Biooxidation of a gold-containing sulfide concentrate in relation to changes in physical and chemical conditions

The growth of a microbial community and the oxidation of iron- and sulfur-containing substrates in batch culture during the leaching/oxidation of the flotation concentrate of refractory gold-arsenic sulfide ore were optimized with respect to the following medium parameters: temperature, pH, and requirement in organic substances. It was revealed that the optimum mode is (i) to maintain the pH at 1.6–1.7 and the temperature at 34–35 and 38°C and (ii) to add Corg in the form of yeast extract (0.02%). Mutually beneficial or competitive relationships among groups of microorganisms of the community were established, depending on the cultivation conditions.     

Accelerated production of 1,3-propanediol from glycerol by <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis> using the method of forced pH fluctuations

1,3-Propanediol (1,3-PD) is a bivalent alcohol, used in a number of chemical syntheses. It could be produced from glycerol in course of microbial fermentation by Klebsiella pneumoniae along with more than five minor liquid products. With the purpose to enhance 1,3-PD production and to eliminate by-products formation, principally new pH control on the process was applied. The method, named “forced pH fluctuations” was realized by consecutive raisings of pH with definite ΔpH amplitude (ranging from 1.0 to 2.0) at time intervals between 2 and 4 h, during a series of fed batch processes. The fermentation performed by forced pH fluctuations with ΔpH = 1.0, risen at every 3 h was evaluated as the most successful. Increase by 10% of the maximal amount of 1,3-PD (g/l), 22% higher productivity [g/(l h)], and 29% increase in 1,3-PD molar yield were achieved, compared to the referent fed batch (with constant pH = 7.0). In addition, significant decrease in by-products formation was obtained. The most important reduction was observed in the lactic and acetic acids yields, where 50 and 70% decrease were reached. The results suggested the potential of pH to manage the share and quantity of product spectrum in mixed diols–acids fermentations. The application of “forced pH fluctuations method” achieves the desirable increase in 1,3-PD formation and decrease in by-products accumulation at the same time by a comparatively simple approach by adjustment of one bioprocess parameter only.     

Production of bioethanol from organic whey using <Emphasis Type="Italic">Kluyveromyces marxianus</Emphasis>

Ethanol production by K. marxianus in whey from organic cheese production was examined in batch and continuous mode. The results showed that no pasteurization or freezing of the whey was necessary and that K. marxianus was able to compete with the lactic acid bacteria added during cheese production. The results also showed that, even though some lactic acid fermentation had taken place prior to ethanol fermentation, K. marxianus was able to take over and produce ethanol from the remaining lactose, since a significant amount of lactic acid was not produced (1–2 g/l). Batch fermentations showed high ethanol yield (~0.50 g ethanol/g lactose) at both 30°C and 40°C using low pH (4.5) or no pH control. Continuous fermentation of nonsterilized whey was performed using Ca-alginate-immobilized K. marxianus. High ethanol productivity (2.5–4.5 g/l/h) was achieved at dilution rate of 0.2/h, and it was concluded that K. marxianus is very suitable for industrial ethanol production from whey.     

A thermophilic and acid stable family-10 xylanase from the acidophilic fungus Bispora sp. MEY-1

A complete gene, xyl10C, encoding a thermophilic endo-1,4-β-xylanase (XYL10C), was cloned from the acidophilic fungus Bispora sp. MEY-1 and expressed in Pichia pastoris. XYL10C shares highest nucleotide and amino acid sequence identities of 57.3 and 49.7%, respectively, with a putative xylanase from Aspergillus fumigatus Af293 of glycoside hydrolase family 10. A high expression level in P. pastoris (73,400 U ml⁻¹) was achieved in a 3.7–l fermenter. The purified recombinant XYL10C was thermophilic, exhibiting maximum activity at 85°C, which is higher than that reported from any fungal xylanase. The enzyme was also highly thermostable, exhibiting ~100% of the initial activity after incubation at 80°C for 60 min and >87% of activity at 90°C for 10 min. The half lives of XYL10C at 80 and 85°C were approximately 45 and 3 h, respectively. It had two activity peaks at pH 3.0 and 4.5–5.0 (maximum), respectively, and was very acid stable, retaining more than 80% activity after incubation at pH 1.5−6.0 for 1 h. The enzyme was resistant to Co²⁺, Mn²⁺, Cr³⁺ and Ag⁺. The specific activity of XYL10C for oat spelt xylan was 18,831 U mg⁻¹. It also had wide substrate specificity and produced simple products (65.1% xylose, 25.0% xylobiose and 9.9% xylan polymer) from oat spelt xylan.     

Biosurfactant production by a thermophilic Bacillus subtilis strain

A strain of Bacillus subtilis was able to grow and produce a biosurfactant on 2% sucrose at 45°C. As a result of biosurfactant synthesis the surface tension of the medium was reduced from 68 dynes cm⁻¹ to 28 dynes cm⁻¹. The strain had the capacity to produce the biosurfactant at high NaCl concentrations (4%) and a wide range of pH (4.5–10.5). The biosurfactant retained its surface-active properties after heating at 100°C for 2 h and at different pH values (4.5–10.5). A maximum amount of biosurfactant was produced when urea or nitrate ions were supplied as nitrogen source. The use of the biosurfactant at high temperatures, acidic, alkaline and saline environments is discussed. As a result of its action, 62% of oil in a sand pack column could be recovered, indicating its potential application in microbiologically enhanced oil recovery. Received 28 March 1996/ Accepted in revised form 16 September 1996     

Distribution of benthic invertebrates in acid,brown water streams in the South Island of New Zealand

Acid, brown water streams are common on the west coast of the South Island, New Zealand. Acid precipitation is not a significant problem in this region where stream water acidity is brought about by high concentrations of humic substances. The interrelationships between pH, alkalinity, conductivity, DOC and total reactive aluminium were investigated at 45 running water sites. pH (range 3.5–8.1) was strongly correlated with alkalinity (range 0–49 g·m⁻³ CaCO₃) and less strongly with conductivity (range 2.0–9.7 mS·m⁻¹). A strong positive correlation was found between DOC and total reactive aluminium concentration both of which were correlated negatively with pH. In all brown water streams, most aluminium was probably in the non-toxic, organically complexed form. Benthic invertebrate assemblages were examined at 34 sites. Taxonomic richness was not correlated with pH and similar numbers of ephemeropteran, plecopteran and trichopteran taxa were taken from acidobiontic (pH ⩽ 5.5), acidophilic (pH 5.6–6.9) and moderately alkaline (pH ⩾ 7.0) groups of streams. Many species occurred over a wide pH range and had a lower limit of about pH 4.5. The mayfly, Deleatidium occurred at 33 sites and was amongst the five most abundant taxa at 32 of them. The stoneflies, Zelandobius confusus, Austroperla cyrene and Stenoperla maclellani, an elmid, Hydora sp. and a caddisfly, Psilochorema sp. also occurred in over half the streams and frequently were abundant. Few habitat specialists were found. Benthic assemblages were not associated strongly with measured physicochemical factors but streams in close proximity tended to have similar faunas. This suggests that the availability of suitable colonizers sets the limits to species richness and is important in determining the composition of benthic assemblages at a particular locality.     

The sulfide tolerance of milkfish and tilapia in relation to fish kills in farms and natural waters in the Philippines

Fish kills of milkfish Chanos chanos and tilapia Oreochromis spp. now occur frequently in brackish, marine, and freshwater farms (ponds, pens, and cages) in the Philippines. Aquafarms with high organic load, limited water exchange and circulation, no aeration, and high stocking and feeding rates can become oxygen-depleted and allow sulfide from the sediments to appear in the water column and poison free-swimming fish. The sulfide tolerance of 2–5 g milkfish and 5–8 g O. mossambicus was determined in 25-liter aquaria with flow-through sea water (100 ml min-1) at 26–30 °C and sulfide stock solutions pumped in at 1ml min-1. Total sulfide concentrations in the aquaria were measured by the methylene blue method and used in the regression against the probits of % survival. Four experiments showed that the two species have similar sulfide tolerance. In sea water of pH 8–8.5, about 163 ± 68 μM or 5.2 ± 2.2 mg l-1 total sulfide (mean ± 2 se) or 10 μM or 313 μg l-1 H2S was lethal to 50% of the fish in 4–8 h, and 61 ± 3 μM total sulfide or 4 μM H2S in 24–96 h (to convert all sulfide concentrations: 1 μM = 32 μg l-1). Earthen pond bottoms had 0–382 μM total dissolved sulfide (mean ± sd = 54 ± 79 μM, n = 76); a tenth of the samples had >200 μM. The water column may have such sulfide levels under hypoxic or anoxic conditions. To simulate some of the conditions during fish kills, 5–12 g milkfish were exposed to an abrupt increase in sulfide, alone or in combination with progressive respiratory hypoxia and decreasing pH. The tests were done in the same flow-through set-up but with sulfide pumped in at 25 ml min-1. The lethal concentration for 50% of the fish was 197 μM total sulfide or 12 μM H2S at 2 h, but 28–53 μM sulfide allowed fish to survive 6–10 h. Milkfish in aquaria with no aeration nor flow-through sea water died of respiratory hypoxia in 5–8 h when oxygen dropped from 6 to 1 mg l-1. Under respiratory hypoxia with 30–115 μM sulfide, the fish died in 2.5–4 h. Tests with low pH were done by pumping a weak sulfuric acid solution at 25 ml min-1 into aquaria with flow-through sea water such that the pH dropped from 8 to 4 in 5 h. Under these conditions, milkfish died in 7–9 h when the pH was 3.5. When 30–93 μM sulfide was pumped in with the acid, the fish died in 2–6 h when the pH was still 4.5–6.3. Thus, sulfide, hypoxia, and low pH are each toxic to milkfish at particular levels and aggravate each other's toxicity. Aquafarms must be well oxygenated to prevent sulfide toxicity and fish kills. This revised version was published online in July 2006 with corrections to the Cover Date.     

Entrapment of β-galactosidase in polyvinylalcohol hydrogel

β-Galactosidase isolated from Aspergillus oryzae was immobilized in lens-shaped polyvinylalcohol capsules (with activity 25 U g⁻¹) giving 32% of its original activity. Immobilization did not change the pH optimum (4.5) of lactose hydrolysis. The relative enzyme activity during product inhibition testing was, in average, 10% higher for immobilized enzyme. No decrease of activity was observed after 35 repeated batch runs and during 530 h of continuous hydrolysis of lactose (10%, w/v) at 45°C. The immobilized enzyme was stable for 14 months without any change of activity during the storage at 4°C and pH 4.5.     

Production of xylanase by Aspergilli using alternative carbon sources: application of the crude extract on cellulose pulp biobleaching

The ability of xylanolytic enzymes produced by Aspergillus fumigatus RP04 and Aspergillus niveus RP05 to promote the biobleaching of cellulose pulp was investigated. Both fungi grew for 4–5 days in liquid medium at 40°C, under static conditions. Xylanase production was tested using different carbon sources, including some types of xylans. A. fumigatus produced high levels of xylanase on agricultural residues (corncob or wheat bran), whereas A. niveus produced more xylanase on birchwood xylan. The optimum temperature of the xylanases from A. fumigatus and A. niveus was around 60–70°C. The enzymes were stable for 30 min at 60°C, maintaining 95–98% of the initial activity. After 1 h at this temperature, the xylanase from A. niveus still retained 85% of initial activity, while the xylanase from A. fumigatus was only 40% active. The pH optimum of the xylanases was acidic (4.5–5.5). The pH stability for the xylanase from A. fumigatus was higher at pH 6.0–8.0, while the enzyme from A. niveus was more stable at pH 4.5–6.5. Crude enzymatic extracts were used to clarify cellulose pulp and the best result was obtained with the A. niveus preparation, showing kappa efficiency around 39.6% as compared to only 11.7% for that of A. fumigatus.     

Effect of medium-pH and MES on adventitious root formation from stem disks of apple

We have examined the effect of medium-pH on rooting using 1-mm slices cut from stems of apple microshoots. Before autoclaving, the pH of the rooting medium was set at various pH values between 4.5 and 8.0. During autoclaving, the pH drifted in particular in the alkaline region. Additional changes occurred during culture and the range set at 4.5–8.0 had shifted to 5.2–6.0 after autoclaving and 3 weeks of culture. When 10 mM 2-(N-morpholino)ethanesulfonic acid (MES) had been added as buffering agent, the pH was stable when set at 5.0–6.5. Highest rooting was achieved at pH ~5.3 with and without MES (pH measured after autoclaving). This maximum did not correlate with highest auxin uptake. MES inhibited adventitious root formation during the initial phase of root formation when the meristemoids are being formed (ca. 30% reduction at 10 mM) but was promotive during outgrowth of the meristemoids to roots (30% increase at 10 mM). Inhibition and promotion by MES were not related to its buffering action as they were observed at all pHs.     

Effects of pH profiles on nisin fermentation coupling with foam separation

Online foam separation was proposed to recover nisin during fermentation of Lactococcus lactis subsp. lactis ATCC 11454. Firstly, the optimal pH profile of nisin fermentation was investigated including different realkalization set values and pH drop gradients. Then the selected pH profiles of 5.75 ± 0.05 and 6.25–5.75 (±0.02) were used to perform nisin fermentation coupling with foam separation. The results showed that pH profile of 5.75 ± 0.05 was better than that of 6.25–5.75 (±0.02) for online foam separation. With the optimal pH profile, an aeration of 20 ml min⁻¹ that started at 8 h of incubation and lasted for 2 h resulted in 6.6 times higher specific productivity than that of the fermentation without aeration. Nisin synthesis was therefore prolonged with low sucrose concentration in the culture broth, which indicated that the feedback inhibition of nisin is more influential than the substrate limitation of sucrose in the late phase of nisin fermentation. Total nisin production (4,870 ± 180 IU ml⁻¹) was increased by 30.3% with online foam separation. This effective online recovery method for nisin production could be easily scaled up due to the facile operation of foaming process.     

Glucoamylase production in batch, chemostat and fed-batch cultivations by an industrial strain of Aspergillus niger

The Aspergillus niger strain BO-1 was grown in batch, continuous (chemostat) and fed-batch cultivations in order to study the production of the extracellular enzyme glucoamylase under different growth conditions. In the pH range 2.5–6.0, the specific glucoamylase productivity and the specific growth rate of the fungus were independent of pH when grown in batch cultivations. The specific glucoamylase producivity increased linearly with the specific growth rate in the range 0–0.1 h⁻¹ and was constant in the range 0.1–0.2 h⁻¹. Maltose and maltodextrin were non-inducing carbon sources compared to glucose, and the maximum specific growth rate was 0.19 ± 0.02 h⁻¹ irrespective of whether glucose or maltose was the carbon source. In fed-batch cultivations, glucoamylase titres of up to 6.5 g l⁻¹ were obtained even though the strain contained only one copy of the glaA gene. Received: 5 May 1999 / Received revision: 7 September 1999 / Accepted: 17 September 1999     

Kinetic models of ribonucleic acid fermentation and continuous culture by <Emphasis Type="Italic">Candida tropicalis</Emphasis> no.121

During ribonucleic acid fermentation, the fermentative processes were researched at pH controlled at 4.0 and under natural conditions. Unstructured models in a 50-L airlift fermentor were established for batch RNA production at pH 4.0 using the Verhulst equation for microbial growth, the Luedeking–Piret equation for product formation and a Luedeking–Piret-like equation for substrate uptake. Parameters of the kinetic models were determined using origin 7.5. Based on the models estimated above, another batch fermentation experiment was conducted in a 300-L airlift fermentor, which demonstrated that the models could be useful for RNA production on an industrial scale. Additionally, continuous fermentation based on kinetic models was proposed to make full use of substrates and reduce the cost of waste water treatment. As a result, although the DCW and RNA concentration were 11.5 and 1.68 g L⁻¹, which were lower than that of batch fermentation, the sugar utilization increased by 14.3%, while the waste water decreased by more than 90%.     

Differential effect of pH on the density and volume of rat reticulocytes and erythrocytes

Rats were injected with⁵⁹Fe-ferrous citrate and bled thereafter at different times (16 h to 49 d). This gave rise to red cell populations in which cells corresponding in age to the time elapsed between injection and bleeding were labeled. The anticoagulant used was either acid-citrate-dextrose (ACD) with a pH adjusted to 7.3 or ACD (pH 5.1). Final pH of the collected blood was about 7.2–7.4 in the former case and 6.4–6.7 in the latter. Red cells were then centrifuged (5) and approximately 7–10% of the packed cells from the top and 7–10% from the bottom of the cell column collected. When reticulocytes are the predominant labeled red cell population, as in blood obtained for about 24 h after isotope injection, a fractionation of these cells and mature erythrocytes is in evidence only when blood is collected at the higher pH. Thus, at pH 7.2–7.4 ratios of specific radioactivities of cells in top fraction/cells in an unfractionated sample are about 3, whereas at pH 6.4–6.7, the analogous ratios are 1 or less. These differences in specific activity ratios, as a function of pH at collection, virtually disappear after about 4 d following isotope injection. The lower pH is known to increase the volume and decrease the density of mature red blood cells. The marked effect of pH on cellular fractionation could be correlated with the smaller change in rat reticulocyte density and volume in acid medium. At pH 6.4–6.7, the densities of mature erythrocytes and reticulocytes are so close that their physical separation by centrifugation is not feasible.     

Extracellular amylase production bySaccharomycopsis capsularis and its evaluation for starch saccharification

A strain of starch-assimilating yeast,Saccharomycopsis capsularis, isolated from Indian cereal-based fermented foods, produced significant levels of extracellular α-amylase and glucoamylase. The enzymes reached their peak activities during the stationary phase at the end of the 5th and 4th day of cultivation, respectively. The amylase yields were maximized by a proper choice of carbon and nitrogen sources, starting pH of the culture medium and growth temperature. High activities of the enzymes were obtained through inexpensive agricultural commodities, such as wheat bran and corn meal as carbon sources, and defatted soybean meal and peanut meal as nitrogen sources. A temperature of 28–32°C and an initial pH of 4.5–5.0 were optimum. The crude amylase mixture could liquefy and saccharify a 1% starch solution completely in 24 h at 50°C.     

Thermolabile xylanase of the Antarctic yeast Cryptococcus adeliae: production and properties

Xylanase production by the Antarctic psychrophilic yeast Cryptococcus adeliae was increased 4.3 fold by optimizing the culture medium composition using statistical designs. The optimized medium containing 24.2 g l⁻¹ xylan and 10.2 g l⁻¹ yeast extract and having an initial pH of 7.5 yielded xylanase activity at 400 nkat (nanokatal) ml⁻¹ after 168-h shake culture at 4°C. In addition, very little endoglucanase, β-mannanase, β-xylosidase, β-glucosidase, α-l-arabinofuranosidase, and no filter paper cellulase activities were detected. Among 12 carbon sources tested, maximum xylanase activity was induced by xylan, followed by lignocelluloses such as steamed wheat straw and alkali-treated bagasse. The level of enzyme activity produced on other carbon sources appeared to be constitutive. Among the complex organic nitrogen sources tested, the xylanase activity was most enhanced by yeast extract, followed by soymeal, Pharmamedia (cotton seed protein), and Alburex (potato protein). A batch culture at 10°C in a 5-l fermenter (3.5-1 working volume) using the optimized medium gave 385 nkat at 111 h of cultivation. The crude xylanase showed optimal activity at pH 5.0–5.5 and good stability at pH 4–9 (21 h at 4°C). Although the enzyme was maximally active at 45°–50°C, it appeared very thermolabile, showing a half-life of 78 min at 35°C. At 40°–50°C, it lost 71%–95% activity within 5 min. This is the first report on the production as well as on the properties of thermolabile xylanase produced by an Antarctic yeast. Received: December 10, 1999 / Accepted: March 23, 2000     

Anaerobic fermentation of gelatinized sago starch-derived sugars to acetone—1-butanol—Ethanol solvent byClostridium acetobutylicum

A study of the kinetics and performance of solvent-yielding batch fermentation of individual sugars and their mixture derived from enzymic hydrolysis of sago starch byClostridium acetobutylicum showed that the use of 30 g/L gelatinized sago starch as the sole carbon source produced 11.2 g/L total solvent,i.e. 1.5–2 times more than with pure maltose or glucose used as carbon sources. Enzymic pretreatment of gelatinized sago starch yielding maltose and glucose hydrolyzates prior to the fermentation did not improve solvent production as compared to direct fermentation of gelatinized sago starch. The solvent yield of direct gelatinized sago starch fermentation depended on the activity and stability of amylolytic enzymes produced during the fermentation. The pH optima for α-amylase and glucoamylase were found to be at 5.3 and 4.0–4.4, respectively. α-Amylase showed a broad pH stability profile, retaining more than 80% of its maximum activity at pH 3.0–8.0 after a 1-d incubation at 37°C. SinceC. acetobutylicum α-amylase has a high activity and stability at low pH, this strain can potentially be employed in a one-step direct solvent-yielding fermentation of sago starch. However, theC. acetobutylicum glucoamylase was only stable at pH 4–5, maintaining more than 90% of its maximum activity after a 1-d incubation at 37°C.     

Effects of temperature on simultaneous nitrification and denitrification via nitrite in a sequencing batch biofilm reactor

A laboratory scale experiment was described in this paper to enhance biological nitrogen removal by simultaneous nitrification and denitrification (SND) via nitrite with a sequencing batch biofilm reactor (SBBR). Under conditions of total nitrogen (TN) about 30 mg/L and pH ranged 7.15–7.62, synthetic wastewater was cyclically operated within the reactor for 110 days. Optimal operation conditions were established to obtain consistently high TN removal rate and nitrite accumulation ratio, which included an optimal temperature of 31 °C and an aeration time of 5 h under the air flow of 50 L/h. Stable nitrite accumulation could be realized under different temperatures and the nitrite accumulation ratio increased with an increase of temperature from 15 to 35 °C. The highest TN removal rate (91.9%) was at 31 °C with DO ranged 3–4 mg/L. Process control could be achieved by observing changes in DO and pH to judge the end-point of oxidation of ammonia and SND.     

Microbial Communities in the World's Largest Acidic Volcanic Lake, Kawah Ijen in Indonesia, and in the Banyupahit River Originating from It

A first study was made on the microbial community composition of the Indonesian crater lake Kawah Ijen (pH < 0.3) and the Banyupahit–Banyuputih river (pH 0.4–3.5) originating from it. Culture-independent, rRNA gene-based denaturing gradient gel electrophoresis was used to profile microbial communities in this natural and ancient, extremely acidic environment. Similarity in community profiles of the different sampling locations was low, indicating heterogeneity in community composition. Archaea were present at all sampling locations; archaeal diversity was low at the most acidic locations and increased at pH >2.6. Bacteria were not detected in the water column of the crater lake, but were found at all locations along the acidic river. Bacterial diversity increased with increasing pH. Eukarya were only present at pH >2.6. Retrieved rRNA gene sequences of Bacteria and Archaea were not closely related to known acidophilic species. It is concluded that tolerance to extreme acidity in this system is developed most extensively among Archaea. The acidity gradient of the Banyupahit–Banyuputih river has a clear effect on microbial community composition and biodiversity.     

Effect of pH on citrinin and red pigments production by Monascus purpureus CCT3802

The production of red pigments and citrinin by Monascus purpureus CCT3802 was investigated in submerged batch cultures performed in two phases: in the first phase, cells were grown on glucose, at pH 4.5, 5.5 or 6.5; after glucose depletion, pH was adjusted, when necessary, to 4.5, 5.5, 6.5, 7.0, 8.0 or 8.5, for a production phase. The highest total red pigments absorbance of 11.3 U was 16 times greater than the lowest absorbance and was achieved with growth at pH 5.5, followed by production at pH 8.5, which causes an immediate reduction of the intra cellular red pigments from 75% to 17% of the total absorbance. The lowest citrinin concentration, 5.5 mg L⁻¹, was verified in the same culture while the highest concentration, 55 mg L⁻¹, was verified in cultures entirely carried out at pH 5.5. An alkaline medium, besides promoting intra cellular red pigments excretion, strongly represses citrinin synthesis.