, a _w = 0·994, T  = 36·3°C) was estimated to be 0·93 h⁻¹. Minimum and maximum estimated pH and temperature for growth were 3·6 and 8·15, and 9·0°C and 47·8°C, respectively, while minimum a _w was 0·918 (equivalent to 12·2% w/v NaCl).
Conclusions:  Weissella cibaria DBPZ1006 is a fast-growing heterofermentative strain, which could be used in a mixed starter culture for making bread.
Significance and Impact of the Study:  This is the first study reporting the modelling of the growth of W. cibaria , a species that is increasingly being used as a starter in sourdough and vegetable fermentations.     

Effect of temperature and water activity on in vitro germination of Monilinia spp.

Aims:  This study evaluated the effect of temperature (0–38°C) and water activity ( a _w: 0·87–0·99) on the lag phase prior to germination and the percentage of germination over time for Monilinia laxa , Monilinia fructicola and Monilinia fructigena .
Methods and Results:  More than 80% of viable conidia germinated at 25°C and 0·99 a _w within 2 h for M. fructicola and M. fructigena and 4 h for M. laxa . There was no germination at 38°C, and all three Monilinia spp. germinated at 0°C. At the lowest a _w (0·87), none of the Monilinia spp. was able to germinate at any of the incubation temperatures studied. Whereas at 0·90 a _w, conidia were only able to germinate at 15, 25 and 30°C for the three species studied, except for M. fructicola at 15°C. In contrast, at 0·95, 0·97 and 0·99 a _w, germination occurred at all studied temperatures less 38°C. Generally, the lag phase was longer at low levels of a _w (0·90–095), and differences were more evident as temperatures were far from the optimum (0–5°C).
Conclusions:  Germination and lag phase period were markedly influenced by temperature and a _w, and in general when conditions of temperature and a _w were suboptimal, the lag phase was longer and the percentage of germination was lower.
Significance and Impact of the Study:  Knowledge of the germination requirements of this fungus is important in order to understand their behaviour in natural situations and to provide baseline data required for the construction of new prediction models. Our study might be used to develop a predictive model to understand and control the disease caused by Monilinia spp.     

Determination of optimal growth parameters for the bioincising fungus Physisporinus vitreus by means of response surface methodology

Aim:  To evaluate the influence of water activity ( a _w), temperature and pH on the radial growth and lag phase of Physisporinus vitreus (E-642), a basidiomycete was used in the biotechnological process of bioincising.
Methods and Results:  Radial growth was monitored for 20 days on malt extract agar medium. Five levels of a _w (0·998, 0·982, 0·955, 0·928, 0·892) were combined with three incubation temperatures (10, 15, 20°C) and three pH values (4, 5, 6). Data analyses showed a highly significant effect of a _w and temperature ( P <  0·0001) and a significant effect of pH ( P <  0·05). The radial growth rate and lag phase of P. vitreus were very sensitive to a _w reduction. Although P. vitreus was able to grow at all the selected temperatures and pH values, the lag phase increased with decreasing a _w and growth became inhibited at a _w = 0·955. Optimal conditions for growth of P. vitreus were a _w = 0·998, 20°C and pH 5. The response surface model provided reliable estimates of these growth parameters and confirmed a greater dependence on a _w than on temperature or pH under in vitro conditions.
Conclusions:  Low levels of a _w can prevent growth of P. vitreus , so wood moisture content should be adjusted accordingly.
Significance and Impact of the Study:  Implementation of these results should contribute towards the optimization and efficiency of bioincising.     

Mycelial growth and ochratoxin A production by Aspergillus section Nigri on simulated grape medium in modified atmospheres

Aims:  To evaluate the impact of modified atmosphere packaging on in vitro growth of Aspergillus carbonarius and Aspergillus niger , and possible effects on ochratoxin A (OTA) biosynthesis.
Methods and Results:  Ochratoxigenic isolates belonging to the species A. carbonarius and A. niger were grown on a synthetic grapejuice medium (SNM) and packaged in combinations of controlled O₂ (1% and 5%) and CO₂ levels (0% and 15%), and in air as a control. Colony diameters were recorded every 3 days up to 21 days, and OTA was analysed after 7, 14 and 21 days. The greatest reductions in mycelial growth rate were observed at 1% O₂ followed by 1% O₂/15% CO₂, whereas 5% O₂ stimulated the growth of all isolates. OTA production by A. carbonarius and A. niger isolates was minimized at 1% O₂/15% CO₂ and 1% O₂, respectively, after 7 days of incubation. Maximal OTA accumulation after 7 days was observed for all isolates in the control pack and at 5% O₂.
Conclusions:  Of the atmospheres tested, only 1% O₂ combined with 15% CO₂ consistently reduced fungal growth and OTA synthesis by A. carbonarius and A. niger .
Significance and Impact of the Study:  Storage under modified atmospheres is unlikely to be suitable as the sole method for OTA minimization and grape preservation; other inhibitory factors are necessary.     

Survival and growth of Cronobacter species (Enterobacter sakazakii) in wheat-based infant follow-on formulas

Aims:  To determine the survival and growth characteristics of Cronobacter species ( Enterobacter sakazakii ) in infant wheat-based formulas reconstituted with water, milk, grape juice or apple juice during storage.
Methods and Results:  Infant wheat-based formulas were reconstituted with water, ultra high temperature milk, pasteurized grape or apple juices. The reconstituted formulas were inoculated with Cronobacter sakazakii and Cronobacter muytjensii and stored at 4, 25 or 37°C for up to 24 h. At 25 and 37°C, Cronobacter grew more (>5 log₁₀) in formulas reconstituted with water or milk than those prepared with grape or apple juices ( c. 2–3 log₁₀). The organism persisted, but did not grow in any formulas stored at 4°C. Formulas reconstituted with water and milk decreased from pH 6·0 to 4·8–5·0 after 24 h, whereas the pH of the formulas reconstituted with fruit juices remained at their initial pH values, c. pH 4·8–5·0.
Conclusions:  Cronobacter sakazakii and C. muytjensii can grow in reconstituted wheat-based formulas. If not immediately consumed, these formulas should be stored at refrigeration temperatures to reduce the risk of infant infection.
Significance and Impact of the Study:  The results of this study will be of use to regulatory agencies and infant formula producers to recommend storage conditions that reduce the growth of Cronobacter in infant wheat-based formulas.     

The survival of Salmonella enterica serovar Typhimurium DT104 and total viable counts on beef surfaces at different relative humidities and temperatures

Aims:  The aim of this study was to investigate changes in Salmonella and total viable count (TVC) survival on beef carcass surfaces stored for 72 h under different combinations of relative humidity (i.e. RH 75% or 96%) and temperature (5°C or 10°C).
Methods and Results:  The influence of low water activity ( a _w) and temperature on the survival and growth of Salmonella enterica serovar Typhimurium DT104 and the aerobic mesophilic flora on meat pieces from different sites on beef carcasses was investigated, under controlled conditions (75% or 96% RH; 5 or 10°C) in an environmental cabinet. Salmonella counts declined during storage at low a _w (75% RH) conditions at 5°C or 10°C. Salmonella counts increased during storage at high a _w (96% RH) at 10°C only. At 5°C, TVCs increased during storage at high a _w, but not at low a _w. TVCs increased on all samples from carcasses stored at high or low a _w at 10°C, except those samples taken from areas of surface fat.
Conclusions:  This suggests that substrate composition dictates growth rates under low a _w conditions. The results are discussed in terms of the possible protective effects of substrate osmolyte accumulation in bacterial survival and/or growth.
Significance and Impact of the Study:  The data obtained in this study provides useful insights on the influence of a _w and temperature on pathogen survival on meat surfaces at chill temperature.     

OTA-producing fungi isolated from stored cocoa beans

Aims:  The aim of this study was to identify fungal populations in unroasted cocoa beans stored in Spain in order to evaluate the ochratoxin A (OTA)-production ability of certain Aspergillus isolates.
Methods and Results:  Twenty batches of cocoa beans from different origins and with different OTA content were selected for this study. Three Aspergillus carbonarius and 13 Aspergillus niger aggregate strains isolated from these cocoa bean samples were selected to evaluate their OTA synthesis ability, being the only A. carbonarius isolates which are OTA producers [−1 culture medium; LOD = 6  μ g kg⁻¹ culture medium].
Conclusions:  No correspondence was found between the OTA levels in cocoa beans and the presence of OTA-producing fungi. Nonetheless, some samples contained A. carbonarius with a high OTA-producing ability and, consequently, specific fungal controls should be set up during storage to avoid this toxin.
Significance and Impact of the Study:  Toxigenic fungi in cocoa beans are not well understood. This study attempted to identify these fungi and evaluate their OTA-producing ability.     

The Effect of Water Activity on the Growth and Respiration of Pseudomonas fluorescens

The growth rate of Pseudomonas fluorescens was greater and continued at lower water activity ( a _w) values when glycerol controlled the a _w of glucose minimal medium than when the a _w was controlled by NaCl and sucrose. Growth was not observed below 0·945, 0·970 and 0·964 a _w when glycerol, sucrose and NaCl respectively controlled the a _w. The catabolism of glucose, Na lactate and DL-arginine as measured by respirometry was completely inhibited at a _w values greater than the minimum for growth when the a _w was controlled with NaCl. When the a _w was controlled with glycerol, catabolism of the three substrates continued at a _w values significantly below the a _w for growth on glucose. Catabolism of glucose in the presence of sucrose occurred at a level below the minimum growth a _w but catabolism of the other two substrates ceased at a _w values greater than the minimum growth a _w. Arrhenius plots between 10° and 34°C of the growth rate in glucose minimal medium at 0·98 a _w showed that the order of inhibition was sucrose > NaCl > glycerol. The order of inhibition differed when Arrhenius plots of catabolism of glucose was examined between 10° and 34 °C, namely NaCl > sucrose > glycerol. The mechanism of action of solutes controlling a _w is discussed.     

Resistance of Enterobacter sakazakii (Cronobacter spp.) to environmental stresses

Aim:  To gain a better understanding of the survival and persistence of Enterobacter sakazakii in severe environments.
Methods and Results:  We evaluated the resistance of Ent. sakazakii to various environmental stresses, including heating, drying, water activity ( a _w), and pH. The resistance of Ent. sakazakii to heat varies widely among strains. Most tested strains of Ent. sakazakii exhibited unusual resistance to dry stress, which depends on drying media. Growth of most strains occurred within 24 h at 37°C when the initial a _w of the medium was adjusted to 0·94 with sucrose or sodium chloride. The minimum pH for growth within 24 h at 37°C was 3·9 or 4·1 for most strains tested. Additionally, there did not appear to be any relationship between resistance to stresses and biofilm-forming ability in Ent. sakazakii planktonic cells.
Conclusions:  These results indicate that Ent. sakazakii is much more resistant than other Enterobacteriaceae to environmental stresses. Moreover, it is likely that Ent. sakazakii has cross-resistance to dry and thermal stresses.
Significance and Impact of the Study:  The findings of this study will contribute to an improved understanding of the survival and behaviour of Ent. sakazakii , which will lead to improved strategies for preventing outbreaks of Ent. sakazakii infection.     

Ontogenetic changes in temperature preference of Atlantic cod

Final thermal preferendum ( T ) experiments were conducted in a horizontal thermal gradient tank from the beginning of August 2001 to mid‐November 2001 using Atlantic cod Gadus morhua from 6·5 to 79·0 cm fork length ( L _F). The value of T varied significantly ( P  < 0·005) with L _F( T  = 7·23–0·054 L _F), with smaller (younger) fish choosing higher temperatures than larger (older) fish. The preferendum varied from 6·9° C for fish of 6·5 cm to 3·0° C for those of 79·0 cm. Experiments comparing fish positions in the gradient tank between thermal gradients of 0·5–11·0 and 4·5–14·5° C demonstrated that fish positions were determined by temperature selection instead of undesirable tank effects. This study is the first to demonstrate the effect of ontogeny on temperature preferences of a marine fish species.     

Anion exchange in the giant erythrocytes of African lungfish

Carbon dioxide transport in African lungfish Protopterus aethiopicus blood conformed to the typical vertebrate scheme, implying a crucial and rate-limiting role of erythrocyte Cl^–/HCO₃^– exchange. The rate coefficient for unidirectional Cl^– efflux via the anion exchanger ( k , s⁻¹) increased with temperature in African lungfish, but values were well below those reported in other species. The erythrocytes of African lungfish were, however, very large (mean cellular volume = 6940 µm³), and the ratio of cell water volume to membrane surface area was high ( V _w A _m⁻¹ = 1·89). Hence, the apparent Cl^– permeability ( P _Cl =  kV _w A _m⁻¹, µm s⁻¹) was close to that in other vertebrates. The plot of ln P _Cl against the inverse absolute temperature was left-shifted in the tropical African lungfish compared to the temperate rainbow trout Oncorhynchus mykiss , which supports the idea that P _Cl is similar among animals when compared at their preferred temperatures. Also, Q ₁₀ for anion exchange calculated from P _Cl values in African lungfish was 2·0, supporting the idea that the temperature sensitivity of erythrocyte anion exchange matches the temperature sensitivity of CO₂ production and transport in ectothermic vertebrates.     

The linamarase of Mucor circinelloides LU M40 and its detoxifying activity on cassava

Mucor circinelloides LU M40 produced 12·2 mU ml⁻¹ of linamarase activity when grown in a 3 l fermenter in the following optimized medium (g l⁻¹ deionized water): pectin, 10·0; (NH₄)₂SO₄,
1·0; KH₂PO₄, 2·0; Na₂HPO₄, 0·7; MgSO₄.7H₂O, 0·5; yeast extract, 1·0; Tween-80,
1·0, added after 48 h of fermentation. The purified linamarase was a dimeric protein with a molecular mass of 210 kDa; the enzyme showed optimum catalytic activity at pH 5·5 and 40 °C and had a wide range (3·0–7·0) of pH stability. The enzyme substrate specificity on plant cyanogenic glycosides was wide; the K_m value for linamarin was 2·93 mmol l⁻¹. The addition, before processing, of the fungal crude enzyme to cassava roots facilitated and shortened detoxification; after 24 h of fermentation, all cyanogenic glycosides were hydrolysed.     

Effects of temperature, body size and feeding on rates of metabolism in young‐of‐the‐year haddock

The mean rate of oxygen consumption (routine respiration rate, R _R, mg O₂ fish⁻¹ h⁻¹), measured for individual or small groups of haddock Melanogrammus aeglefinus (3–12 cm standard length, L _S) maintained for 5 days within flow‐through respiratory chambers at four different temperatures, increased with increasing dry mass ( M _D). The relationship between R _R and M _D was allometric ( R _R = α  M ^b ) with b values of 0·631, 0·606, 0·655 and 0·650 at 5·0, 8·0, 12·0 and 15·0° C, respectively. The effect of temperature ( T ) and M _D on mean R _R was described by     indicating a Q ₁₀ of 2·27 between 5 and 15° C. Juvenile haddock routine metabolic scope, calculated as the ratio of the mean of highest and lowest deciles of R _R measured in each chamber, significantly decreased with temperature such that the routine scope at 15° C was half that at 5° C. The cost of feeding ( R _SDA) was c . 3% of consumed food energy, a value half that found for larger gadoid juveniles and adults.     

Protective effect of salicylates against hydrogen peroxide stress in yeast

Aims:  To investigate the effects of salicylates in Saccharomyces cerevisiae exposed to oxidative stress induced by hydrogen peroxide (H₂O₂).
Methods and Results:  Saccharomyces cerevisiae was cultured through to the postlogarithmic phase of growth. Stress was induced by the addition of 1·5 mmol l⁻¹ H₂O₂ for 1 h, while N-acetyl-l-cysteine (NAC) and glutathione (GSSG) were used as control agents that affect the redox balance. Sodium salicylate, at 0·01–10 mmol l⁻¹or acetylsalicylic acid, at 0·02–2·5 mmol l⁻¹ was administered at various times before hydrogen peroxide stress. Both agents conferred resistance to a subsequent hydrogen peroxide stress, similarly to the induction of the adaptive response observed upon pretreatment with NAC and GSSG. Sodium salicylate was more potent as a short-term, but not as a long-term pretreatment agent, compared to acetylsalicylic acid.
Conclusions:  Pharmacological pretreatment with salicylates resulted in dose related increases in cell survival, indicating the induction of the protective response in yeast.
Significance and Impact of the study:  The possible role of salicylates in the modulation of the hydrogen peroxide stress response in eukaryotic cells address questions on the effects of these commonly used therapeutic agents in a number of disorders exhibiting an oxidative stress component.     

Polyaluminum chloride-enhanced concentration efficiency of poliovirus and f2 phage from sewage water

Aims:  To enhance the recovery of f₂ bacteriophage and poliovirus by an established method based on the adsorption to and elution from positively-charged Al(OH)₃-treated silica gel.
Methods and Results:  Polyaluminum Chloride (PAC) was added to water samples to neutralize the negatively charged materials, which can reduce virus recovery by providing a competing adsorption mode on the media surface. Using this improved process (PAC 30 mg l⁻¹, pH 6·5, temperature 20∼30°C), the recoveries of Poliovirus I and f₂ from small-volume sewage (100 ml) were 110·76 ± 36·0% and 92·06 ± 8·65%, respectively ( P  < 0·05 vs. traditional methods). Recovery from a 20-L volume of sewage averaged 85·65 ± 4·43% for f₂ and 88·73 ± 9·76% for poliovirus, significantly higher than the recoveries in the traditional methods ( P  < 0·05).
Conclusions:  PAC could enhance concentration efficiency of poliovirus and f₂ phage from sewage water.
Significance and Impact of the Study:  This method should significantly improve the recovery of viruses from sewage.     

Sucrose phosphorylase of the rumen bacterium Pseudobutyrivibrio ruminis strain A

Aims:  To verify the taxonomic affiliation of bacterium Butyrivibrio fibrisolvens strain A from our collection and to characterize its enzyme(s) responsible for digestion of sucrose.
Methods and Results:  Comparison of the 16S rRNA gene of the bacterium with GenBank showed over 99% sequence identity to the species Pseudobutyrivibrio ruminis . Molecular filtration, native electrophoresis on polyacrylamide gel, zymography and thin layer chromatography were used to identify and characterize the relevant enzyme. An intracellular sucrose phosphorylase with an approximate molecular mass of 52 kDa exhibiting maximum activity at pH 6·0 and temperature 45°C was identified. The enzyme was of inducible character and catalysed the reversible conversion of sucrose to fructose and glucose-1-P. The reaction required inorganic phosphate. The K _m for glucose-1-P formation and fructose release were 3·88 × 10⁻³ and 5·56 × 10⁻³ mol l⁻¹ sucrose, respectively – while the V _max of the reactions were −0·579 and 0·9  μ mol mg protein⁻¹ min⁻¹. The enzyme also released free glucose from glucose phosphate.
Conclusion:  Pseudobutyrivibrio ruminis strain A utilized sucrose by phosphorolytic cleavage.
Significance and Impact of the Study:  Bacterium P. ruminis strain A probably participates in the transfer of energy from dietetary sucrose to the host animal.     

Optimization of medium constituents for improved chitinase production by Paenibacillus sp. D1 using statistical approach

Aims:  Statistical optimization of medium components for improved chitinase production by Paenibacillus sp. D1.
Methods and Results:  Urea, K₂HPO₄, chitin and yeast extract were identified as significant components influencing chitinase production by Paenibacillus sp. D1 using Plackett–Burman method. Response surface methodology (central composite design) was applied for further optimization. The concentrations of medium components for improved chitinase production were as follows (g l⁻¹): urea, 0·33; K₂HPO₄, 1·17; MgSO₄, 0·3; yeast extract, 0·65 and chitin, 3·75. This statistical optimization approach led to the production of 93·2 ± 0·58 U ml⁻¹ of chitinase.
Conclusions:  The important factors controlling the production of chitinase by Paenibacillus sp. D1 were identified as urea, K₂HPO₄, chitin and yeast extract. Statistical approach was found to be very effective in optimizing the medium components in manageable number of experimental runs with overall 2·56-fold increase in chitinase production.
Significance and Impact of the Study:  The present investigation provides a report on statistical optimization of medium components for improved chitinase production by Paenibacillus sp. D1. Paenibacillus species are gram-positive, spore-forming bacteria with several PGPR and biocontrol potentials. However, only few reports concerning mycolytic enzyme production especially chitinases are available. Chitinase produced by Paenibacillus sp. D1 represents new source for biotechnological and agricultural use.     

The effect of temperature and fish size on growth and feed efficiency ratio of juvenile spotted wolffish Anarhichas minor

The growth properties of juvenile spotted wolffish Anarhichas minor reared at 4, 6, 8 and 12° C, and a group reared under 'temperature steps', (T‐step) i.e . with temperature reduced successively from 12 to 9 and 6° C were investigated. Growth rate and feed efficiency ration was significantly influenced by temperature and fish size. Overall growth rate was highest at 6° C (0·68% day⁻¹) and lowest at 12° C (0·48% day⁻¹), while the 4 and 8° C, and the T‐step groups had similar overall growth rates, i.e . 0·59, 0·62 and 0·51% day⁻¹ respectively. Optimal temperature for growth ( T _{opt G} ) and feed efficiency ratio (T_{opt FCE}) decreased as fish size increased, indicating an ontogenetic reduction in T _{opt G} and T _{opt FCE}. The results suggest a T _{opt G} of juvenile spotted wolffish in the size range 135–380 g, dropping from 7·9° C for 130–135 g to 6·6° C for 360–380 g juveniles. The T _{opt FCE} dropped from 7·4° C for 120–150 g to 6·5° C for 300–380 g juveniles. A wider parabolic regression curve between growth, feed efficiency ratio and temperature as fish size increased, may indicate increased temperature tolerance with size. Individual growth rates varied greatly at all time periods within the experimental temperatures, but at the same time significant size rank correlations were maintained and this may indicate stable size hierarchies in juvenile spotted wolffish.     

Growth studies with fish—overcoming the problems of size variation

The relationship between growth rate and fish size is described by the equation log_e G _w= a —0·4 log_e W, where G_w is the specific growth rate and W is fish weight. Since the intercept (a) represents the log_e G_w of a fish unit size, the relationship presents a method allowing comparison of data from experiments involving fish of different sizes. The application of the method is demonstrated by examining the effects of environmental temperature on growth rates of cod, Gadus morhua , and it is suggested that the optimum temperature for growth of cod is 13–15° C.     

Acrebol, a novel toxic peptaibol produced by an Acremonium exuviarum indoor isolate

Aims:  To identify a toxin and its producer isolated from woody material in a building where the occupants experienced serious ill health symptoms.
Methods and Results:  Hyphal extracts of an indoor fungus, identified as the cycloheximide-tolerant species Acremonium exuviarum , inhibited motility of boar spermatozoa (EC₅₀ 5 ± 2 μg of crude solids ml⁻¹) and caused cytolysis of murine neuroblastoma cells (MNA) and feline fetal lung cells (FL). The responsible substances were purified and identified as two structurally similar, heat-stable, novel, toxic peptaibols, 1726 Da and 1740 Da, respectively, with amino acid sequences of Acetyl-Phe-Iva/Val-Gln-Aib-Ile-Thr-Leu-Aib-Pro-Aib-Gln-Pro-Aib-(X-X-X)-SerOH and Acetyl-Phe-Iva/Val-Gln-Aib-Ile-Thr-Leu-Val-Pro-Aib-Gln-Pro-Aib-(X-X-X)-SerOH. Purified acrebol inhibited motility of boar sperm, depleted ATP half-content in 1 day (EC₅₀ of 0·1 μg ml⁻¹, 60 nmol l⁻¹) depolarised the mitochondria after 2 days, but did not affect the cellular content in NADH. This indicates mitochondrial toxicity. Plate-grown biomass of A. exuviarum BMB4 contained 0·1–1% (w/w) of acrebol, depending on the culture medium.
Conclusions:  Acrebol paralysed the energy generation of mammalian cells suggesting that mitochondria were its target of action.
Significance and Impact of the Study:  Acremonium exuviarum, as an indoor fungus, is potentially hazardous to health because of the toxic peptaibols that it produces.