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There is a progressive increase in numbers of bacteria along the jejunum and ileum, from approximately 10⁴ in the jejunum to 10⁷ colony-forming units per gram of contents at the ileal end, with a predominance of Gram-negative aerobes and some obligate anaerobes [8]. In contrast, the large intestine is heavily populated by anaerobes and bacteria counts reach densities around 10¹² colony-forming units per gram of luminal contents because transit time is slow and microorganisms have the opportunity to proliferate by fermenting available substrates derived from either the diet or endogenous secretions. Because bacteria represent the sole source of gut H₂ and CH₄, fasting breath H₂ and CH₄ gases have been used as markers of colonic fermentation [12, 13]. As H₂ production increases when a small amount of carbohydrate is supplied to colonic bacteria, the measurement of breath H₂ concentration has been proposed as an indicator of carbohydrate malabsorption [14]. H₂ gas is produced at a rate of 4 liters for every 12.5 g of undigested carbohydrate. Since it has been reported that 2-20% of carbohydrates escape small intestinal absorption [16] and men in their 40s consume 428 ± 72 g of carbohydrates [17], the calculated amount of H₂ produced in the colon is 2.7-27 liters/day (428 g × 0.2 × 4 liters/12.5 g = 27.4). Approximately 20% of all H₂ ingested or produced is eliminated via the lungs; the rest is either consumed or expelled via the rectum.

Several mechanisms of H₂ utilization have been reported in the human large intestine including methanogenesis, dissimilating sulfate reduction, and acetogenesis. The latter process corresponds to the reduction of 2 mol of CO₂ by 4 mol of H₂ to form 1 mol of acetate [18]. This process greatly decreases colonic gas volume. Methanobrevibacter smithii, which uses H₂ to reduce CO₂ to CH₄, is responsible for almost all the CH₄ produced in the intestine [19]. Since CH₄ production occurs primarily in the left colon whereas H₂ is produced primarily in the right colon [20], H₂ produced in the left colon may be rapidly converted to CH₄. CH₄ appears in the breath only when the numbers of methanogenic bacteria reach a critical level, about 10⁸/g dry weight counts [19].