Electronic Thesis and Dissertation Repository


Doctor of Philosophy


Chemical and Biochemical Engineering


Nakhla, George

2nd Supervisor

Hafez, Hisham


3rd Supervisor

Nasr, Fayza A. (National Research Center, Egypt)



The growing concerns regarding climate change, population growth, depletion of fossil fuel, and pollution arising from the combustion of petroleum-based fuel can be identified as the most important factors driving the urgent need for environmentally friendly renewable energy. Among all the recognized alternatives to gasoline-based fuel, hydrogen is not only considered as a clean energy but also it has a high energy content of 142 kJ/g which is almost three times higher compared to other fossil fuels. Only water and heat are the by-products of hydrogen combustion. Dark fermentative hydrogen production is a feasible option in which inexpensive, low-grade, carbohydrate-rich, and renewable lignocellulosic biomass can be used as a substrate and anaerobic digester sludge (ADS) as a seed for biohydrogen production.

Lignocellulosic substances are abundant in nature and are suitable for dark fermentative hydrogen production. Pretreatment of these carbohydrate-rich materials is required to get rid of lignin and increase the readily biodegradable sugars required for fermentation. There are several methods to break down the rigid structure of lignin and increase the fermentable sugar content. Although chemical treatment may be appropriate, it produces not only readily biodegradable sugars but also other by-products which inhibit microbial growth.

The main purpose of this study was to assess the significance of acclimatization and the impact of furfural inhibition in both batches and continuous-flow systems for biohydrogen production from synthetic lignocellulosic hydrolysates. First, acclimatization of ADS was tested for biohydrogen production in a patented continuous-flow system known as integrated biohydrogen reactor clarifier systems (IBRCS), and in batches. IBRCS, R1, was fed initially with glucose at a concentration of 10 g/L (phase 1) and then the feed was switched to a mixture of C6 and C5 sugars: glucose, cellobiose, xylose, arabinose at a concentration of 2.5 g/L each (phase 2) and then the feed reverted to glucose at the same concentration of 10 g/L (phase 3). The results showed that hydrogen production yields were negatively affected by changing the feed substrates, despite their biodegradability. Additionally, propionate, which is not favorable for both biohydrogen and biomethane production, was predominant as a result of feed changes. This was evident by microbial community analysis which revealed that the propionate-producing Megasphaera were predominant while the hydrogen and acetate-producing bacteria i.e. Clostridium were washed out after switching substrates in phases 2 and 3. On the other hand, neither hydrogen yields nor volatile fatty acids (VFAs) distribution was negatively affected in the batch study, but rather changing the feed from mono substrate to co-substrate enhanced the hydrogen production yields. A confirmation experiment has been conducted in IBRCS, R2, to investigate the effect of feed changes on the acclimatized anaerobic hydrogen-producing mesophilic mixed cultures where the system was initially fed with a mixture of C5 and C6 sugars similar in concentration and composition to R1 in the second phase of this project. The results showed a significantly higher hydrogen production yields in R2 compared to R1 phase 2 (1.9 mol H2/mol sugar vs 1.1 mol H2/mol sugar) verifying that the reduction in hydrogen yields resulted from feed changes.

Second, the impact of furfural inhibition on biohydrogen production was investigated in both continuous-flow systems and batch studies. In the continuous-flow systems, IBRCS were used to test glucose and xylose individually in presence of gradual increase of furfural concentrations from 0-4 g/L for mesophilic biohydrogen production. The results of this study showed that the biohydrogen-producing microorganisms in both glucose-fed reactor and xylose-fed reactor behaved similarly towards furfural inhibition. The acclimatized anaerobic mesophilic hydrogen-producing cultures could tolerate furfural inhibition up to 2 g/L with 29% percent reduction of the hydrogen yields compared to the control phase with 0 g/L furfural in the feed. However, the furfural inhibition threshold level ranged from 2-4 g/L. The revivability of the inhibited cultures from the glucose-fed reactor at 4 g/L furfural was assessed by removing furfural from the feed. The revivability of the inhibited cultures was proven as evident by the 95% recovery of the specific hydrogen production rate. On the other hand, synthetic lignocellulosic hydrolysate comprised mainly of 76% (by weight) xylose, 10% glucose, 9% arabinose, and the rest a mixture of other sugars i.e. galactose and mannose was investigated as a substrate at concentrations of 2-32 g/L in the presence of furfural at concentrations of 0, 1, and 2 g/L. The results showed that furfural completely inhibits the biohydrogen producers at 2 g/L and the optimum substrate concentration tested was 16 g/L.