A strict anaerobic extreme thermophilic hydrogen-producing culture enriched from digested household waste

Aims:  The aim of this study was to enrich, characterize and identify strict anaerobic extreme thermophilic hydrogen (H₂) producers from digested household solid wastes.
Methods and Results:  A strict anaerobic extreme thermophilic H₂ producing bacterial culture was enriched from a lab-scale digester treating household wastes at 70°C. The enriched mixed culture consisted of two rod-shaped bacterial members growing at an optimal temperature of 80°C and an optimal pH 8·1. The culture was able to utilize glucose, galactose, mannose, xylose, arabinose, maltose, sucrose, pyruvate and glycerol as carbon sources. Growth on glucose produced acetate, H₂ and carbon dioxide. Maximal H₂ production rate on glucose was 1·1 mmol l⁻¹ h⁻¹ with a maximum H₂ yield of 1·9 mole H₂ per mole glucose. 16S ribosomal DNA clone library analyses showed that the culture members were phylogenetically affiliated to the genera Bacillus and Clostridium. Relative abundance of the culture members, assessed by fluorescence in situ hybridization, were 87 ± 5% and 13 ± 5% for Bacillus and Clostridium , respectively.
Conclusions:  An extreme thermophilic, strict anaerobic, mixed microbial culture with H₂-producing potential was enriched from digested household wastes.
Significance and Impact of the Study:  This study provided a culture with a potential to be applied in reactor systems for extreme thermophilic H₂ production from complex organic wastes.     

Survival of Lactobacillus helveticus entrapped in Ca-alginate in relation to water content, storage and rehydration

Lactobacillus helveticus CNRZ 303 entrapped in Ca-alginate gel beads was investigated for improved survival and stability during fluidized-bed drying, storage and rehydration. Addition of protective solutes was very important. Studies of the conditions showed that inactivation of entrapped L. helveticus started when the water content exceeded 0.3–0.4 g H₂O (g dry wt)⁻¹ for adonitol, glycerol and reconstituted non fat milk solids (NFSM). With Ringer’s solution (control) and betaine, the fall in viability was evident above 1 g H₂O (g dry wt)⁻¹. Drying down to 0.2 g H₂O (g dry wt)⁻¹ required the removal of 98.5–98.9% of the water. The best survival rate with the least injured cells among survivors was experienced with adonitol and NFMS, respectively, 71% and 57% (compared to the initial) immediately after dehydration. Adonitol and NFMS were also best for survival during storage. The highest cell recovery was obtained by rehydrating the cells in cheese whey permeate between 20–30°C done at pH 6.0–7.0, satisfying the demands for cell survival, repair and slow swelling (adaptions). Received 04 January 1999/ Accepted in revised form 29 April 1999     

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.     

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.     

The interaction of temperature and fish size on growth of juvenile halibut

Growth rate of individually tagged juvenile halibut was influenced significantly by the interaction of temperature and fish size. The results suggest an optimum temperature for growth of juvenile halibut in the size range 5–70 g between 12 and 15° C. Overall growth rate was highest at 13° C (1·62% day ⁻¹). At c. 5 g at the beginning of the experiment, fish at 16° C had the highest growth rate (3·2% day ⁻¹), but reduced this rate as they grew bigger. At 9 and 11°p C, growth rates were equal or only slightly lower during the later stages of the experiment, while the fish at 6° C showed significantly lower overall growth rate (0·87% day⁻¹). Optimal temperature for growth decreased rapidly with increasing size, indicating an ontogenetic reduction in optimum temperature for growth. Moreover, a more flattened parabolic regression curve between growth and temperature as size increased indicated reduced temperature dependence with size. Although individual growth rates varied significantly at all times within the experimental temperatures, significant size rank correlations were maintained during the experiment. This indicated an early establishment of a stable size hierarchy within the fish groups. Haematocrit was highest at the highest temperature while Na⁺/K⁺-ATPase activity was inversely related to temperature. There was no difference in plasma Na⁺, Cl⁻ and K⁺ concentrations among the temperature groups.     

Growth, CO2 consumption and H2 production of Anabaena variabilis ATCC 29413-U under different irradiances and CO2 concentrations

Aims:  The objective of this study is to develop kinetic models based on batch experiments describing the growth, CO₂ consumption, and H₂ production of Anabaena variabilis ATCC 29413-U^TM as functions of irradiance and CO₂ concentration.
Methods and Results:  A parametric experimental study is performed for irradiances from 1120 to 16100 lux and for initial CO₂ mole fractions from 0·03 to 0·20 in argon at pH 7·0 ± 0·4 with nitrate in the medium. Kinetic models are successfully developed based on the Monod model and on a novel scaling analysis employing the CO₂ consumption half-time as the time scale.
Conclusions:  Monod models predict the growth, CO₂ consumption and O₂ production within 30%. Moreover, the CO₂ consumption half-time is an appropriate time scale for analysing all experimental data. In addition, the optimum initial CO₂ mole fraction is 0·05 for maximum growth and CO₂ consumption rates. Finally, the saturation irradiance is determined to be 5170 lux for CO₂ consumption and growth whereas, the maximum H₂ production rate occurs around 10 000 lux.
Significance and Impact of the Study:  The study presents kinetic models predicting the growth, CO₂ consumption and H₂ production of A. variabilis . The experimental and scaling analysis methods can be generalized to other micro-organisms.     

SULPHIDE PRODUCTION FROM SULPHATE-ENRICHED SEWAGE SLUDGES

SUMMARY: Sterilized raw sewage sludge enriched with sulphate and inoculated with pure strains of Desulphovibrio desulphuricans produced negligible sulphide. Unsterilized sludge supplemented with 7% (w/v) CaSO₄.2H₂O and inoculated with crude cultures of sulphate-reducing bacteria obtained from sewage yielded 1·0% S^2- (wt S^2- produced as H₂S/vol. of raw sludge) in 6 months at 30°. By repeated subculture more active cultures developed which produced 1% S^2- in 7 days and 1·2–1·9% in 28 days. Digested sludge yielded only 0·1% S^2-. In semicontinuous fermentations at 30°, raw sludge without added sulphate produced 20 times its own volume of gas containing 70% CH₄ and 30% CO₂. When 5% CaSO₄.2H₂O and an active crude culture of sulphate reducers were added, gas production decreased steadily to zero. There were no differences in pH, temperature and redox potential in sludges producing methane or sulphide. The chief cause of inhibition appeared to be the action of sulphide: 0·02% soluble sulphide (S^2-) totally inhibited methane formation; 0·01% S^2- initially decreased gas production by one-quarter but there was a slow recovery to normal, suggesting acclimatization of the methane-producing organisms to sulphide.
Linked fermentations, in which gas from a methane fermentation swept H₂S from a sulphide fermentation, gave a final gas mixture of about 60% CH₄, 30% CO₂ and 5–10% H₂S. The yield of sulphide depended on the rate of sweeping.     

Factors affecting the production of hydrogen sulphide by Lactobacillus sake L13 growing on vacuum-packaged beef

Lactobacillus sake L13 produced hydrogen sulphide during growth at 0°C on vacuum-packaged beef of normal pH (5·6–5·8) when the packaging films used had oxygen permeabilities as high as 200 ml/m²/24 h/atm (measured at 25°C and 98% relative humidity. No hydrogen sulphide was detected when the film permeability was 300 ml/m²/24 h/atm. Sulphmyoglobin was formed whenever hydrogen sulphide was present except when the film permeability was very low (1 ml of oxygen/m²/24 h/atm). Lactobacillus sake L13 also produced hydrogen sulphide when grown on beef under anaerobic conditions at 5°C. When meat pH was high (6·4–6·6) hydrogen sulphide was first detected after incubation for 9 d. When 250 μg of glucose was added to each g of high pH meat, or when meat pH was normal (5·6–5·8), hydrogen sulphide was first detected after incubation for 18 d. The spoilage of beef by hydrogen sulphide-producing lactobacilli is more rapid when the pH of the meat is high because high-pH meat contains less glucose. Sulphmyoglobin formation and greening can be prevented by the use of packaging films of very low oxygen permeability.     

Isolation of Yersinia enterocolitica-resembling Organisms and Alteromonas putrefaciens from Vacuum-Packed Chilled Beef Cuts

Yersinia enterocolitica -resembling organisms were found at levels of 10⁷/g on a high pH (pH ≧ 6·0) vacuum-packaged beef striploin held for 6 weeks at 0·2°C, but did not exceed 10⁵/g on normal pH (pH < 6·0) striploins held for 10 weeks. Gram negative bacteria that produced H₂S on peptone iron agar were isolated from high pH vacuum packed striploins. These organisms were identified as Alteromonas putrefaciens . They attained levels of about 10⁷/g in 6 weeks at 0–2°C, at which time greening of the fat surface and 'drip'had occurred. On meat of normal pH, counts of A. putrefaciens were less than 10⁴/g after 6 weeks and no greening was evident.     

Persistence of Shiga toxin-producing Escherichia coli O26 in various manure-amended soil types

Aims:  To evaluate the behaviour of Shiga toxin-producing Escherichia coli (STEC) O26 strains inoculated in manure-amended soils under in vitro conditions.
Methods and Results:  Four green fluorescent protein (GFP)-labelled STEC O26 strains were inoculated in duplicate (at 10⁶ CFU g⁻¹) in three different manure-amended soil types, including two loam soils (A and B) and one clay loam soil (C), and two incubation temperatures (4 and 20°C) were tested. STEC counts and soil physical parameters were periodically monitored. STEC O26 cells were able to persist during extended periods in soil even in the presence of low moisture levels, i.e. less than 0·08 g H₂O g⁻¹ dry soil. At 4 and 20°C, STEC could be detected in soil A for 288 and 196 days, respectively, and in soils B and C for at least 365 days postinoculation at both temperatures. The ambient temperature (i.e. 20°C) was significantly associated with the highest STEC count decline in all soils tested.
Conclusions:  The temperature and soil properties appear to be contributory factors affecting the long-term survival of STEC O26 in manure-amended soils.
Significance and Impact of the Study:  This study provides useful information regarding the ecology of STEC O26 in manure-amended soils and may have implications for land and waste management.     

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.     

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.     

Hydrogen-dependent control of the continuous thermophilic anaerobic digestion process using membrane inlet mass spectrometry

The control of a thermophilic continuous anaerobic digestion system when subjected to potential inhibitory shock loadings was achieved through the regulation of dissolved H₂, monitored using membrane inlet mass spectrometry, by the controlled addition of carbon source. At a feed pump switching threshold equivalent to 1 μmol/1 H₂ a steady state rate of methanogenesis of approximately 40 μmol/1/min was obtained. Higher H₂ thresholds resulted in an inhibition of methanogenesis, but precise control of H₂ concentration was demonstrated with an oscillatory response of period 2·5–5·0 min.     

Impact of temperature on food intake and growth in juvenile burbot

The effect of temperature on food consumption, food conversion and somatic growth was investigated with juvenile burbot Lota lota (age 0 years). Juvenile burbot showed a significant dome shaped relationship between relative daily food consumption ( C _R) and temperature ( T ) with C _R = − 0·00044 T ² + 0·01583 T  − 0·06010; ( n  = 90, r ² = 0·61). Maximum C _R was at 17·9° C (95% CL 17·2–18·6° C). The temperature related instantaneous growth rate ( G ) also followed a dome shaped function with G  = − 0·000063 T ² + 0·002010 T  − 0·007462; ( n  = 95, r ² = 0·57), with maximum growth rate at 16·0° C (95% CL 15·3–16·6° C). A significant linear relationship was found between the water temperature and the conversion coefficient ( C _C) with C _C = − 1·63 T  + 59·04; ( n  = 80, r ² = 0·74). The results indicate that juvenile burbot in large lakes benefit from higher water temperatures in the littoral zone, by increased food uptake and growth, especially during the warm summer months. Because profundal water temperatures do not reflect the optimal temperature for food consumption in large burbot, temperature is unlikely to be the main proximate factor for the obligate littoral‐profundal migration of juvenile burbot observed in many lake populations.     

Comparison of quantitative and qualitative methods of detecting hydrogen peroxide produced by human vaginal strains of lactobacilli

A quantitative method was developed for the measurement of micromolar quantities of H₂O₂ produced in Rogosa broth and peptonized milk broth by vaginal strains of lactobacilli isolated from women. The production of substantial amounts reproducibly was dependent on the growth of the organisms in acid media (pH ≤6.0) under anaerobic or micro-aerophilic conditions with continuous agitation. The addition to the media of the enzyme inhibitor, 3-amino-l,2,4-triazole, with or without catalase sometimes induced the production of H₂O₂ especially in non-agitated cultures. However, other agents such as concanavalin and o -dianisidine had no enhancing effect, and catalase or peroxidase alone completely inhibited H₂O₂ production.
The H₂O₂ produced in the acid media was stable for more than a month at 5°C but not in media at pH ≥ 7.0. Of five strains of lactobacilli tested by the quantitative method and by a chromogenic qualitative method (Rogosa-catalase or -peroxidase agar), three consistently produced H₂O₂ measurable by the former method, but none did so after growth of the organisms on Rogosa-catalase/peroxidase agar which suggested that the qualitative method was unreliable. The fact that H₂O₂ was produced in substantial quantities by some strains and not at all by others enabled H₂O₂-producers and non-producers to be distinguished easily.     

Performance and diacetyl production of commercial strains of malolactic bacteria in wine

This study compares 11 commercial cultures of Leuconostoc oenos and Lactobacillus plantarum in Cabernet Sauvignon, Pinot Noir and Chardonnay wines. Performance of the cultures was found to be greatly influenced by wine type. Better survival of the bacteria was observed in Cabernet Sauvignon and Pinot Noir wines. The time necessary to complete malolactic fermentation (MLF) was 65 ± 14 d for Chardonnay, 71 ± 3 d for Cabernet Sauvignon, and 25 ± 8 d for Pinot Noir. The maximal rate of malate utilization was 0·4 g d^-1 for Pinot Noir, and 0·2 g d^-1 for the two other wine types. Final diacetyl concentration was lower in Chardonnay wines (highest 0·58 mg l^-1) compared to the other wines (highest 5·8 mg l^-1). Malic and citric acid were co-metabolized by all strains. None of the strains metabolized glycerol. Significant differences in final diacetyl concentration of wine vinified with the different strains were found. Panelists could reliably differentiate MLF wines from non-MLF wines, irrespective of their diacetyl content, indicating that diacetyl is not the only important MLF flavour.     

Research note : Unsuitability of the MRS medium for the screening of hydrogen peroxide-producing lactic acid bacteria

The effect of MRS broth on the stability of hydrogen peroxide (H₂O₂) has been studied. Known concentrations (1–100 μg ml⁻¹) of H₂O₂ were prepared in distilled water, phosphate buffer (pH 7·0) and MRS broth (pH 6·2 and 3·9). H₂O₂ was very stable in aqueous and buffer solutions but it was rapidly degraded in MRS broth (pH 3·9). The presence of H₂O₂ in MRS broth (pH 6·2) could not be detected.     

Isolation and characterization of an intracellular esterase from Lactobacillus casei subsp. casei IFPL731

An intracellular esterase from Lactobacillus casei subsp. casei IFPL731 was purified 1000-fold by ion exchange chromatography and gel filtration chromatography. The relative molecular mass of the native enzyme was 105 kDa, while the subunit molecular mass was estimated to be 38 kDa. The esterase hydrolysed tributyrin and had a preference for esters of short-chain fatty acids (butyrate, caproate and caprylate), while it did not hydrolyse palmitate and sterate esters. The apparent Michaelis-Menten constant of the enzyme on p -nitrophenyl butyrate was 0·3 mmol l⁻¹ while on p -nitrophenyl caprylate, it was 0·04 mmol l⁻¹. The esterase was active over a broad range of pH and temperature values, and retained about 50% of maximal activity at pH 5·0 and 12 °C. Activity was strongly inhibited by 5 mmol l⁻¹ phenylmethylsulphonyl fluoride, β-mercaptoethanol and N -ethylmaleimide, and was stimulated by Zn²⁺ at 1 mmol l⁻¹.     

Survival and growth of Bacillus cereus in bread

Bread doughs were artificially inoculated with spores of six Bacillus cereus strains at different inoculum levels and counts of survivors in bread determined during storage at 27·5°C. No B. cereus were isolated from the centre crumb of 400 g loaves when the dough contained less than 10⁴ spores/g whereas with 800 g loaves survival occurred with doughs containing 5·0 times 10³ spores/g. With all strains there was a period of at least 24 h before multiplication took place in the bread. The inclusion in dough of 0·2% of calcium propionate, based on flour, effectively delayed germination and subsequent multiplication of B. cereus spores. It is concluded that the risk of food poisoning due to the presence of B. cereus in bread is minimal.     

Age and growth of Anguilla anguilla in the Camargue lagoons

Age and total length ( L _T) data from a 11 year monitoring of the Anguilla anguilla eel population of the Camargue lagoons (Rhône delta, southern France) were collected for glass, yellow and silver eels. Three distinct models were calibrated to describe the growth process of undifferentiated eels, females and males, respectively. Uncertainty of parameter estimates was evaluated by bootstrapping. Females were characterized by larger asymptotic body size ( L _T) than males (580 ± 50 v . 388 ± 13 mm) and faster growth, whilst the Brody growth coefficient was larger for males than for females (means ±  s . d . 3·00 10⁻³ ± 1·68 10⁻³ v . 1·73 10⁻³ ± 0·50 10⁻³). Sexual differentiation was estimated to begin at 204 ± 38 mm mean ±  s . d ., i.e . at the end of the second year in the lagoons, well before the L _T at which macroscopic differentiation became possible ( c . 300 mm). Males probably leave the lagoon or die (due to either natural or fishing mortality) within the first 3 years, whilst females can remain up to 5 years. Sexual differentiation and maturation have a major role in shaping the L _T structure of the population. The L _T and mass ( M ) data were fitted by allometric curves     . The calibration of distinct curves for data from different years indicated that the allometric coefficient a was subject to wider interannual fluctuations than the allometric exponent b . A negative correlation linked the average L _T and the allometric exponent ( r  = −0·58, P  < 0·01).