A novel two-phase bioprocess for the production of Arthrospira (Spirulina) maxima LJGR1 at pilot plant scale during different seasons and for phycocyanin induction under controlled conditions.

A two-phase outdoor cultivation bioprocess for Arthrospira maxima LJGR1 combined with phycocyanin induction in concentrated cultures under controlled conditions was evaluated using a modified low-cost Zarrouk medium. Growth was monitored during 4 cycles in 2018 and 4 cycles in 2019. Biomass was harvested and concentrated using membrane technology at the end of each cycle for further phycocyanin induction using blue LED light (controlled conditions, 24 h). The highest biomass productivity was observed during spring and summer cycles (13.63-18.97 gDWm-2 d-1); during mid-fall and mid-end fall, a decrease was observed (9.93-7.76 gDWm-2 d-1). Under favorable growth conditions, phycocyanin induction was successful. However, during cycles with unfavorable growth condition, phycocyanin induction was not observed. Reactive-grade phycocyanin (3.72 ± 0.14) was recovered and purified using microfiltration and ultrafiltration technologies.

Recovery of volatile fatty acids (VFA) from complex waste effluents using membranes.

Waste effluents from anaerobic digesters of agricultural waste were treated with a range of membranes, including microfiltration and nanofiltration (NF), to concentrate volatile fatty acids (VFA). Microfiltration was applied successfully to produce sterile, particle-free solutions with a VFA concentration of 21.08 mM of acetic acid and 15.81 mM of butyric acid. These were further treated using a variety of NF membranes: NF270 (Dow Chemicals, USA), HL, DL, DK (Osmonics, USA) and LF10 (Nitto Denko, Japan), achieving retention ratios of up to 75%, and giving retentates of up to 53.94 mM of acetate and 28.38 mM of butyrate. DK and NF270 membranes were identified as the best candidates for VFA separation and concentration from these multicomponent effluents, both in terms of retention and permeate flux. When the effluents are adjusted to alkali conditions, the highest productivity, retention and flux were achieved at pH 7. At higher pH there was a significant reduction in flux.

Strategies for the recovery of nutrients and metals from anaerobically digested dairy farm sludge using cross-flow microfiltration.

This work reports on the recovery of nutrients and metals from anaerobically digested manure sludge using a pilot scale microfiltration membrane system. Soluble nitrogen (N), phosphorous (P) and metals are valuable commodities which exist in high concentration in anaerobically digested manure sludge. The typical disposal of sludge on farmland can cause serious harm to the ecosystem due to eutrophication. The recovery of these materials in clarified solutions represents an added value product and a less contaminated sludge that is environmentally less hazardous. The objective of this study was to investigate the recovery of nutrients and metals using a pilot scale cross-flow membrane filtration system. A ceramic membrane of 0.22 m(2) and 0.2 µm pore size was used to perform solid-liquid separations and soluble materials were recovered in particle and bacteria free solutions. Strategies such as batch diafiltration (DF) and acid pre-treatment were investigated and the fractions collected compared against the initial permeate containing 686.2 mg NH3-N L(-1) and 41.51 mg PO4-P L(-1). Clarified fractions obtained through DF with no acid pre-treatment yielded N:P ratios of around 30 and relatively low levels of P (364.24 mg NH3-N L(-1) and 25.60 mg PO4-P L(-1)) and metals. Acid pre-treatment of the sludge resulted in a two-fold increase of P extracted (271.11 mg NH3-N L(-1) and 71.60 mg PO4-P L(-1)), altering N:P ratios to 8. Depending on the metal species, a 2-9 fold increase in concentration was also observed. Thus it has been demonstrated that different treatment strategies influence the removal and recovery of nutrients and metals from sludge. The best treatment conditions therefore depend on the targeted materials to be recovered. By careful manipulation of the treatment processes the production of specific nutrient compositions in terms of N:P ratios is possible.

Investigation of Shelf Life of Potency and Activity of the Lactobacilli Produced Bacteriocins Through Their Exposure to Various Physicochemical Stress Factors.

Three Lactobacilli strains, Lactobacillus casei NCIMB 11970, Lactobacillus plantarum NCIMB 8014, Lactobacillus lactis NCIMB 8586 have been used for the production of bacteriocins. Though, their production phase, their biochemical nature, their mode of activity even their genetic structure have been widely investigated, there are hardly any studies investigating their potency and activity in depth of time, in other words their shelf life under several physicochemical conditions that may occur during their production in large scale. As such, the effect of several factors influencing the activity and the potency of bacteriocins when produced in large scale was examined as due to bacteriocins peptide nature degradation or denaturation might occur, under extreme physicochemical conditions. During scale-up process, differences between the output data may occur, such as concerning biomass, metabolic by-products and limiting substrate concentrations. These may affect negatively the activity and the potency of the bacteriocins. For investigating these effects and minimizing them, numerous studies were conducted, which were related to the exact phase of the production of these substances, the effect of dilution and temperature changes. These studies could be used in order to minimize the scaling-up effect when decided to produce these peptides in large scale.

Leaching techniques to remove metals and potentially hazardous nutrients from trout farm sludge.

A fish farm sludge high in P (2-6% w/w as dry matter), Fe (5-7%), C (40-50%) and N (0.8-4%) was subjected to a series of acid leaching treatments using HCl, organic acids, and biologically mediated acid production. Additions of biodegradable organic acid solubilized heavy metals better than HCl, while additions of 1.5% w/v glucose followed by 7 day incubation stabilized the sludge releasing 92% P, 100% Fe. The use of homo-lactic Lactobacillus plantarum starter cultures were more effective than hetero-lactic Lactobacillus buchneri, solubilizing 81.9% P, 92.2% Fe, 93.0% Zn and 96.4% Ca in the sludge. The anaerobic sludge-glucose fermentation using L. plantarum produced a leached sludge that has low heavy metal and nutrient content while affording the recovery of nutrients. The potential of these methods for practical application are briefly discussed.

A comparative study of an intensive malolactic transformation of cider using Lactobacillus brevis and Oenococcus oeni in a membrane bioreactor.

The aim of this study was to investigate the secondary fermentation of alcoholic green cider by Lactobacillus brevis and Oenococcus oeni in a membrane bioreactor so as to compare the performance of the two organisms to rapidly carry out the malolactic fermentation (MLF), an important step in reducing acidity and enhancing the flavor characteristics of the beverages. First, the growth of both organisms was intensified by using perfusion culture in a membrane bioreactor (MBR). O. oeni and L. brevis were grown up to 12.8 g dry cell weight (DCW) l(-1) and 15.5 g DCW l(-1) in the MBR. Secondly, the resultant cells were then used for the malolactic transformation of green cider in the MBR. The influences of the residence time in the MBR and the ethanol concentration of the green cider on the organic acid transformation were investigated. Both organisms showed a good tolerance against the acidic conditions (pH 3.0-4.0) and ethanol (90 g l(-1)). Good levels of malate removal in the MBR were achieved by both organisms but O. oeni was more tolerant to high ethanol concentrations and was capable of growth and malate removal in 130 g ethanol l(-1) green cider. L. brevis malate removal was significantly inhibited above 110 g ethanol l(-1). The MBR allowed the development of high concentrations of active cells capable of rapid MLF and could be achieved over a prolonged period and over a wide range of conditions thus allowing the control of malate transformation rate. Organism selection for the transformation will be governed by the desired beverage characteristics. There is considerable scope to optimize the process further both with the choice of organisms and the design and operation of the reactor. Rapid beverage maturation on a commercial scale may be possible using MBR and pure cultures of MLF lactic acid bacteria.

Placing microalgae on the biofuels priority list: a review of the technological challenges.

Microalgae provide various potential advantages for biofuel production when compared with 'traditional' crops. Specifically, large-scale microalgal culture need not compete for arable land, while in theory their productivity is greater. In consequence, there has been resurgence in interest and a proliferation of algae fuel projects. However, while on a theoretical basis, microalgae may produce between 10- and 100-fold more oil per acre, such capacities have not been validated on a commercial scale. We critically review current designs of algal culture facilities, including photobioreactors and open ponds, with regards to photosynthetic productivity and associated biomass and oil production and include an analysis of alternative approaches using models, balancing space needs, productivity and biomass concentrations, together with nutrient requirements. In the light of the current interest in synthetic genomics and genetic modifications, we also evaluate the options for potential metabolic engineering of the lipid biosynthesis pathways of microalgae. We conclude that although significant literature exists on microalgal growth and biochemistry, significantly more work needs to be undertaken to understand and potentially manipulate algal lipid metabolism. Furthermore, with regards to chemical upgrading of algal lipids and biomass, we describe alternative fuel synthesis routes, and discuss and evaluate the application of catalysts traditionally used for plant oils. Simulations that incorporate financial elements, along with fluid dynamics and algae growth models, are likely to be increasingly useful for predicting reactor design efficiency and life cycle analysis to determine the viability of the various options for large-scale culture. The greatest potential for cost reduction and increased yields most probably lies within closed or hybrid closed-open production systems.

Studies on growth and metabolism of Oenococcus oeni on sugars and sugar mixtures.

AIMS: To study the effect of sugars and sugar mixtures on the growth kinetics of Oenococcus oeni NCIMB 11648 in batch culture with the aim of producing a high cell productivity system for starter cultures. METHODS AND RESULTS: The growth of O. oeni was investigated on single sugars (glucose, fructose or sucrose) and their mixtures (glucose-fructose, glucose-sucrose or fructose-sucrose). Better growth was obtained on sugar mixtures compared with growth on a single sugar. The production system of O. oeni biomass was investigated in batch culture with or without pH control with respect to kinetics, specific growth rate and biomass yield. The effect of pH and substrate concentration on fermentation balances and ATP yield were determined. The optimal growth of O. oeni was achieved on the glucose-fructose mixture (9 g l(-1), 1 : 1) at pH 4.5 and 25 degrees C with pH control, with highest cell volumetric productivity (7.9 mg cell l(-1) h(-1)), biomass yield (0.041 g cell g(-1) sugar) and specific growth rate (0.066 h(-1)). CONCLUSIONS: The limitations to the growth of O. oeni were pH and inhibition by end product resulting in poor utilization of the medium with low cell yields. The cell productivity of the system can be improved by the appropriate use of mixed sugar growth medium. SIGNIFICANCE AND IMPACT OF THE STUDY: This study uniquely showed that appropriate sugar mixtures with the correct environmental conditions can significantly improve the productivity of O. oeni cultures.

Formulation of defined media for carbon monoxide fermentation by Eubacterium limosum KIST612 and the growth characteristics of the bacterium.

Phosphate-buffered (PBBM) and carbonate-buffered (CBBM) basal media were used in the formulation of defined media for the cultivation of Eubacterium limosum KIST612 with carbon monoxide (CO) as the sole energy source. The bacterium was adapted to the minimal media by sequential passage in media containing casamino acids and those containing ammonium chloride in the place of yeast extract. Biological growth was slower with a lower growth yield in the defined minimal media than in PBBM or CBBM. More butyrate was produced in phosphate-buffered media than in carbonate-buffered media. The bacteria grew without any organic nitrogen in the presence of trace quantities of biotin and pantothenic acid. Anaerobic digester fluid stimulated bacterial growth.

Ethanol production by thermophilic bacteria: biochemical basis for ethanol and hydrogen tolerance in Clostridium thermohydrosulfuricum.

The metabolic and enzymatic bases for growth tolerance to ethanol (4%) and H2 (2 atm [1 atm = 101.29 kPa]) fermentation products in Clostridium thermohydrosulfuricum were compared in a sensitive wild-type strain and an insensitive alcohol-adapted strain. In the wild-type strain, ethanol (4%) and H2 (2 atm) inhibited glucose but not pyruvate fermentation parameters (growth and end product formation). Inhibition of glucose fermentation by ethanol (4%) in the wild-type strain was reversed by addition of acetone (1%), which lowered H2 and ethanol production while increasing isopropanol and acetate production. Pulsing cells grown in continuous culture on glucose with 5% ethanol or 1 atm of H2 significantly raised the NADH/NAD ratio in the wild-type strain but not in the alcohol-adapted strain. Analysis of key oxidoreductases demonstrated that the alcohol-adapted strain lacked detectable levels of reduced ferredoxin-linked NAD reductase and NAD-linked alcohol dehydrogenase activities which were present in the wild-type strain. Differences in the glucose fermentation product ratios of the two strains were related to differences in lactate dehydrogenase and hydrogenase levels and sensitivity of glyceraldehyde 3-phosphate dehydrogenase activity to NADH inhibition. A biochemical model is proposed which describes a common enzymatic mechanism for growth tolerance of thermoanaerobes to moderate concentrations of both ethanol and hydrogen.

The growth and nutrition of Clostridium sporogenes NCIB 8053 in defined media.

Various defined and minimal media are described for the growth of Clostridium sporogenes NCIB 8053. The organism requires 10 amino acids and one vitamin for growth, whilst three other vitamins are growth stimulatory. L-alpha-hydroxy acid analogues can replace eight, and fatty acid analogues four, of these amino acids. The organism may generate free energy by a variety of Stickland reactions. Most Stickland acceptors, but not glycine, stimulate the growth of this organism on glucose. Nonetheless, cells grown in the presence of glycine will reductively deaminate it. The media described support the growth of several other strains of this species. The simplified growth media which we have developed permit quantitative studies of the physiology of this organism.

The physiology of Clostridium sporogenes NCIB 8053 growing in defined media.

The physiology of Clostridium sporogenes was investigated in defined, minimal media. In batch culture, the major end products of glucose dissimilation were acetate, ethanol and formate. When L-proline was present as an electron acceptor, acetate production was strongly enhanced at the expense of ethanol. As judged by assay of the relevant enzymes, glucose was metabolized via the Embden-Meyerhof-Parnas pathway. The growth energetics of Cl. sporogenes were investigated in glucose- or L-valine-limited chemostat cultures. In the former case, the addition of L-proline to the medium caused a significant increase in the molar growth yield (as calculated by extrapolation to infinite dilution rate). This finding adds weight to the view that the reduction of L-proline by Cl. sporogenes is coupled to the conservation of free energy.

Ethanol Production by Thermophilic Bacteria: Physiological Comparison of Solvent Effects on Parent and Alcohol-Tolerant Strains of Clostridium thermohydrosulfuricum.

The effects of temperature, solvents, and cultural conditions on the fermentative physiology of an ethanol-tolerant (56 g/liter at 60 degrees C) and parent strain of Clostridium thermohydrosulfuricum were compared. An ethanol-tolerant mutant was selected by successive transfer of the parent strain into media with progressively higher ethanol concentrations. Physiological differences noted in the mutant included enhanced growth, tolerance to various solvents, and alterations in the substrate range and the fermentation end product ratio. Ethanol tolerance was temperature dependent in the mutant but not in the parent strain. The mutant grew with ethanol concentrations up to 8.0% (wt/vol) at 45 degrees C, but only up to 3.3% (wt/vol) at 68 degrees C. Low ethanol concentration (0.2 to 1.6% [wt/vol]) progressively inhibited the parent strain to where glucose was not fermented at 2.0% (wt/vol) ethanol. Both strains grew and produced alcohols on glucose complex medium at 60 degrees C in the presence of either 5% methanol or acetone, and these solvents when added at low concentration stimulated fermentative metabolism. The mutant produced ethanol at high concentrations and displayed an ethanol/glucose ratio (mole/mole) of 1.0 in media where initial ethanol concentrations were

The gradostat: a bidirectional compound chemostat and its application in microbiological research.

A multistage continuous culture system is described in which solutes are transferred between vessels in opposite directions simultaneously. The system, called a gradostat, produces opposing solute gradients and is a good laboratory model of many natural microbial ecosystems in which solute gradients are important. Theoretical predictions concerning solute transfer were confirmed under steady-state and non steady-state conditions, using a coloured dye. Paracoccus denitrificans grew anaerobically in the gradostat at the intersection between opposing gradients of succinate and nitrate. Opposing gradients of glucose and oxygen separated the growth of a Bacillus sp. (a facultative anaerobe) and Clostridium butyricum (an obligate anaerobe). Viable counts for both species fell exponentially away from their growth positions at the ends of the gradostat. The potential value of the gradostat and possible alternative conformations are discussed.

Physiological behaviour of Escherichia coli grown in opposing gradients of oxidant and reductant in the gradostat.

Gradients of nutrients are extremely common in nature, and this paper describes changes in the physiology of Escherichia coli grown in the gradostat, a series of five linked vessels with opposing gradients of glucose and of oxygen plus nitrate. Most growth occurred at the aerobic and anaerobic ends of the system. High rates of respiration, high energy charge and high activities of various oxidative enzymes were seen in the two most aerobic vessels; however, oxygen provision was presumably poor, because nitrate reductase activities were also high in this region. Vessels 3 and 4 showed the lowest values for respiration rate, enzyme activity and energy charge, and cells here were both nutrient starved and possibly inhibited by nitrite. Vessel 5 was highly anaerobic, resulting in the presence of hydrogenase activity. It was concluded that cells found in different regions of the gradostat had undergone biochemical differentiation in spatial gradients of electron donors and acceptors.