In this study, a range of nutritional supplements including twenty amino acids, major vitamins and four nucleic acid bases were exploited as added-value supplements for the growth of a lactate-minus ( ldh ) mutant Bacillus stearothermophilus LLD-16 under anaerobic environment. The chemostat studies revealed that five amino acids that includes aspartate, glutamate, isoleucine, methionine, and serine were essential for persuaded growth of B. stearothermophilus LLD-16. The anaerobic batch studies showed that a number of nutritional supplements, such as, p -aminobenzoic acid (PABA), folic acid, pantothenic acid, adenine, glycine, leucine, tryptophan, proline, alanine and α-ketoglutarate, when added individually, improved the biomass levels. In contrast, the higher concentrations of cyanocobalamine or biotin, guanine, uracil and isoleucine were found inhibitory. Furthermore, the study explains why the highest biomass formation cannot necessarily be achieved on the richest mixture of amino acids, and the inadequacy of the biosynthetic machinery is very much dependent on the growth conditions of the microorganism.

Bacillus stearothermophilus LLD-R is a prototype facultative anaerobic thermophile which predominantly produces lactate while its lactate-minus mutant, B. stearothermophilus LLD-16 mainly produces ethanol ( Javed, 1993 ). Both strains can grow on a wide range of hexoses and pentoses carbon substrates. More attention, therefore, has been given on its growth on pentose sugars, such as xylose because it is one of the major components of hemicelluosic sugars and is relatively recalcitrant. As a part of these studies, this paper describes the nutritional requirements of this microorganism in the medium with xylose as the sole carbon and energy source, since a knowledge of these growth requirements, for physiological studies, is desirable.

The nutritional requirements of several strains of B. stearothermophilus have been studied by various research workers ( Campbell and Williams, 1953 , Rowe et al., 1975 and Lee et al., 1982 Amartey et al., 1991 and San Martin et al., 1992 ). These workers showed that different strains had different growth requirements. However, they rarely exploited the nutritional requirements in the presence of xylose as sole carbon and energy source. LLD-15 is another ethanol producing mutant of B. stearothermophilus LLD-R strain ( Payton & Hartley, 1985 ) and their nutritional requirements are quite similar to each other ( Javed, 1993 ). A defined medium for the anaerobic growth of LLD-15 strain on sucrose has been described by San Martin et al. (1992) . However, this medium does not support the growth of either LLD-15 or LLD-16 strains on xylose as the nutritional requirements vary with different carbon sources, because different carbon substrates enter the central metabolic pathway by various routes ( Kotte et al., 2014 and Hollinshead et al., 2015 ).

All microorganisms synthesize their cellular protein from 20 different amino acids which are either synthesized by the cells or must be provided in the growth medium. The biosynthesis of these amino acids starts at five different points in central metabolic pathway and this is divided into five different amino acid families: aromatic, serine, pyruvate, glutamate and aspartate amino acid families, as shown in Fig. 1 ( Cooper, 2000 and Barton, 2005 ). Although histidine is generally shown outside of these families, but for this study it has been considered as a part of aromatic amino acid family ( Chen et al., 2001 and Betts and Russell, 2003 ). Occasionally, provision of all 20 amino acids in a growth medium can support only a week growth. However, higher biomass is achieved in the complex medium because microorganisms show requirement for peptides for higher growth rates ( Currell & Dam-Mieras, 2014 ).

Biosynthetic routes of amino acids.

Since performing such studies in chemostat culture are very time consuming, growth requirements were initially determined in shake bottles and later on by using a technique known as ‘Pulse and Shift technique’ originally developed by Mateles and Battat (1974) and later on modified by Kuhn et al. (1979) . Therefore, to gain an insight into the utilization of representative nutritional supplements, this paper explores some of the physiological responses by examining the fate of these supplements after their addition to the culture of B. stearothermophilus .

All of the chemicals/reagents, amino acids, vitamins and four nucleic acid bases were obtained from used in the current work were mainly purchased from Sigma–Aldrich Company Ltd. (Gillingham, Dorset, United Kingdom). For quantitative and qualitative assays, analytical grade reagents and ultra-pure water were used.

In the present study, a pure bacterial culture i.e. , B. stearothermophilus (Strain-LLD-16) was obtained from the bacterial culture collection unit of the Department of Biochemistry, Imperial College of Science, Technology & Medicine, London, UK. Initially, the LLD-16 was grown in a temperature controlled incubator at 70 °C in a sterile mineral salt (MS) medium additionally supplemented with 1% xylose and 0.1% yeast extract for the development of a homogenous inoculum suspension. The main constituents of the MS medium were as follows (g/L): Citric acid, 0.32; Disodium hydrogen orthophosphate (anhydrous) 0.5; Magnesium sulphate (heptahydrate) 0.27; Potassium sulphate 0.5; Ammonium chloride 2.0; Calcium chloride (hexahydrate) 0.5 mL from stock (10 g/L in water); Manganese chloride (tetrahydrate) 0.25 mL from stock (12 g/L in water); Trace elements solution (TE) 1.0 mL from the stock. All solutions were prepared with distilled water and autoclaved at 121 °C for 20 min. The pH of the medium was adjusted to 7.0 prior to sterilization. After the stipulated incubation time period (24 h), the incubated bacterial culture was used subsequently for further experimental work to interrogate the nutritional potential of added supplements on the growth of B. stearothermophilus LLD-16 and ethanol production.

Each of the solution was prepared as stock solutions in concentrations 20 times higher than those mentioned here. Each solution was prepared separately in deionized water and warmed, if necessary, to dissolve all of the added supplements. All the solutions were filter-sterilized. Following final compositions were used in the medium: all amino acids used were 50.0 mg/L except Asparagine (Asn) 25.0 mg/L; Cysteine (Cys) 25.0 mg/L; Glutamate (Glu) 100.0 mg/L and Isoleucine (Ile) 25.0 mg/L. Vitamins compositions used were as: Biotin 1.0 mg/L; Nicotinic acid (Vit. B₃) 1.5 mg/L; Pyridoxine HCl 0.5 mg/L; Riboflavin 0.5 mg/L; Thiamine HCl (Vit. B₁) 1.0 mg/L, p -aminobenzoic acid (PABA) 0.1 mg/L; Folic acid 0.05 mg/L; Pantothenic acid (Pant. Acid) 0.2 mg/L; Cyanocobalamine (Vit. B₁₂) 0.02 mg/L.

For batch growth studies, the cells were initially grown in the medium containing mineral salts, five vitamins (biotin, nicotinic acid, pyridoxine, riboflavin, thiamine HCl), and all amino acids of five families ( Fig. 1 ). The amino acids of one family were removed in the presence of all other amino acids. Once it was known which amino acid families were required for the growth, then essential amino acids of each of these families were determined. This was done by putting the amino acids of one family in various combinations in the medium in the presence of all amino acids of other required families. Finally, all the required families were replaced by their respective essential amino acids. Once this was worked out in the batch studies, their requirement was studied in chemostat by Pulse and Shift technique as described below.

The chemostat was set at a dilution rate of 0.2 h⁻¹ with a medium whose compositions was arbitrarily determined by shake bottle experiments. The cells were allowed to grow for 3 to 5 volume changes. Then production of carbon dioxide from the exit gases and change in OD were observed by methods as mentioned earlier. The concentrations nutritional supplements were then reduced stepwise until the steady state biomass concentration started to decrease. Then at these amino acids and vitamins concentrations when steady state was obtained, one of the amino acids was injected (6 mg/L) and the response was observed by noting the OD over 6–8 h.