Institute for Environmental Biotechnology

It has been established that dissolved heavy metals escaping into the environment pose a serious health hazard. As a result, there is an urgent need for controlling metal emissions into the environment. The aims of this study were to purify and biochemically characterise hydrogenase(s) from sulphate reducing consortium (SRB) and investigate the potential of the purified enzyme(s) in the recovery of platinum from wastewaters. A hydrogenase from sulphate reducing consortium was purified by a combination of PEG 20000 concentration, ion exchange (Toyopearl-Super Q 650 S) and size exclusion (Sephacryl S-200) chromatographies. SDS-PAGE analysis revealed a distinct protein band with a molecular mass of 58 kDa. The investigation of enzymatic platinum (IV) reduction in vitro, showed highest hydrogen-dependent platinum (IV) reducing activity in the presence of hydrogenase and its physiological electron carrier, cytochrome c 3. When the purified hydrogenase enzyme (with and without cytochrome c 3) was used with the industrial effluent, containing 7.9 mg.l-1 platinum, only 10-15% recovery was noted pointing to a suppression of enzyme activity due to the low pH (0.38) of the effluent. Bioremediation studies on industrial effluent using resting SRB cells showed a 34% platinum removal from the effluent while growing SRB cells, within a sulphidogenic reactor, gave a platinum removal of 78%, with the pH of the system fluctuating at around 5.6. Evidence of sulphate reduction and sulphide generation were not observed during this treatment process suggesting that platinum sulphide was not formed and supporting the argument that the increased amount (78%) of platinum removal from the industrial wastewater by the growing SRB cells was due to more hydrogenase/cytochrome c 3 enzyme complex being available.

Fundamental processes involved in the microbial degradation of coal and its derivatives have been well investigated and documented over the past two decades. However, limited progress in industrial application has been identified as bottleneck in further active development of the field. The sporadic and unanticipated growth of Cynodon dactylon (Bermuda grass) has been observed on the surface of some coal dumps in the Witbank coal mining area of South Africa. Preliminary investigations showed the formation of a humic soil-like material from the breakdown of hard coal in the root zone of these plants. The potential of this system to contribute to industrial scale bioprocessing of hard coal was investigated.
This study involved an investigation of the C. dactylon/coal rhizosphere environment and demonstrated the presence of fungal species with known coal bioconversion capability.
Amongst these Neosartorya fischeri was identified and its activity in coal bioconversion was described for the first time. Cynodon dactylon plant roots were also shown to be colonized by mycorrhizal fungi including Glomus, Paraglomus and Gigaspora species. The role of plant photosynthate translocation into the root zone, providing organic carbon supplementation of fungal coal bioconversion was investigated in deep liquid culture with the N. fischeri isolate used as the biocatalyst. Organic acids, sugars and complex organic carbon sources were investigated and it was shown that glutamate provided significant enhancement of bioconversion activity in this system.
The performance of N. fischeri in coal bioconversion was compared with Phanaerochaete chrysosporium and Trametes versicolor, both previously described fungal species in the coal bioconversion application. Fourier transform infrared spectroscopy indicated more pronounced oxidation and introduction of nitro groups in the matrix of the humic acid product of coal bioconversion in N. fischeri and P. chrysosporium than for T. versicolor. Macro-elemental analysis of biomass-bound humic acid obtained from the N. fischeri catalyzed reaction showed an increase in the oxygen and nitrogen components and coupled with a reduction in carbon and hydrogen. Pyrolysis gas chromatography mass spectroscopy further supported the proposal that the mechanism of bioconversion involves oxygen and nitrogen insertion into the coal structure.
The C. dactylon bituminous hard coal dump environment was simulated in a fixed-bed perfusion column bioreactor in which the contribution of organic supplement by the plant/mycorrhizal component of the system was investigated. The results enabled the proposal of a descriptive model accounting for the performance of the system in which the plant/mycorrhizal component introduces organic substances into the root zone. The non-mycorrhizal fungi utilize the organic carbon supplement in its attack on the coal substrate, breaking it down, and releasing plant nutrients and a soil-like substrate which in turn enables the growth of C. dactylon in this hostile environment.
Based on these results, the Stacked Heap Coal Bioreactor concept was developed as a large-scale industrial bioprocess application based on heap-leach mineral processing technology. Field studies have confirmed that bituminous hard coal can be converted to a humic acid rich substrate in a stacked heap system inoculated with mycorrhizal and N. fischeri cultures and planted with C. dactylon.

African Journal of Biotechnology Vol. 2 (9), pp. 264-270, September 2003 Available online at http://www.academicjournals.org/AJB ISSN 1684–5315 © 2003 Academic Journals ... Mevastatin-induced inhibition of cell growth in avocado ... Dudley I. Mitchell and A. Keith ...

The biochemical basis of color as an aesthetic quality in mature fruit of navel and Valencia orange (Citrus sinensis) was determined. Saponification of the two major color-imparting components resolved by thin-layer chromatography, followed by reversed-phase high-performance liquid chromatography, revealed that these comprised acyl esters of (9Z)-violaxanthin and -citraurin. Identification of the chromophores was based on cochromatography and online spectral analysis. The color quality of flavedo of mature fruit was dependent on the content and relative amounts of (9Z)-violaxanthin and -citraurin. Quantitative results revealed that increased color intensity was associated with a decline in the (9Z)-violaxanthin: -citraurin ratio from greater than 50 to below 10, an increase in flavedo (9Z)-violaxanthin and -citraurin content, and that measurement of the mass and ratio of these carotenoids can be used to accurately color-grade orange fruit for local and export markets.

xanthine dehydrogenase (XDH) activity and a model illustrating the proposed metabolic interrelationship is The penultimate step in abscisic acid (ABA) biosynpresented. thesis involves oxidation of xanthoxal (XAN) catalysed by a molybdenum-cofactor (MoCo)-containing alde-Key words: Abscisic acid, aldehyde oxidase, allopurinol, hyde oxidase (AO) and represents one potential site avocado, cytokinin, molybdenum cofactor, tungstate, of regulation of ABA in plant tissues. In an attempt xanthoxal. to understand the biochemical basis for cytokininabscisic acid (CK-ABA) antagonism the effect of sev-

Cell-free extracts prepared from an acetone powder derived from the flavedo of mature, coloured fruits of Citrus sinensis L. cv. Midknight transformed mevalonic acid, isopentenyl pyrophosphate, all-rrans-/?-carotene and 1 ,4 -frans-ABAdiol into ABA. This is the first report of a cell-free system capable of producing ABA from both a terpenyl pyrophosphate and carotenoid origin. ABA biosynthesizing activity was stimulated by reduced nicotinamide nucleotides, molybdate, AMO1618 and a cold-pool trap of (±)-ABA, but was inhibited by FAD. Addition of FAD caused accumulation of label from isopentenyl pyrophosphate in a compound with similar chromatographic and spectrophotometric properties to those of ^-carotene. A ,4-Trans-ABAdiol, ABA and PA were unequivocally characterized as products of ^-carotene metabolism in this cell-free system, a process that was markedly improved by extraction of enzyme in the presence of detergents. Dithiothreitol enhanced ABA biosynthesis from allfrans-/?-carotene. Stigmasterol, and to a lesser extent cholesterol reduced conversion of ^-carotene to ABA, but did not influence transformation of 1 ,4-frans-ABAdiol to ABA. AM01618 stimulated formation of ABA and appeared to exert its effect at the level of conversion of 1 ,4'-frans-ABAdiol to ABA. Ancymidol and zeatin reduced incorporation of label from aW-trans-flcarotene into ABA suggesting involvement of a mixed function oxidase in this reaction sequence. Sodium dodecylsulphate polyacrylamide gel electrophoresis of the enzyme extract derived from Citrus flavedo revealed the presence of a 53 kDa protein with peroxidase activity characteristic of a cytochrome P-450.

A cell-free system prepared from Citrus sinensis exocarp transformed R-[2-14 C}mevalonic acid into abscisic acid, phaseic acid and 1',4'-trans-abscisic acid diol. 1',4'-Trans-abscisic acid diol was unequivocally characterized as the major product of mevalonic acid metabolism in the Citrus cell-free system. The addition of (R,S)-abscisic acid as a «cold-pool trap» increased incorporation of radioactivity into putative abscisic acid but reduced the levels of [HC] associated with 1',4'-trans-abscisic acid diol, negating a role for this metabolite as a precursor to abscisic acid.

The contribution of xanthine dehydrogenase (XDH, EC 1.1.1.204) to fruit size was investigated using the normal and small-fruit variants of Persea americana Mill. cv. 'Hass'. Inhibition of XDH by treatment of normal fruit, in the linear phase of growth (phase II), with allopurinol (Allo) arrested fruit growth. Adenine (Ade), a less effective inhibitor of this enzyme, also arrested fruit growth when applied in phase II and slowed fruit growth when applied in phase III. A time-course study on the activity of XDH in mesocarp tissue from normal and small fruit showed that maximum activity occurred late in phase II and that the peak in activity was absent in mesocarp of the small fruit. Feeding Ade to growing fruit in phase III caused a transient decline in fruit growth (measured as change in fruit length). Thereafter, growth resumed although fruit size was irreversibly affected. Treatment of fruit with Ade and Adecontaining cytokinins altered activity of another molybdenum enzyme, aldehyde oxidase (EC 1.2.3.1). Cytokinin oxidase was induced by cytokinin and auxin. Purine catabolism via hypoxanthine/xanthine was operative in normal fruit and in mesocarp from the small-fruit variant and as expected, Allo treatment caused accumulation of xanthine and adenine. In the absence of an increase in XDH during growth of the small-fruit phenotype, low levels of Ade were interpreted as resulting from respiration-enhanced adenylate depletion. Stress and/or pathogen induction of the alternative oxidase pathway is proposed as a possible cause.

respiration and both seed and mesocarp tissue showed increased Carbohydrate and abscisic acid (ABA) metabolism were inves-ABA metabolism. Applied ABA caused an increase in insoluble tigated in normal and phenotypically small 'Hass' avocado (Persea americana Mill.) fruit in an attempt to link alterations acid invertase activity in seed tissue of normal fruit while mevastatin reduced HMGR activity in this tissue, caused in sugar and ABA content with changes in 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR, EC 1.1.1.34) activity sucrose depletion and increased the proportion of glucose from 5 to 57% of total soluble sugars. Exogenous glucose suppressed and fruit size. The small-fruit phenotype was characterized by reduced seed HMGR activity, increased seed insoluble acid HMGR activity in seed tissue whereas in mesocarp tissue, invertase (-D-fructofuranosidase, EC 3.2.1.26), decreased su-HMGR activity was reduced to 38% of the control after 6 h but enhanced by 46% by 48 h. Glucose increased ABA crose synthase (SS; UDP-D-glucose:D-fructose-2--glucosyltransferase, EC 2.4.1.13) activity, decreased sucrose content, biosynthesis and turnover in competent tissues. These results and increased glucose as a proportion of the total soluble sugar.

non-fermenting 1-related protein kinase (SnRK1) complex. It biochemical and molecular processes at any stage during fruit growth will impact on final fruit size. Because fruit size is a is argued that sugar content and composition of sink cells impact on SnRK1 (and hexokinase) to modulate expression of function of cell number rather than cell size, factors affecting sugar-metabolizing enzymes, HMGR and molybdenum cofac-cell division cycle activity assume importance. In this paper, we focus attention on the metabolic control of fruit growth using tor (MoCo)-containing enzymes. These changes, in turn, impact on hormone metabolism by affecting allocation of the avocado as a model system. Three areas of current interest are purine-derived MoCo to aldehyde oxidase and thus the endoge-highlighted, viz. the contribution by isoprenoid metabolism in the control of cell proliferation, the role played by carbohydrate nous concentration of indole-3-acetic acid, abscisic acid and cytokinin (CK) to alter plant hormone homeostasis. These content and composition in signalling changes in metabolite status and gene expression and maintenance of plant hormone aspects are integrated into a model to explain the metabolic homeostasis. Central to the process of fruit growth and control control of avocado fruit growth and final fruit size.

In this paper the potential to use phospholipids and lysophospholipids as plant growth regulators is discussed. Recent evidence shows that phospholipids and phospholipases play an important signalling role in the normal course of plant development and in the response of plants to abiotic and biotic stress. It is apparent that phospholipase A (PLA), C (PLC) and D (PLD), lysophospholipids, and phosphatidic acid (PA) are key components of plant lipid signalling pathways. By comparison, there is very little information available on the effect of exogenously applied phospholipids on plant growth and development. This paper serves to introduce phospholipids as a novel class of plant growth regulator for use in commercial plant production. The biochemistry and physiology of phospholipids is discussed in relation to studies in which phospholipids and lysophospholipids have been applied to plants and plant parts. Implicit in the observations is that phospholipids impact the hypersensitive response and systemic acquired resistance in plants to improve crop performance and product quality. Based on published data, a scheme outlining a possible mode of action of exogenously applied phospholipids is proposed.

Experiments were carried out to evaluate the effect of glucose on ripening and ethylene biosynthesis in tomato fruit (Lycopersicon esculentum Mill.). Fruit at the light-red stage were vacuum infiltrated with glucose solutions post-harvest and changes in 1-aminocyclopropane-1-carboxylic acid (ACC) synthase, ACC, ACC oxidase, and ethylene production monitored over time. ACC oxidase activity was also measured in pericarp discs from the same fruits that were treated either with glucose, fructose, mannose, or galactose. While control fruit displayed a typical peak of ethylene production, fruit treated with glucose did not. Glucose appeared to exert its effect on ethylene biosynthesis by suppressing ACC oxidase activity. Fructose, mannose, and galactose did not inhibit ACC oxidase activity in tomato pericarp discs. Glucose treatment inhibited ripening-associated colour development in whole fruit. The extent of inhibition of colour development was dependent upon the concentration of glucose. These results indicate that glucose may play an important role in ethylene-associated regulation of fruit ripening.

Lyso-phosphatidylethanolamine (LPE) is a minor membrane glycerolipid and egg-derived 18:0-LPE is used commercially as a plant bio-regulator to improve plant product quality. Physiological responses initiated by LPE treatment included delayed senescence in leaves and fruits, improved shelf-life of products post harvest, and mitigation of ethylene-induced process. However, the biochemical and molecular mechanisms underlying LPE-induced responses in plants and harvested plant parts remain unclear. In this paper, commentary is presented on the effects of LPE at the biochemical level in an effort to develop a mode of action. Implications, although tentative, are that LPE exerts its effect via lipid-protein interaction to attenuate ethylene (ETH)-mediated responses and impact pathogenesis-related proteins which together delay senescence progression.

Senescence and reserve mobilization are integral components of plant development, are basic strategles in stress mitigation, and regulated at least in part by cytokinin. In the present study the effect of altered cytokinin metabolism caused by senescence-specific autoregulated expression of the Agrobacterium tumefaciens IPT gene under control of the P SAG12 promoter (P SAG12 -IPT) on seed germination and the response to a water-deficit stress was studied in tobacco (Nicotiana tabacum L.). Cytokinin levels, sugar content and composition of the leaf strata within the canopy of wild-type and P SAG12 -IPT plants confirmed the reported altered source-sink relations. No measurable difference in sugar and pigment content of discs harvested from apical and basal leaves was evident 72 h after incubation with (+)-ABA or in darkness, indicating that expression of the transgene was not restricted to senescing leaves. No difference in quantum efficiency, photosynthetic activity, accumulation of ABA, and stomatal conductance was apparent in apical, middle and basal leaves of either wild-type or P SAG12 -IPT plants after imposition of a mild water stress. However, compared to wild-type plants, P SAG12 -IPT plants were slower to adjust biomass allocation. A stress-induced increase in root:shoot ratio and specific leaf area (SLA) occurred more rapidly in wild-type than in P SAG12 -IPT plants reflecting delayed remobilization of leaf reserves to sink organs in the transformant. P SAG12 -IPT seeds germinated more slowly even though abscisic acid (ABA) content was 50% that of the wild-type seeds confirming cytokinin-induced alterations in reserve remobilization. Thus, senescence is integral to plant growth and development and an increased endogenous cytokinin content impacts sourcesink relations to delay ontogenic transitions wherein senescence in a necessary process.

The "subtropical" avocado tree evolved in a cool, mesic highland rainforest with a decomposing litter layer, in which its feeder roots proliferate. We reasoned that under more stressful orchard conditions it should benefit from mulching with suitable materials to reinforce the natural dead leaf layer. We also hypothesized that the resultant improved root health and growth would partly ameliorate the 'Hass' small fruit syndrome, which is associated with "stress". An ongoing mulching trial with composted pine bark in the cool, mesic summer rainfall mistbelt of KwaZulu-Natal has led to an average increase form mulching (over 3 seasons) of 7% in fruit size, 15% in fruit number and 23% in yield. Mulching reduced measured components of tree stress, including a lowering of leaf canopy temperature, photoinhibition of photosynthesis, premature seed coat abortion and pedicel ringneck. Phenotypically small fruits were higher in flesh abscisic acid and lower in cyt...