ARTICLE On-Line Near Infrared Monitoring of Glycerol-Boosted Anaerobic Digestion Processes: Evaluation of Process Analytical Technologies Jens Bo Holm-Nielsen, Carina Juel Lomborg, Piotr Oleskowicz-Popiel, Kim H. Esbensen Applied Chemometrics, Analytical Chemistry, Acoustic Chemometrics, Applied Biotechnology, Bioenergy and Sampling Research Group, Esbjerg Institute of Technology, Aalborg University, Niels Bohrs Vej 8, 6700 Esbjerg, Denmark; telephone: þ45-7912-7715; fax: þ45-6650-1091; e-mail: jhn@aaue.dk Received 29 March 2007; revision received 7 June 2007; accepted 26 June 2007 Published online 11 July 2007 in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/bit.21571 ABSTRACT: A study of NIR as a tool for process monitoring of thermophilic anaerobic digestion boosted by glycerol has been carried out, aiming at developing simple and robust Process Analytical Technology modalities for on-line sur- veillance in full scale biogas plants. Three 5 L laboratory fermenters equipped with on-line NIR sensor and special sampling stations were used as a basis for chemometric multivariate calibration. NIR characterisation using Trans- flexive Embedded Near Infra-Red Sensor (TENIRS) equip- ment integrated into an external recurrent loop on the fermentation reactors, allows for representative sampling, of the highly heterogeneous fermentation bio slurries. Glycerol is an important by-product from the increasing European bio-diesel production. Glycerol addition can boost biogas yields, if not exceeding a limiting 5–7 g L 1 concentration inside the fermenter—further increase can cause strong imbalance in the anaerobic digestion process. A secondary objective was to evaluate the effect of addition of glycerol, in a spiking experiment which introduced increas- ing organic overloading as monitored by volatile fatty acids (VFA) levels. High correlation between on-line NIR deter- minations of glycerol and VFA contents has been documen- ted. Chemometric regression models (PLS) between glycerol and NIR spectra needed no outlier removals and only one PLS-component was required. Test set validation resulted in excellent measures of prediction performance, precision: r 2 ¼ 0.96 and accuracy ¼ 1.04, slope of predicted versus reference fitting. Similar prediction statistics for acetic acid, iso-butanoic acid and total VFA proves that process NIR spectroscopy is able to quantify all pertinent levels of both volatile fatty acids and glycerol. Biotechnol. Bioeng. 2008;99: 302–313. ß 2007 Wiley Periodicals, Inc. KEYWORDS: anaerobic digestion (AD); near infrared spectroscopy (NIR); on-line measurement; volatile fatty acids (VFA); glycerol; process analytical technologies (PAT) Introduction Bioethanol and biodiesel are the main biofuels utilized in the transportation sector world-wide, comprising of a total volume of 23.3 million tonnes in 2003, of which 6.4% was biodiesel (Mandil, 2004). The largest contributors of biofuels Brazil and the US produce mainly bioethanol. In Europe the production of biodiesel in the same year comprised 78% of the total amount of biofuels produced, resulting in 65% of the total world capacity. Further significant increase is planned for biofuels in Europe. A likely vision for year 2030 will be to exchange one fourth of the EU transportation fossil fuels by biofuels according to the Biofuels Research Advisory Council the predicted energy demand for transport is estimated to be 440 million tonnes of oil equivalents. Biodiesel is produced by a direct trans-esterification of vegetable oils: rapeseed, palm, soybean, sunflower and other organic oil products or by-products. The main by-product from biodiesel production is glycerol with varying purity. When the conventional catalytic processes using caustic soda or sodium methylate process, known as the Fatty Acid Methyl Ester (FAME), is applied, purity falls between 80% and 95%. When applying a new heterogeneous process, consisting of mixtures of oxides of zinc and aluminium, a purity of 98% can be reached (Bournay et al., 2005). The glycerol helps to make the biodiesel production more economically feasible as it is a valuable by-product used by the cosmetic, medicine, and food industries. The industrial glycerol market is limited, so when the biodiesel production capacity is expanding glycerol ends up as a relatively low value energy by-product raising the cost of the biodiesel production (Mandil, 2004). Correspondence to: J.B. Holm-Nielsen 302 Biotechnology and Bioengineering, Vol. 99, No. 2, February 1, 2008 ß 2007 Wiley Periodicals, Inc.